TWI885227B - Nucleic acid detection kit and nucleic acid detection method - Google Patents

Abstract

A GAPDH nucleic acid detection kit includes a primer set for detecting GAPDH nucleic acid. The primer set for detecting GAPDH nucleic acid includes a forward internal primer for GAPDH nucleic acids, a forward outer primer for GAPDH nucleic acids, a backward internal primer for GAPDH nucleic acids and a backward outer primer for GAPDH nucleic acids. The primer set for detecting GAPDH nucleic acid is used in a loop-mediated isothermal amplification (LAMP).

Description

Nucleic acid detection kit and nucleic acid detection method

The present disclosure relates to a nucleic acid detection kit and a nucleic acid detection method, and in particular to a GAPDH nucleic acid detection kit, a target nucleic acid detection kit or a novel coronavirus nucleic acid detection kit and a detection method.

COVID-19 is caused by the novel coronavirus (SARS-CoV-2). As of February 20, 2021, 110 million people have been infected worldwide and more than 2.46 million people have died. The clinical symptoms of COVID-19 vary, ranging from severe pneumonia, respiratory distress, loss of taste, to asymptomatic infection.

The current detection method for the novel coronavirus mainly includes collecting upper respiratory tract specimens (nasopharyngeal or throat swabs) or lower respiratory tract specimens (sputum, endotracheal extracts or bronchoalveolar lavage fluid), extracting nucleic acids from the specimens, and then testing them with quantitative reverse transcription polymerase reaction (RT-qPCR, Quantitative reverse transcription PCR), and the screening time is about 4 hours. The tester must wear protective measures such as gloves, isolation clothing and goggles, and this process requires a lot of manpower and supplies. In addition, the collection of upper respiratory tract specimens (nasopharyngeal or throat swabs) or lower respiratory tract specimens is invasive, which can easily cause discomfort to the testee, causing sneezing or coughing and other droplets, and thus creating a potential risk of virus spread.

Therefore, there is an urgent need for a novel nucleic acid detection kit and method, which not only has a good detection sensitivity but also can use non-invasively collected samples for testing.

The present disclosure provides a GAPDH nucleic acid detection kit, including a primer set for detecting GAPDH nucleic acid. The primer set for detecting GAPDH nucleic acid includes: a forward inner primer for GAPDH nucleic acid; a forward outer primer for GAPDH nucleic acid; a reverse inner primer for GAPDH nucleic acid; and a reverse outer primer for GAPDH nucleic acid. The forward inner primer for GAPDH nucleic acid is composed of a first fragment and a second fragment, and the 3' end of the first fragment is connected to the 5' end of the second fragment, or the forward inner primer for GAPDH nucleic acid is composed of a first fragment, a first linker and a second fragment, and the 3' end of the first fragment is connected to the 5' end of the first linker, and the 3' end of the first linker is connected to the 5' end of the second fragment. The first fragment has about 10-30 nucleotides and is composed of a complementary strand of a first sequence segment, and the first sequence segment is located between the 134th position and the 175th position of the nucleotide sequence of sequence identification number: 1. The second fragment has 10-30 nucleotides and is composed of a second sequence segment, and the second sequence segment is located between the 77th position and the 115th position of the nucleotide sequence of sequence identification number: 1, and the first linker is composed of 1-6 thymines (thymine, T) or peptide nucleic acid (peptide nucleic acid, PNA). The forward outer primer for GAPDH nucleic acid has about 10-30 nucleotides and is composed of a third sequence segment, and the third sequence segment is located between the 42nd position and the 79th position of the nucleotide sequence of sequence identification number: 1. The reverse inner primer for GAPDH nucleic acid is composed of a third segment and a fourth segment, and the 3' end of the third segment is connected to the 5' end of the fourth segment, or the reverse inner primer for GAPDH nucleic acid is composed of a third segment, a second linker and a fourth segment, and the 3' end of the third segment is connected to the 5' end of the second linker, and the 3' end of the second linker is connected to the 5' end of the fourth segment. The third fragment has about 10-30 nucleotides and is composed of a fourth sequence segment, and the fourth sequence segment is located between the 156th position and the 207th position of the nucleotide sequence of sequence identification number: 1. The fourth fragment has about 10-30 nucleotides and is composed of a complementary strand of a fifth sequence segment, and the fifth sequence segment is located between the 211th position and the 250th position of the nucleotide sequence of sequence identification number: 1, and the second linker is composed of 1-6 thymines or peptide nucleic acid. The reverse outer primer for GAPDH nucleic acid has about 10-30 nucleotides and is composed of a complementary strand of a sixth sequence segment, and the sixth sequence segment is located between the 238th position and the 275th position of the nucleotide sequence of sequence identification number: 1. Furthermore, the primer set for detecting GAPDH nucleic acid is used in a loop-mediated isothermal amplification (LAMP) method, and the loop-mediated isothermal amplification method includes a standard loop-mediated isothermal amplification method or a reverse transcription loop-mediated isothermal amplification (RT LAMP) method.

The present disclosure also provides a target nucleic acid detection kit, including a primer set for detecting GAPDH nucleic acid and a primer set for detecting target nucleic acid. The primer set for detecting GAPDH nucleic acid includes: a forward inner primer for GAPDH nucleic acid; a forward outer primer for GAPDH nucleic acid; a reverse inner primer for GAPDH nucleic acid; and a reverse outer primer for GAPDH nucleic acid. The forward inner primer for GAPDH nucleic acid is composed of a first segment and a second segment, and the 3' end of the first segment is connected to the 5' end of the second segment, or the forward inner primer for GAPDH nucleic acid is composed of a first segment, a first linker and a second segment, and the 3' end of the first segment is connected to the 5' end of the first linker, and the 3' end of the first linker is connected to the 5' end of the second segment, wherein the first segment has about 10-30 nucleotides , and is composed of a complementary strand of a first sequence segment, and the first sequence segment is located between the 134th position and the 175th position of the nucleotide sequence of sequence identification number: 1, and the second fragment has about 10-30 nucleotides and is composed of a second sequence segment, and the second sequence segment is located between the 77th position and the 115th position of the nucleotide sequence of sequence identification number: 1, and the first linker is composed of 1-6 thymines or peptide nucleic acid. The forward outer primer for GAPDH nucleic acid has about 10-30 nucleotides and is composed of a third sequence segment, and the third sequence segment is located between the 42nd position and the 79th position of the nucleotide sequence of sequence identification number: 1. The reverse inner primer for GAPDH nucleic acid is composed of a third segment and a fourth segment, and the 3' end of the third segment is connected to the 5' end of the fourth segment, or the reverse inner primer for GAPDH nucleic acid is composed of a third segment, a second linker and a fourth segment, and the 3' end of the third segment is connected to the 5' end of the second linker, and the 3' end of the second linker is connected to the 5' end of the fourth segment, wherein the third segment has about 10-30 nucleotides , and is composed of a fourth sequence segment, and the fourth sequence segment is located between the 156th position and the 207th position of the nucleotide sequence of sequence identification number: 1, and the fourth segment has about 10-30 nucleotides and is composed of a complementary strand of a fifth sequence segment, and the fifth sequence segment is located between the 211th position and the 250th position of the nucleotide sequence of sequence identification number: 1, and the second linker is composed of 1-6 thymines or peptide nucleic acid. The reverse exon primer for GAPDH nucleic acid has about 10-30 nucleotides and is composed of a complementary strand of a sixth sequence segment, and the sixth sequence segment is located between the 238th position and the 275th position of the nucleotide sequence of sequence identification number: 1. Furthermore, the primer set for detecting the target nucleic acid includes: a forward inner primer for the target nucleic acid; a forward outer primer for the target nucleic acid; a reverse inner primer for the target nucleic acid; and a reverse outer primer for the target nucleic acid. The detection target of the primer set for detecting the target nucleic acid is different from the detection target of the primer set for detecting the GAPDH nucleic acid. The primer set for detecting GAPDH nucleic acid and the primer set for detecting target nucleic acid are used in a first constant temperature circular nucleic acid amplification method and a second constant temperature circular nucleic acid amplification method, respectively, and the first constant temperature circular nucleic acid amplification method and the second constant temperature circular nucleic acid amplification method independently include a standard constant temperature circular nucleic acid amplification method or a reverse transcription constant temperature circular nucleic acid amplification method. In addition, the result of the first constant temperature circular nucleic acid amplification method can be used as an internal control group.

The present disclosure also provides a novel coronavirus detection kit, including: a primer set for detecting the nucleic acid of the novel coronavirus. The primer set for detecting the nucleic acid of the novel coronavirus includes: a forward inner primer for the nucleic acid of the novel coronavirus; a forward outer primer for the nucleic acid of the novel coronavirus; a reverse inner primer for the nucleic acid of the novel coronavirus; and a reverse outer primer for the nucleic acid of the novel coronavirus. The forward inner primer for the nucleic acid of the novel coronavirus is composed of a fifth segment and a sixth segment, and the 3' end of the fifth segment is connected to the 5' end of the sixth segment, or the forward inner primer for the target nucleic acid is composed of a fifth segment, a third linker and a sixth segment, and the 3' end of the fifth segment is connected to the 5' end of the third linker, and the 3' end of the third linker is connected to the 5' end of the sixth segment. The fifth segment has about 10-30 nucleotides and is composed of a complementary strand of a seventh sequence segment, and the seventh sequence segment is located between the 90th and 134th positions of the nucleotide sequence of sequence identification number: 11, and the sixth segment has about 10-30 nucleotides and is composed of an eighth sequence segment, and the eighth sequence segment is located between the 45th and 82nd positions of the nucleotide sequence of sequence identification number: 11, and the third linker is composed of 1-6 thymines or peptide nucleic acids. The forward outer primer for the nucleic acid against the novel coronavirus has about 10-30 nucleotides and is composed of a ninth sequence segment, and the ninth sequence segment is located between the 27th and 64th positions of the nucleotide sequence of sequence identification number: 11. The reverse inner primer for the nucleic acid of the novel coronavirus is composed of a seventh segment and an eighth segment, and the 3' end of the seventh segment is connected to the 5' end of the eighth segment, or the reverse inner primer for the target nucleic acid is composed of a seventh segment, a fourth linker and an eighth segment, and the 3' end of the seventh segment is connected to the 5' end of the fourth linker, and the 3' end of the fourth linker is connected to the 5' end of the eighth segment. The seventh fragment has about 10-30 nucleotides and is composed of a tenth sequence segment, and the tenth sequence segment is located between the 123rd and 165th positions of the nucleotide sequence of sequence identification number: 11. The eighth fragment has about 10-30 nucleotides and is composed of a complementary strand of an eleventh sequence segment, and the eleventh sequence segment is located between the 170th and 208th positions of the nucleotide sequence of sequence identification number: 11, and the fourth linker is composed of 1-6 thymines or peptide nucleic acids. The reverse outer primer for the nucleic acid of the novel coronavirus has about 10-30 nucleotides and is composed of a complementary strand of a twelfth sequence segment, and the twelfth sequence segment is located between the 226th position and the 263rd position of the nucleotide sequence of sequence identification number: 11. In addition, the primer set for detecting the nucleic acid of the novel coronavirus is used in a constant temperature circular nucleic acid amplification method, and the constant temperature circular nucleic acid amplification method includes a standard constant temperature circular nucleic acid amplification method or a reverse transcription constant temperature circular nucleic acid amplification method.

In addition, the present disclosure provides a method for detecting GAPDH nucleic acid, comprising: (a) providing a sample to be tested; and (b) subjecting the sample to be tested to the constant temperature circular nucleic acid amplification method using the primer set for detecting GAPDH nucleic acid in the above-mentioned GAPDH nucleic acid detection kit. If the sample to be tested contains GAPDH nucleic acid, a GAPDH nucleic acid amplification product is obtained from the constant temperature circular nucleic acid amplification method. The constant temperature circular nucleic acid amplification method includes a standard constant temperature circular nucleic acid amplification method or a reverse transcription constant temperature circular nucleic acid amplification method.

The present disclosure also provides a method for detecting a novel coronavirus, comprising: (a) providing a sample to be tested; and (b) subjecting the sample to be tested to a constant temperature circular nucleic acid amplification method using the primer set for detecting the nucleic acid of the novel coronavirus in the novel coronavirus detection kit described above. If the sample to be tested contains the novel coronavirus, a nucleic acid amplification product of the novel coronavirus is obtained from the constant temperature circular nucleic acid amplification method. Furthermore, the constant temperature circular nucleic acid amplification method includes a standard constant temperature circular nucleic acid amplification method or a reverse transcription constant temperature circular nucleic acid amplification method.

In order to make the above and other purposes, features, and advantages of the present invention more clearly understood, the following is a detailed description of the preferred embodiments with reference to the accompanying drawings as follows:

The present disclosure may provide a GAPDH nucleic acid detection kit, which may include a primer set for detecting GAPDH nucleic acid, but is not limited thereto.

The primer set for detecting GAPDH nucleic acid in the GAPDH nucleic acid detection kit disclosed above can be used in a loop-mediated isothermal amplification (LAMP) method to confirm whether GAPDH nucleic acid exists in a sample to be tested.

The above-mentioned constant temperature circular nucleic acid amplification method may include, but is not limited to, a standard constant temperature circular nucleic acid amplification method or a reverse transcription loop-mediated isothermal amplification method (RT LAMP).

The sample to be tested may be a sample that has not been subjected to any purification treatment, such as a sample that has not been subjected to nucleic acid purification treatment. That is, by using the primer set for detecting GAPDH nucleic acid in the GAPDH nucleic acid detection kit disclosed above, a nucleic acid amplification reaction can be performed on a biological sample that has not been subjected to any purification treatment to obtain accurate GAPDH nucleic acid detection results, thereby achieving the effect of reducing or eliminating the need to process the sample to be tested.

The sources of the above-mentioned samples to be tested may include, but are not limited to, saliva samples, sputum samples, nose swab samples, throat swab samples, nasopharyngeal samples, urine samples, stool samples, rectal swab samples, cerebrospinal fluid (CSF) samples, body fluid samples, etc.

In one embodiment, the source of the sample to be tested can be a sample obtained by non-invasive sampling, such as a saliva sample, a sputum sample, a urine sample, a feces sample, etc., but not limited thereto. In this embodiment, the GAPDH nucleic acid detection kit disclosed herein can be used in conjunction with a thermostatic reaction machine and a simple analysis strip, such as a lateral flow immunoassay test strip, to achieve home testing.

Furthermore, the primer set for detecting GAPDH nucleic acid in the GAPDH nucleic acid detection kit disclosed herein can be used for the constant temperature circular nucleic acid amplification method using single strand RNA or first strand cDNA as the starting template, but is not limited thereto.

The design of the primer set for detecting GAPDH nucleic acid in the GAPDH nucleic acid detection kit disclosed herein is based on a constant temperature cyclic amplification method, but the designed primer set is not only applicable to the constant temperature cyclic amplification method. The design and operation principle of the primer set for detecting GAPDH nucleic acid in the GAPDH nucleic acid detection kit disclosed herein are shown in FIG. 1A, FIG. 1B and FIG. 1C.

See Figure 1A, Figure 1B and Figure 1C. As shown in Figure 1A, six regions are selected from a target nucleic acid (such as an RNA sequence) 100, namely, the F1 region, the F2 region, the F3 region, the B1c region, the B2c region and the B3c region (the complementary strands are the F1c region, the F2c region, the F3c region, the B1 region, the B2 region and the B3 region). In this method, four primers are used, namely, a specially designed forward internal primer (FIP) 101, a forward outer primer (forward outer primer) 103, a backward internal primer (BIP) 105 and a backward outer primer (backward outer primer) 107, wherein the sequence of the forward inner primer 101 is composed of a first fragment (the complementary strand of the sequence of the F1 region, i.e., the sequence of the F1c region) and a second fragment (the sequence of the F2 region), the sequence of the forward outer primer 103 is the sequence of the F3 region, and the sequence of the reverse inner primer 105 is composed of a third fragment (the sequence of the B1c region, i.e., the complementary strand of the sequence of the B1 region) and a fourth fragment (the complementary strand of the sequence of the B2c region, i.e., the sequence of the B2 region), and the sequence of the reverse outer primer 107 is the complementary strand of the sequence of the B3c region (i.e., the sequence of the B3 region).

During the isothermal cyclic amplification method, the second fragment (sequence of the F2 region) along the inner primer 101 will adhere to the F2c region of the complementary strand (e.g., cDNA) of the target nucleic acid 100 to undergo complementary strand synthesis reaction, thereby synthesizing a first strand having the sequence of the first fragment (the complementary strand of the sequence of the F1 region, i.e., the sequence of the F1c region), the second fragment (the sequence of the F2 region), the F1 region, the B1c region, the B2c region, and the B3c region. The outer primer 103 will move this strand away, thereby synthesizing a second strand having the sequence of the F3 region, the F2 region, the F1 region, the B1c region, the B2c region, and the B3c region. Next, the fourth fragment (sequence of the B2 region) of the reverse inner primer 105 adheres to the B2c region of the first strand, and a third strand having the sequence of the B1c region, the B2 region, the B1 region, the F1c region, the F2c region, and the F1 region is synthesized using the first strand as a template. Afterwards, the reverse outer primer 107 removes the third strand and synthesizes a fourth strand having the sequence of the B3 region, the B2 region, the B1 region, the F1c region, the F2c region, and the F1 region. The B1c region and the B1 region of the third strand will self-adhere, and similarly, the F1 region and the F1c region will also self-adhere, so that the third strand becomes a strand with stem loops formed at both ends. Then, the forward inner primer and the reverse inner primer sequentially carry out complementary strand synthesis reaction according to this strand and/or its complementary strand synthesis product as a template, thereby forming a double-stranded product with multiple stem loops (see Figure 1A and Figure 1B again).

In addition, a forward loop primer (FLP) 109 may be further designed between the F1 region and the F2 region, and a backward loop primer (BLP) 111 may be further designed between the B1c region and the B2c region to enhance the efficiency of the constant temperature circular nucleic acid amplification method (see FIG. 1C ).

Therefore, the primer set for detecting GAPDH nucleic acid in the GAPDH nucleic acid detection kit disclosed above may include at least a forward inner primer for GAPDH nucleic acid, a forward outer primer for GAPDH nucleic acid, a reverse inner primer for GAPDH nucleic acid and a reverse outer primer for GAPDH nucleic acid, but is not limited thereto.

The sequence of the GAPDH nucleic acid used to design the above primer set may be the nucleotide sequence of sequence identification number: 1, which is a part of a GAPDH mRNA sequence (NCBI accession number NM_001256799), but is not limited thereto.

The forward inner primer for GAPDH nucleic acid may be composed of a first segment and a second segment, and the 3' end of the first segment is connected to the 5' end of the second segment, or the forward inner primer for GAPDH nucleic acid may be composed of a first segment, a first linker and a second segment, and the 3' end of the first segment is connected to the 5' end of the first linker, and the 3' end of the first linker is connected to the 5' end of the second segment. The first segment may have about 10-30 nucleotides and may be composed of a complementary strand of a first sequence segment, and the first sequence segment may be located between the 134th position and the 175th position of the nucleotide sequence of sequence identification number: 1, but is not limited thereto. The second fragment may have about 10-30 nucleotides and may be composed of a second sequence segment, and the second sequence segment may be located between the 77th position and the 115th position of the nucleotide sequence of sequence identification number: 1, but is not limited thereto. The first linker may include about 1-6 thymines (T) or peptide nucleic acids (PNA), but is not limited thereto.

The forward outer primer for GAPDH nucleic acid may have about 10-30 nucleotides and may be composed of a third sequence segment, and the third sequence segment may be located between the 42nd position and the 79th position of the nucleotide sequence of sequence identification number: 1, but is not limited thereto.

The reverse inner primer for GAPDH nucleic acid may be composed of a third segment and a fourth segment, and the 3' end of the third segment is connected to the 5' end of the fourth segment, or the reverse inner primer for GAPDH nucleic acid may be composed of a third segment, a second linker and a fourth segment, and the 3' end of the third segment is connected to the 5' end of the second linker, and the 3' end of the second linker is connected to the 5' end of the fourth segment. The third segment may have about 10-30 nucleotides and may be composed of a fourth sequence segment, and the fourth sequence segment may be located between the 156th position and the 207th position of the nucleotide sequence of sequence identification number: 1, but is not limited thereto. The fourth segment may have about 10-30 nucleotides and may be composed of a complementary strand of a fifth sequence segment, and the fifth sequence segment may be located between the 211th position and the 250th position of the nucleotide sequence of sequence identification number: 1, but is not limited thereto. The second linker may include about 1-6 thymines or peptide nucleic acids, but is not limited thereto.

The reverse outer primer for GAPDH nucleic acid may have about 10-30 nucleotides and may be composed of a complementary strand of a sixth sequence segment, and the sixth sequence segment may be located between the 238th position and the 275th position of the nucleotide sequence of SEQ ID No.: 1, but is not limited thereto.

In one embodiment, in the primer set for detecting GAPDH nucleic acid in the GAPDH nucleic acid detection kit disclosed herein, the first sequence segment may be located between the 139th position and the 170th position of the nucleotide sequence of sequence identification number: 1, the second sequence segment may be located between the 82nd position and the 110th position of the nucleotide sequence of sequence identification number: 1, and the third sequence segment may be located between the 82nd position and the 110th position of the nucleotide sequence of sequence identification number: 1. The fourth sequence segment may be located between the 47th position and the 74th position of the nucleotide sequence of sequence identification number: 1, the fifth sequence segment may be located between the 216th position and the 245th position of the nucleotide sequence of sequence identification number: 1, and the sixth sequence segment may be located between the 243rd position and the 270th position of the nucleotide sequence of sequence identification number: 1.

Furthermore, in a specific embodiment, in the primer set for detecting GAPDH nucleic acid in the GAPDH nucleic acid detection kit disclosed above, the sequence of the forward inner primer for GAPDH nucleic acid may include the nucleotide sequence of sequence identification number: 2, the sequence of the forward outer primer for GAPDH nucleic acid may include the nucleotide sequence of sequence identification number: 3, the sequence of the reverse inner primer for GAPDH nucleic acid may include the nucleotide sequence of sequence identification number: 4, the nucleotide sequence of sequence identification number: 6 or the nucleotide sequence of sequence identification number: 7, and the sequence of the reverse outer primer for GAPDH nucleic acid may include the nucleotide sequence of sequence identification number: 5.

In another embodiment, in the primer set for detecting GAPDH nucleic acid in the GAPDH nucleic acid detection kit disclosed above, at least one of the forward inner primer for GAPDH nucleic acid, the forward outer primer for GAPDH nucleic acid, the reverse inner primer for GAPDH nucleic acid, and the reverse outer primer for GAPDH nucleic acid, starting from any one of the 4th to 14th positions from the 3' end thereof, 1 to 10 nucleotides can be independently substituted by inosine (I), guanine (G), uracil (U), etc., but is not limited thereto. For example, in one embodiment, in the primer set for detecting GAPDH nucleic acid in the GAPDH nucleic acid detection kit disclosed herein, at least one of the forward inner primer for GAPDH nucleic acid, the forward outer primer for GAPDH nucleic acid, the reverse inner primer for GAPDH nucleic acid, and the reverse outer primer for GAPDH nucleic acid may include, but is not limited to, the following substitutions: 2 to 7 nucleotides starting from any one of the 5th to 9th positions from the 3' end thereof may be independently substituted by inosine, guanine, uracil, etc., 3 to 5 nucleotides starting from the 7th position from the 3' end thereof may be independently substituted by inosine, or 3 to 5 nucleotides starting from the 9th position from the 3' end thereof may be independently substituted by inosine. For example, in the primer set for detecting GAPDH nucleic acid in the above-mentioned GAPDH nucleic acid detection kit disclosed herein, the sequence of the above-mentioned reverse inner primer for GAPDH nucleic acid may include the nucleotide sequence of sequence identification number: 8, or, for example, in the primer set for detecting GAPDH nucleic acid in the above-mentioned GAPDH nucleic acid detection kit disclosed herein, the sequence of the above-mentioned reverse outer primer for GAPDH nucleic acid may include the nucleotide sequence of sequence identification number: 9. In addition, in a specific embodiment, in the primer set for detecting GAPDH nucleic acid in the GAPDH nucleic acid detection kit disclosed above, the sequence of the forward inner primer for GAPDH nucleic acid may include the nucleotide sequence of sequence identification number: 2; the sequence of the forward outer primer for GAPDH nucleic acid may include the nucleotide sequence of sequence identification number: 3; the sequence of the reverse inner primer for GAPDH nucleic acid may include the nucleotide sequence of sequence identification number: 4, and the sequence of the reverse outer primer for GAPDH nucleic acid may include the nucleotide sequence of sequence identification number: 9. In another specific embodiment, in the primer set for detecting GAPDH nucleic acid in the GAPDH nucleic acid detection kit disclosed above, the sequence of the forward inner primer for GAPDH nucleic acid may include the nucleotide sequence of sequence identification number: 2, the sequence of the forward outer primer for GAPDH nucleic acid may include the nucleotide sequence of sequence identification number: 3, the sequence of the reverse inner primer for GAPDH nucleic acid may include the nucleotide sequence of sequence identification number: 8, and the sequence of the reverse outer primer for GAPDH nucleic acid may include the nucleotide sequence of sequence identification number: 9.

Please refer to FIG. 2A, which is a schematic diagram of the mechanism of action of the lateral flow immunoassay test strip in one embodiment of the present disclosure. In FIG. 2A, the size, quantity, shape and/or structure of each element shown are only used for illustration and convenient description, and are not used to represent the actual size, quantity, shape and/or structure of each element. In one embodiment, in the primer set for detecting GAPDH nucleic acid in the GAPDH nucleic acid detection kit disclosed above, the 5' end of the reverse inner primer BIP-G for GAPDH nucleic acid can be marked with a first marker M1, and the 5' end of the forward inner primer FIP-G for GAPDH nucleic acid can be marked with a second marker M2, and wherein the first marker M1 is different from the second marker M2. The first marker M1 may include, but is not limited to, biotin, avidin, streptavidin (SA), digoxigenin (DIG), fluorescein (FAM), etc. The second marker M2 may also include, but is not limited to, biotin, avidin, streptavidin, digoxigenin, fluorescein, etc. In addition, in this embodiment, the GAPDH nucleic acid detection kit disclosed herein may further include a lateral flow immunoassay test strip 200 in addition to the primer set for detecting GAPDH nucleic acid, but is not limited to this. The material of the lateral flow immunoassay test strip 200 may include, but is not limited to, nitrocellulose membrane, nylon membrane, polyvinylidene fluoride (PVDF) membrane, polyethersulfone (PES) membrane, etc.

Refer to Figure 2A again. The lateral flow immunoassay test strip 200 may include, in order according to the flow direction of the analyte: an analyte addition zone 201, a binding zone 203, a GAPDH detection zone 205 and a test strip control zone 207. The binding zone 203 has a first binding particle 203BP, which has a first binding molecule 203B and a particle 203P connected to the first binding molecule 203B, wherein the first binding molecule 203B has the ability to bind to the first marker M1. The material of the particle 203P may include, but is not limited to, gold, carbon, latex, magnetic substances, etc. Alternatively, the binding region 203 may further include a control particle CP (not shown) coated with a specific substance in addition to the first binding particle 203BP. The material of the control particle CP may refer to the material of the particle 203P described above, but on the lateral flow immunoassay test strip 200, the materials of the control particle CP and the particle 203P may be the same or different. In addition, the specific substance is not particularly limited, as long as there is a molecule that can bind to it, and the example of the specific substance may include, but is not limited to, serum (such as mouse serum, but not limited thereto). The GAPDH detection region 205 is fixed with a second binding molecule 205B, which has the ability to bind to the second marker M2. Furthermore, the test strip control area 207 is fixed with a third binding molecule 207B, which has the ability to bind to the first binding molecule 203B of the first binding particle 203BP, wherein the third binding molecule 207B and the first marker M1 may be the same or different. Alternatively, when the binding area 203 includes a control particle CP (not shown) in addition to the first binding particle 203BP, the third binding molecule 207B fixed to the test strip control area 207 may be the ability to bind to the specific substance of the control particle CP. For example, when the specific substance coating the control particle is mouse serum, the third binding molecule 207B may be an anti-mouse serum antibody.

The following describes the reactions occurring in various regions of the lateral flow immunoassay test strip 200 shown in the schematic diagram of FIG. 2A during operation. See FIG. 2A. When a sample to be tested contains GAPDH nucleic acid, the sample to be tested is subjected to a constant temperature cyclic nucleic acid amplification method using the primer set for detecting GAPDH nucleic acid in the GAPDH nucleic acid detection kit disclosed above to obtain a reaction solution containing a GAPDH nucleic acid amplification product, and the GAPDH nucleic acid amplification product has a first marker M1 and a second marker M2. After the reaction solution is added to the analyte addition area 201 of the lateral flow immunoassay test strip 200, the reaction solution moves to the binding area 203, so that the first marker M1 of the GAPDH nucleic acid amplification product binds to the first binding molecule 203B of a portion of the first binding particle 203BP in the binding area 203, and moves to the GAPDH detection area 205 together with the remaining first binding particle 203BP that is not bound to any amplification product. In the GAPDH detection area 205, the second marker M2 of the GAPDH nucleic acid amplification product that has bound to the first binding molecule 203B of the first binding particle 203BP will bind to the second binding molecule 205B fixed to the GAPDH detection area 205, so that the GAPDH nucleic acid amplification product stays in the GAPDH detection area 205 and presents the color of the first binding particle 203BP, while the remaining first binding particle 203BP that has not bound to any amplification product continues to move to the test piece control area 207. In the above-mentioned test chip control area 207, the first binding molecule 203B of the remaining first binding particle 203BP that has not been bound to any amplification product will bind to the third binding molecule 207B fixed to the above-mentioned test chip control area 207, so that the remaining first binding particle 203BP that has not been bound to any amplification product will stay in the test chip control area 207 and present the color of the first binding particle 203BP. In contrast, when a sample to be tested does not contain GAPDH nucleic acid, the sample to be tested is subjected to the constant temperature cycle nucleic acid amplification method using the primer set for detecting GAPDH nucleic acid in the above-mentioned GAPDH nucleic acid detection kit disclosed in the present invention to obtain a reaction solution that does not contain GAPDH nucleic acid amplification products. After the reaction solution is added to the analyte addition area 201 of the lateral flow immunoassay test strip 200, the reaction solution will move to the binding area 203. However, since there is no amplification product in the reaction solution, the first binding particle 203BP in the binding area 203 will not bind to any amplification product (such as the amplification product with the first marker M1 and the second marker M2 at the same time), but will move to the GAPDH detection area 205. Similarly, since the first binding particle 203BP does not bind to any amplified product (such as the amplified product with the first marker M1 and the second marker M2 at the same time), it does not bind to the second binding molecule 205B fixed to the GAPDH detection area 205 and stays in the GAPDH detection area 205 and displays its color. Afterwards, the first binding particle 203BP continues to move to the test chip control area 207. In the test chip control area 207, the first binding molecule 203B of the first binding particle 203BP binds to the third binding molecule 207B fixed to the test chip control area 207, so that the first binding particle 203BP stays in the test chip control area 207 and displays the color of the first binding particle 203BP.

In a specific embodiment, in the primer set for detecting GAPDH nucleic acid in the GAPDH nucleic acid detection kit disclosed herein, the 5' end of the reverse inner primer BIP-G for GAPDH nucleic acid is labeled with biotin, and the 5' end of the forward inner primer FIP-G for GAPDH nucleic acid is labeled with foxgloveine. Furthermore, in this specific embodiment, the GAPDH nucleic acid detection kit disclosed herein, in addition to the primer set for detecting GAPDH nucleic acid, further includes the lateral flow immunoassay test strip 200, and in the lateral flow immunoassay test strip 200, the binding region 203 has a first binding particle 203BP, and the first binding molecule 203B of the first binding particle is avidin, the second binding molecule 205B of the GAPDH detection region 205 is an antibody that can recognize digitalis, and the third binding molecule 207B in the test strip control region 207 is biotin or an antibody that can recognize avidin.

Furthermore, in one embodiment, the GAPDH nucleic acid detection kit disclosed herein may further include a polymerase and/or a nucleotide substrate in addition to the primer set for detecting GAPDH nucleic acid, but is not limited thereto. The polymerase may have the function of a reverse transcriptase, but is not limited thereto. In a specific embodiment, the polymerase is a Bst DNA polymerase, for example, a Bst DNA polymerase whose sequence includes the amino acid sequence of sequence identification number: 10, but is not limited thereto.

Furthermore, in one embodiment, the GAPDH nucleic acid detection kit disclosed herein may further include a reverse transcriptase and/or a nucleotide substrate in addition to the primer set for detecting GAPDH nucleic acid, but is not limited thereto. The reverse transcriptase may have the function of a ribonuclease (RNase), but is not limited thereto. In a specific embodiment, the reverse transcriptase may have the function of ribonuclease H (RNase H).

The present disclosure may also provide a target nucleic acid detection kit, which may include, but is not limited to, a primer set for detecting GAPDH nucleic acid and a primer set for detecting target nucleic acid, wherein the detection target of the primer set for detecting target nucleic acid is different from the detection target of the primer set for detecting GAPDH nucleic acid.

The primer set for detecting GAPDH nucleic acid and the primer set for detecting target nucleic acid in the target nucleic acid detection kit disclosed above can be used in a first constant temperature cyclic nucleic acid amplification method and a second constant temperature cyclic nucleic acid amplification method, respectively, to confirm whether the target nucleic acid exists in a sample to be tested.

The first constant temperature circular nucleic acid amplification method and the second constant temperature circular nucleic acid amplification method may include, but are not limited to, a standard constant temperature circular nucleic acid amplification method or a reverse transcription constant temperature circular nucleic acid amplification method.

The sample to be tested may be a sample that has not been subjected to any purification treatment, such as a sample that has not been subjected to nucleic acid purification treatment. That is, by using the primer set for detecting GAPDH nucleic acid and the primer set for detecting target nucleic acid in the target nucleic acid detection kit disclosed above, a constant temperature cycle nucleic acid amplification method can be performed on a biological sample that has not been subjected to any purification treatment to obtain accurate target nucleic acid detection results, thereby achieving the effect of reducing or eliminating the need to process the sample to be tested.

The sources of the above-mentioned samples to be tested may include, but are not limited to, saliva samples, sputum samples, nasal swab samples, throat swab samples, nasopharyngeal samples, urine samples, stool samples, rectal swab samples, cerebrospinal fluid samples, body fluid samples, etc.

In one embodiment, the source of the sample to be tested can be a sample obtained by non-invasive sampling, such as a saliva sample, a sputum sample, a urine sample, a feces sample, etc., but not limited thereto. In this embodiment, the nucleic acid detection kit disclosed herein can be used in conjunction with a constant temperature reaction machine, a constant temperature heating machine, or a simple analysis test strip, such as a lateral flow immunoassay test strip, to achieve the purpose of home testing.

In addition, the primer set for detecting GAPDH nucleic acid and the primer set for detecting target nucleic acid in the target nucleic acid detection kit disclosed above can use single-stranded RNA or first-stranded cDNA as the starting template to perform the first constant temperature circular nucleic acid amplification method and the second constant temperature circular nucleic acid amplification method, respectively, but are not limited to this.

The design of the primer set for detecting GAPDH nucleic acid and the primer set for detecting target nucleic acid in the target nucleic acid detection kit of the present disclosure is also based on a constant temperature cyclic amplification method, but the designed primer set is not only applicable to the constant temperature cyclic amplification method. The design and operation principle of the primer set for detecting GAPDH nucleic acid and the primer set for detecting target nucleic acid in the target nucleic acid detection kit of the present disclosure are also shown in Figures 1A, 1B and 1C. For detailed description of the design and operation principle of the primer set for detecting GAPDH nucleic acid and the primer set for detecting target nucleic acid in the target nucleic acid detection kit of the present disclosure, please refer to the description of the design and operation principle of the primer set for detecting GAPDH nucleic acid in the GAPDH nucleic acid detection kit of the present disclosure in the previous paragraph, and therefore it will not be elaborated here.

Therefore, the primer set for detecting GAPDH nucleic acid in the target nucleic acid detection kit disclosed above can be any primer set for detecting GAPDH nucleic acid in the target nucleic acid detection kit disclosed above, and the primer set for detecting target nucleic acid in the target nucleic acid detection kit disclosed above can include a forward inner primer for the target nucleic acid, a forward outer primer for the target nucleic acid, a reverse inner primer for the target nucleic acid, and a reverse outer primer for the target nucleic acid, but is not limited thereto.

The detection target of the primer set for detecting the target nucleic acid in the target nucleic acid detection kit of the present disclosure is not particularly limited, as long as it is different from the primer set for detecting GAPDH nucleic acid in the target nucleic acid detection kit of the present disclosure.

In one embodiment, the target of the primer set for detecting the target nucleic acid in the target nucleic acid detection kit disclosed herein may be a nucleic acid of an RNA virus. Examples of the above RNA viruses may include, but are not limited to, coronavirus, influenza virus, human immunodeficiency virus (HIV), Ebola virus, and hepatitis C virus (HCV).

The above-mentioned coronaviruses may include, but are not limited to, severe acute respiratory syndrome coronavirus (SARS-CoV), severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) (novel coronavirus), Middle East respiratory syndrome coronavirus (MERS-CoV), etc.

In addition, the nucleic acid of the above-mentioned severe acute respiratory syndrome coronavirus type 2 (novel coronavirus) may include, but is not limited to, nucleic acid within the range of ORF1ab (such as nucleic acid of RdRp gene, but not limited to this), nucleic acid of spike protein (S) gene, nucleic acid of envelope (E) gene, nucleic acid of membrane protein (M) gene, nucleic acid of nucleoprotein (N) gene, etc.

In one embodiment, the detection target of the primer set for detecting the target nucleic acid in the target nucleic acid detection kit disclosed herein may be the nucleic acid of the RdRp gene of severe acute respiratory syndrome coronavirus type 2 (novel coronavirus).

In the embodiment in which the detection target of the primer set for detecting the target nucleic acid in the target nucleic acid detection kit disclosed herein may be the nucleic acid of the RdRp gene of severe acute respiratory syndrome coronavirus type 2 (novel coronavirus), the sequence of the nucleic acid of the RdRp gene used to design the above-mentioned primer set may be a sequence identification number: 11, which is a part of the nucleic acid sequence of ORF1ab of severe acute respiratory syndrome coronavirus type 2 (NCBI accession number MN908947), but is not limited thereto.

In an embodiment in which the detection target of the primer set for detecting the target nucleic acid in the target nucleic acid detection kit disclosed herein may be a nucleic acid of the RdRp gene of severe acute respiratory syndrome coronavirus type 2 (novel coronavirus), the forward inner primer for the target nucleic acid may be composed of a fifth segment and a sixth segment, and the 3' end of the fifth segment is connected to the 5' end of the sixth segment, or the forward inner primer for the target nucleic acid is composed of a fifth segment, a third linker and a sixth segment, and the 3' end of the fifth segment is connected to the 5' end of the third linker, and the 3' end of the third linker is connected to the 5' end of the sixth segment. The fifth segment may have about 10-30 nucleotides and may be composed of a complementary strand of a seventh sequence segment, and the seventh sequence segment may be located between the 90th position and the 134th position of the nucleotide sequence of SEQ ID NO: 11, but not limited thereto. The sixth segment may have about 10-30 nucleotides and may be composed of an eighth sequence segment, and the eighth sequence segment may be located between the 45th position and the 82nd position of the nucleotide sequence of SEQ ID NO: 11, but not limited thereto. The third linker may include about 1-6 thymines or peptide nucleic acid, but not limited thereto.

Furthermore, the forward outer primer targeting the target nucleic acid may have about 10-30 nucleotides and may be composed of a ninth sequence segment, and the ninth sequence segment may be located between the 27th position and the 64th position of the nucleotide sequence of sequence identification number: 11, but is not limited thereto.

The reverse inner primer for the target nucleic acid may be composed of a seventh segment and an eighth segment, and the 3' end of the seventh segment is connected to the 5' end of the eighth segment, or the reverse inner primer for the target nucleic acid is composed of a seventh segment, a fourth linker and an eighth segment, and the 3' end of the seventh segment is connected to the 5' end of the fourth linker, and the 3' end of the fourth linker is connected to the 5' end of the eighth segment. The seventh segment may have about 10-30 nucleotides and may be composed of a tenth sequence segment, and the tenth sequence segment may be located between the 123rd position and the 165th position of the nucleotide sequence of sequence identification number: 11, but is not limited thereto. The eighth segment may have about 10-30 nucleotides and may be composed of a complementary strand of an eleventh sequence segment, and the eleventh sequence segment may be located between the 170th position and the 208th position of the nucleotide sequence of sequence identification number: 11, but is not limited thereto. The fourth linker may include about 1-6 thymines or peptide nucleic acids, but is not limited thereto.

The reverse exon primer for the target nucleic acid may have about 10-30 nucleotides and may be composed of a complementary strand of a twelfth sequence segment, and the twelfth sequence segment may be located between the 226th position and the 263rd position of the nucleotide sequence of sequence identification number: 11, but is not limited thereto.

In the embodiment in which the detection target of the primer set of the target nucleic acid detection kit of the present disclosure may be the nucleic acid of the RdRp gene of severe acute respiratory syndrome coronavirus type 2 (novel coronavirus), in a specific embodiment, in the primer set of the target nucleic acid detection kit of the present disclosure, the seventh sequence segment may be located between the 95th position and the 129th position of the nucleotide sequence of sequence identification number: 11, and the eighth sequence segment may be located at the 50th position of the nucleotide sequence of sequence identification number: 11. The ninth sequence segment may be located between the 32nd and 59th positions of the nucleotide sequence of sequence identification number: 11, the tenth sequence segment may be located between the 128th and 150th positions of the nucleotide sequence of sequence identification number: 11, the eleventh sequence segment may be located between the 175th and 203rd positions of the nucleotide sequence of sequence identification number: 11, and the twelfth sequence segment may be located between the 231st and 258th positions of the nucleotide sequence of sequence identification number: 11.

Alternatively, in an embodiment in which the detection target of the primer set for detecting the target nucleic acid in the target nucleic acid detection kit disclosed herein may be the nucleic acid of the RdRp gene of severe acute respiratory syndrome coronavirus type 2 (novel coronavirus), for a specific embodiment, in the primer set for detecting the target nucleic acid in the above-mentioned target nucleic acid detection kit disclosed herein, the sequence of the forward inner primer for the target nucleic acid may include a nucleotide sequence of sequence identification number: 12, the sequence of the forward outer primer for the target nucleic acid may include a nucleotide sequence of sequence identification number: 13, the sequence of the reverse inner primer for the target nucleic acid may include a nucleotide sequence of sequence identification number: 14, and the sequence of the reverse outer primer for the target nucleic acid may include a nucleotide sequence of sequence identification number: 15.

In an embodiment in which the detection target of the primer set for detecting the target nucleic acid in the target nucleic acid detection kit disclosed herein may be the nucleic acid of the RdRp gene of severe acute respiratory syndrome coronavirus type 2 (novel coronavirus), for a specific embodiment, the primer set for detecting the target nucleic acid may further include a forward loop primer for the target nucleic acid and a reverse loop primer for the target nucleic acid, but is not limited to this.

The forward loop primer for the target nucleic acid may have about 10-30 nucleotides and may consist of a thirteenth sequence segment, and the thirteenth sequence segment may be located between the 63rd position and the 104th position of the nucleotide sequence of sequence identification number: 11. The reverse loop primer for the target nucleic acid may have about 10-30 nucleotides and may consist of a fourteenth sequence segment, and the fourteenth sequence segment may be located between the 146th position and the 187th position of the nucleotide sequence of sequence identification number: 11. Alternatively, the thirteenth sequence segment may be located between the 68th position and the 99th position of the nucleotide sequence of sequence identification number: 11, and the fourteenth sequence segment may be located between the 151st position and the 182nd position of the nucleotide sequence of sequence identification number: 11.

Alternatively, in the above-mentioned specific embodiment, the sequence of the forward inner primer for the target nucleic acid in the primer set for detecting the target nucleic acid in the target nucleic acid detection kit of the present disclosure may include a nucleotide sequence with sequence identification number: 12, the sequence of the forward outer primer for the target nucleic acid may include a nucleotide sequence with sequence identification number: 13, the sequence of the forward loop primer for the target nucleic acid may include a nucleotide sequence with sequence identification number: 16, the sequence of the reverse inner primer for the target nucleic acid may include a nucleotide sequence with sequence identification number: 14, the sequence of the reverse outer primer for the target nucleic acid may include a nucleotide sequence with sequence identification number: 15, and the sequence of the reverse loop primer for the target nucleic acid may include a nucleotide sequence with sequence identification number: 17.

In another embodiment, in the primer set for detecting target nucleic acid in the kit for detecting target nucleic acid disclosed above, at least one of the forward inner primer for target nucleic acid, the forward outer primer for target nucleic acid, the reverse inner primer for target nucleic acid, and the reverse outer primer for target nucleic acid, starting from any one of the 4th to 14th positions from the 3' end thereof, 1 to 10 nucleotides can be independently replaced by inosine (I), guanine (G), uracil (U), etc., but is not limited thereto. For example, in one embodiment, in the primer set for detecting target nucleic acid in the above-mentioned kit for detecting target nucleic acid of the present disclosure, at least one of the above-mentioned forward inner primer for target nucleic acid, the above-mentioned forward outer primer for target nucleic acid, the above-mentioned reverse inner primer for target nucleic acid and the above-mentioned reverse outer primer for target nucleic acid may include, but is not limited to, the following substitutions: 2 to 7 nucleotides starting from any one of the 5th to 9th positions from its 3' end may be independently substituted by inosine, guanine, uracil, etc., 3 to 5 nucleotides starting from the 7th position from its 3' end may be independently substituted by inosine, or 3 to 5 nucleotides starting from the 9th position from its 3' end may be independently substituted by inosine.

FIG. 2B and FIG. 2C are schematic diagrams of the mechanism of action of the lateral flow immunoassay test strip in different embodiments. In FIG. 2B and FIG. 2C, the size, quantity, shape and/or structure of each component shown are only used for illustration and convenience of description, and do not represent the actual size, quantity, shape and/or structure of each component.

In one embodiment, in the kit for detecting target nucleic acid disclosed above, the 5' end of the reverse inner primer BIP-G for GAPDH nucleic acid and the 5' end of the reverse inner primer BIP-T for target nucleic acid are marked with a first marker M1, the 5' end of the forward inner primer FIP-G for GAPDH nucleic acid is marked with a second marker M2, and the 5' end of the forward inner primer FIP-T for target nucleic acid is marked with a third marker M3, wherein the first marker M1, the second marker M2 and the third marker M3 are all different. The first marker M1 may include biotin, avidin, streptavidin, luciferin, fluorescein, etc., but is not limited thereto. The second marker M2 may also include, but not limited to, biotin, avidin, streptavidin, glutinosin, luciferin, etc. Similarly, the third marker M3 may also include, but not limited to, biotin, avidin, streptavidin, glutinosin, luciferin, etc. Furthermore, in this embodiment, the target nucleic acid detection kit disclosed herein may further include a lateral flow immunoassay test strip 200' or 200'' in addition to the primer set for detecting GAPDH nucleic acid and the primer set for detecting target nucleic acid, but not limited thereto. The material of the lateral flow immunoassay test strip 200' or 200'' may include, but not limited to, nitrocellulose membrane, nylon membrane, polyvinylidene fluoride membrane, polyether sulfide membrane, etc.

Please refer to Figure 2B and Figure 2C again. The lateral flow immunoassay test strip 200' or 200'' may include, according to the flow direction of the analyte, an analyte addition zone 201, a binding zone 203, a GAPDH detection zone 205, a target nucleic acid detection zone 206, and a test strip control zone 207 (lateral flow immunoassay test strip 200'), or may include, according to the flow direction of the analyte, an analyte addition zone 201, a binding zone 203, a target nucleic acid detection zone 206, a GAPDH detection zone 205, and a test strip control zone 207 (lateral flow immunoassay test strip 200''). The binding region has a first binding particle, wherein the first binding particle 203BP has a first binding molecule 203B and a particle 203P connected to the first binding molecule 203B, and the first binding molecule 203B has the ability to bind to the first marker M1. The material of the particle 203P may include, but is not limited to, gold, carbon, latex, magnetic material, etc. Alternatively, the binding region 203 may include, in addition to the first binding particle 203BP, a control particle CP (not shown) coated with a specific substance. The material of the control particle CP can refer to the material of the particle 203P described above, but on the lateral flow immunoassay test strip 200' or 200'', the materials of the control particle CP and the particle 203P may be the same or different. Furthermore, the above-mentioned specific substance is not particularly limited, as long as there is a molecule that can bind to it, and examples of the above-mentioned specific substance may include, but are not limited to, serum (such as mouse serum, but not limited thereto). The above-mentioned GAPDH detection area 205 is fixed with a second binding molecule 205B, which has the ability to bind to the above-mentioned second marker M2. The above-mentioned test piece control area 207 is fixed with a third binding molecule 207B, which has the ability to bind to the above-mentioned first binding molecule 203B of the above-mentioned first binding particle 203BP, wherein the above-mentioned third binding molecule 207B and the above-mentioned first marker M1 may be the same or different. Alternatively, in the case where the above-mentioned binding area 203 may further include a control particle CP (not shown) in addition to the first binding particle 203BP, the third binding molecule 207B fixed to the above-mentioned test piece control area 207 may be the above-mentioned specific substance that has the ability to bind to the above-mentioned control particle CP. For example, when the specific substance coating the control particle is mouse serum, the third binding molecule 207B can be an anti-mouse serum antibody. In addition, the target nucleic acid detection region 206 is fixed with a fourth binding molecule 206B, which has the ability to bind to the third marker M3.

The following describes the reactions occurring in various regions of the lateral flow immunoassay test strip 200' shown in the schematic diagram of FIG2B during operation. See FIG2B. When a sample to be tested contains GAPDH nucleic acid and target nucleic acid, the sample to be tested is subjected to the first constant temperature cyclic nucleic acid amplification method and the second constant temperature cyclic nucleic acid amplification method respectively using the primer set for detecting GAPDH nucleic acid and the primer set for detecting target nucleic acid in the target nucleic acid detection kit disclosed herein to obtain a first reaction solution containing a GAPDH nucleic acid amplification product and a second reaction solution containing a target nucleic acid amplification product, wherein the GAPDH nucleic acid amplification product has a first marker M1 and a second marker M2, and the target nucleic acid amplification product has a first marker M1 and a third marker M3. The first reaction solution and the second reaction solution are mixed to obtain a mixed reaction solution. After the mixed reaction solution is added to the analyte addition area 201 of the lateral flow immunoassay test strip 200', the reaction solution moves to the binding area 203, so that the first marker M1 of the GAPDH nucleic acid amplification product and the first marker M1 of the target nucleic acid amplification product are respectively bound to the first binding molecule 203B of the first binding particle 203BP in the binding area 203, and move to the GAPDH detection area 205 together with the remaining first binding particle 203BP that is not bound to any amplification product. In the GAPDH detection region 205, the second marker M2 of the GAPDH nucleic acid amplification product that has bound to the first binding molecule 203B of the first binding particle 203BP will bind to the second binding molecule 205B fixed to the GAPDH detection region 205, so that the GAPDH amplification product stays in the GAPDH detection region 205 and presents the color of the first binding particle 203BP, while the target nucleic acid amplification product that has bound to the first binding molecule 203B of the first binding particle 203BP and the remaining first binding particle 203BP that has not bound to any amplification product continue to move to the target nucleic acid detection region 206. In the target nucleic acid detection area 206, the third marker M3 of the target nucleic acid amplification product that has bound to the first binding molecule 203B of the first binding particle 203BP will bind to the fourth binding molecule 206B fixed to the target nucleic acid detection area 206, so that the target nucleic acid amplification product stays in the target nucleic acid detection area 206 and presents the color of the first binding particle 203BP, while the remaining first binding particle 203BP that has not bound to any amplification product continues to move to the test strip control area 207. In the above-mentioned test chip control area 207, the first binding molecule 203B of the remaining first binding particle 203BP that has not been bound to any amplified product will bind to the third binding molecule 207B fixed on the above-mentioned test chip control area 207, so that the remaining first binding particle 203BP that has not been bound to any amplified product will stay in the test chip control area 207 and show the color of the first binding particle 203BP. In contrast, when a sample to be tested contains GAPDH nucleic acid but does not contain target nucleic acid, the sample to be tested is subjected to the first constant temperature cyclic nucleic acid amplification method and the second constant temperature cyclic nucleic acid amplification method respectively with the primer set for detecting GAPDH nucleic acid and the primer set for detecting target nucleic acid in the target nucleic acid detection kit disclosed herein to obtain a first reaction solution containing a GAPDH nucleic acid amplification product and a second reaction solution containing no target nucleic acid amplification product, and the GAPDH nucleic acid amplification product has a first marker M1 and a second marker M2. The first reaction solution and the second reaction solution are mixed to obtain a mixed reaction solution. After the mixed reaction solution is added to the analyte addition area 201 of the lateral flow immunoassay test strip 200', the reaction solution moves to the binding area 203, so that the first marker M1 of the GAPDH nucleic acid amplification product binds to the first binding molecule 203B of part of the first binding particle 203BP in the binding area 203, and moves to the GAPDH detection area 205 together with the remaining first binding particle 203BP that is not bound to any amplification product. In the GAPDH detection region 205, the second marker M2 of the GAPDH nucleic acid amplification product that has bound to the first binding molecule 203B of the first binding particle 203BP will bind to the second binding molecule 205B fixed to the GAPDH detection region 205, so that the GAPDH amplification product stays in the GAPDH detection region 205 and presents the color of the first binding particle 203BP, while the remaining first binding particle 203BP that has not bound to any amplification product continues to move to the first target nucleic acid detection region 206. Since the target nucleic acid amplification product (the amplification product with the first mark M1 and the third mark M3) does not exist in the mixed reaction solution, the remaining first binding particles 203BP that have not been bound to any amplification product will not be bound to the target nucleic acid amplification product (the amplification product with the first mark M1 and the third mark M3), and will not be bound to the fourth binding molecule 206B fixed to the target nucleic acid detection area 206, but will stay in the target nucleic acid detection area 206 and show its color. Afterwards, the remaining first binding particles 203BP continue to move to the test chip control area 207. In the above-mentioned test chip control area 207, the first binding molecule 203B of the remaining first binding particle 203BP will bind to the third binding molecule 207B fixed on the above-mentioned test chip control area 207, so that the remaining first binding particle 203BP stays in the test chip control area 207 and presents the color of the first binding particle 203BP.

The reaction principles of each area of the lateral flow immunoassay test strip 200'' shown in the schematic diagram of Figure 2C are similar to those of the lateral flow immunoassay test strip 200' shown in the schematic diagram of Figure 2B, and therefore will not be described in detail here.

In a specific embodiment, in the primer set for detecting GAPDH nucleic acid and the primer set for detecting target nucleic acid in the above-mentioned kit for detecting target nucleic acid of the present disclosure, the 5' end of the reverse inner primer BIP-G for GAPDH nucleic acid and the 5' end of the reverse inner primer BIP-T for target nucleic acid are labeled with biotin, and the 5' end of the forward inner primer FIP-G for GAPDH nucleic acid is labeled with foxgloveine, and the 5' end of the forward inner primer FIP-T for target nucleic acid is labeled with fluorescein. Furthermore, in this specific embodiment, the target nucleic acid detection kit disclosed herein, in addition to the primer set for detecting GAPDH nucleic acid and the primer set for detecting target nucleic acid, may further include the lateral flow immunoassay test strip 200' or 200'', and in the lateral flow immunoassay test strip, the binding region 203 has a first binding particle 203BP, and the first binding molecule 203B of the first binding particle is avidin, the second binding molecule 205B of the GAPDH detection region 205 is an antibody that can recognize digitalis, the fourth binding molecule 206B of the target nucleic acid detection region 206 is an antibody that can recognize fluorescein, and the third binding molecule 207B in the test strip control region 207 is biotin or an antibody that can recognize avidin.

Furthermore, in one embodiment, the target nucleic acid detection kit disclosed herein may further include a polymerase and/or a nucleotide substrate in addition to the primer set for detecting GAPDH nucleic acid and the primer set for detecting target nucleic acid, but is not limited thereto. The polymerase may have the function of a reverse transcriptase, but is not limited thereto. In a specific embodiment, the polymerase is a Bst DNA polymerase, for example, a Bst DNA polymerase whose sequence includes the amino acid sequence of sequence identification number: 10, but is not limited thereto.

Furthermore, in one embodiment, the target nucleic acid detection kit disclosed herein may further include a reverse transcriptase and/or a nucleotide substrate in addition to the primer set for detecting GAPDH nucleic acid and the primer set for detecting target nucleic acid, but is not limited thereto. The reverse transcriptase may have the function of a ribonuclease (RNase), but is not limited thereto. In a specific embodiment, the reverse transcriptase may have the function of ribonuclease H (RNase H).

According to the above content, the present disclosure can also provide a novel coronavirus detection kit, which may include a primer set for detecting nucleic acid of the novel coronavirus, but is not limited thereto.

The primer set for detecting nucleic acid of the novel coronavirus in the novel coronavirus detection kit disclosed above may be any primer set for detecting target nucleic acid described in all the paragraphs above regarding the embodiments of the primer set for detecting target nucleic acid in the target nucleic acid detection kit disclosed above, wherein the detection target may be the nucleic acid of the RdRp gene of severe acute respiratory syndrome coronavirus type 2 (novel coronavirus), but is not limited thereto.

The primer set for detecting novel coronavirus nucleic acid in the novel coronavirus detection kit disclosed above can be used in a constant temperature cycle nucleic acid amplification method to confirm whether a novel coronavirus is present in a sample to be tested.

The above-mentioned constant temperature circular nucleic acid amplification method may include, but is not limited to, a standard constant temperature circular nucleic acid amplification method or a reverse transcription constant temperature circular nucleic acid amplification method.

The sample to be tested may be a sample that has not been subjected to any purification treatment, such as nucleic acid purification treatment. That is, by using the primer set for detecting nucleic acid of the novel coronavirus in the novel coronavirus detection kit disclosed above, a constant temperature cycle nucleic acid amplification method can be performed on a biological sample that has not been subjected to any purification treatment to obtain accurate novel coronavirus detection results, thereby achieving the effect of reducing or eliminating the need to process the sample to be tested.

The sources of the above-mentioned samples to be tested may include, but are not limited to, saliva samples, sputum samples, nasal swab samples, throat swab samples, nasopharyngeal samples, urine samples, stool samples, rectal swab samples, cerebrospinal fluid samples, body fluid samples, etc.

In one embodiment, the source of the sample to be tested can be a sample obtained by non-invasive sampling, such as a saliva sample, a sputum sample, a urine sample, a feces sample, etc., but not limited thereto. In this embodiment, the novel coronavirus detection kit disclosed herein can be used in conjunction with a constant temperature reaction machine, a constant temperature heating machine, or a simple analysis test strip, such as a lateral flow immunoassay test strip, to achieve the purpose of home testing.

In addition, the primer set for detecting the nucleic acid of the novel coronavirus in the novel coronavirus detection kit disclosed above can use single-stranded RNA or first-stranded cDNA as a starting template to perform the above-mentioned constant temperature circular nucleic acid amplification method, but is not limited to this.

In addition, in one embodiment, in the primer set for detecting the nucleic acid of the novel coronavirus in the novel coronavirus detection kit disclosed above, the 5' end of the reverse inner primer for the nucleic acid of the novel coronavirus may be marked with a first marker, and the 5' end of the forward inner primer for the nucleic acid of the novel coronavirus may be marked with a second marker, and wherein the first marker is different from the second marker. The first marker may include, but is not limited to, biotin, avidin, streptavidin, luteolin, luciferin, etc. The second marker may also include, but is not limited to, biotin, avidin, streptavidin, luteolin, luciferin, etc.

Furthermore, in this embodiment, the novel coronavirus detection kit disclosed herein may include, in addition to the primer set for detecting the nucleic acid of the novel coronavirus, a lateral flow immunoassay test strip, but is not limited thereto. The material of the lateral flow immunoassay test strip may include, but is not limited to, nitrocellulose membrane, nylon membrane, polyvinylidene fluoride membrane, polyether sulfide membrane, etc.

The lateral flow immunoassay test strip may include an analyte addition zone, a binding zone, a novel coronavirus detection zone and a test strip control zone in sequence according to the flow direction of the analyte. The binding zone has a first binding particle, which has a first binding molecule and a particle connected to the first binding molecule, wherein the first binding molecule has the ability to bind to the first marker. The material of the particles may include, but is not limited to, gold, carbon, latex, magnetic materials, etc. Alternatively, in addition to the first binding particles, the binding zone may further include a control particle coated with a specific substance, and examples of the material of the control particles can refer to the materials of the particles described above, but on the lateral flow immunoassay test strip, the materials of the control particles and the particles may be the same or different. Furthermore, there is no special limitation on the above-mentioned specific substance, as long as there is a molecule that can bind to it, and examples of the above-mentioned specific substance may include, but are not limited to, serum (such as mouse serum, but not limited to this). The above-mentioned novel coronavirus detection area is fixed with a second binding molecule, which has the ability to bind to the above-mentioned second marker. Furthermore, the above-mentioned test piece control area is fixed with a third binding molecule, which has the ability to bind to the above-mentioned first binding molecule in the above-mentioned first binding particle, wherein the above-mentioned third binding molecule and the above-mentioned first marker may be the same or different. Alternatively, in the case where the above-mentioned binding area may further include a control particle in addition to the first binding particle, the third binding molecule fixed by the above-mentioned test piece control area may be the ability to bind to the above-mentioned specific substance of the above-mentioned control particle. For example, when the specific substance coating the control particle is mouse serum, the third binding molecule may be an anti-mouse serum antibody. In a specific embodiment, in the primer set for detecting the nucleic acid of the novel coronavirus in the novel coronavirus detection kit disclosed herein, the 5' end of the reverse inner primer for the nucleic acid of the novel coronavirus is labeled with biotin, and the 5' end of the forward inner primer for the nucleic acid of the novel coronavirus is labeled with fluorescein. Furthermore, in this specific embodiment, the novel coronavirus detection kit disclosed herein, in addition to the primer set for detecting the nucleic acid of the novel coronavirus, further includes the lateral flow immunoassay test strip, and in the lateral flow immunoassay test strip, the first binding molecule in the binding region is avidin, the second binding molecule in the novel coronavirus detection region is an antibody that can recognize fluorescein, and the third binding molecule in the control region of the test strip is biotin or an antibody that can recognize avidin.

Furthermore, in one embodiment, the novel coronavirus detection kit disclosed herein may further include a polymerase and/or a nucleotide substrate in addition to the primer set for detecting the nucleic acid of the novel coronavirus, but is not limited thereto. The polymerase may have the function of a reverse transcriptase, but is not limited thereto. In a specific embodiment, the polymerase is a Bst DNA polymerase, for example, a Bst DNA polymerase whose sequence includes an amino acid sequence of sequence identification number: 10, but is not limited thereto.

In addition, in one embodiment, the novel coronavirus detection kit disclosed herein may further include a reverse transcriptase and/or a nucleotide substrate in addition to the primer set for detecting the nucleic acid of the novel coronavirus, but is not limited thereto. The reverse transcriptase may have the function of a ribonuclease (RNase), but is not limited thereto. In a specific embodiment, the reverse transcriptase may have the function of ribonuclease H (RNase H).

In addition, according to the above, the present disclosure can also provide a method for detecting GAPDH nucleic acid. The method for detecting GAPDH nucleic acid disclosed herein may include, but is not limited to, the following steps.

First, a sample to be tested is provided. The description of the source of the sample to be tested described herein may be the same as the description of the source of the sample to be tested described in the above paragraph regarding the GAPDH nucleic acid detection kit disclosed herein, and therefore will not be repeated here. In one embodiment, the source of the sample to be tested may be a saliva sample.

Next, the above-mentioned sample to be tested is subjected to a constant temperature cycle nucleic acid amplification method using any of the above-mentioned primer sets for detecting GAPDH nucleic acid in the disclosed GAPDH nucleic acid detection kit. If the above-mentioned sample to be tested contains GAPDH nucleic acid, a GAPDH nucleic acid amplification product is obtained from the above reaction.

Furthermore, in one embodiment, in the method for detecting GAPDH nucleic acid disclosed above, the method for confirming the presence of the amplification product of the GAPDH nucleic acid is not particularly limited, as long as the presence of the amplification product of the GAPDH nucleic acid can be confirmed. For example, the method for confirming the presence of the amplification product of the GAPDH nucleic acid may include, but is not limited to, a gel electrophoresis analysis, a lateral flow immunoassay, etc.

In the method for detecting GAPDH nucleic acid disclosed above, the constant temperature circular nucleic acid amplification method can use the single strand RNA or the first strand cDNA in the sample to be tested as a starting template, but is not limited thereto.

In one embodiment, in the method for detecting GAPDH nucleic acid disclosed above, the sample to be tested may be a sample that has not been subjected to any purification treatment, such as nucleic acid purification treatment. That is, the method for detecting GAPDH nucleic acid disclosed above may directly perform the above constant temperature cycle nucleic acid amplification method on a biological sample that has not been subjected to any purification treatment, and obtain accurate GAPDH nucleic acid detection results, thereby achieving the effect of reducing or eliminating the need to process the sample to be tested. In a specific embodiment, the sample to be tested may be an original saliva sample.

In one embodiment, the method for detecting GAPDH nucleic acid disclosed herein may further include pre-treating a biological sample to obtain the sample before providing the sample. The above-mentioned pre-treatment may include, but is not limited to, one of the following steps: (i) subjecting the biological sample to a heat treatment step; (ii) subjecting the above-mentioned biological sample to a nucleic acid purification step; (iii) subjecting the above-mentioned biological sample to a reverse transcription step; (iv) subjecting the above-mentioned biological sample to a nucleic acid purification step and then to a reverse transcription step; (v) subjecting the above-mentioned biological sample to a reverse transcription step and then to an RNA removal step; and (vi) subjecting the above-mentioned biological sample to a nucleic acid purification step and then to a reverse transcription step and then to an RNA removal step.

The temperature of the heat treatment step may be about 45-100°C, such as about 50-95°C, about 55-90°C, about 60°C, about 70°C, about 80°C, about 90°C, about 95°C, etc., but is not limited thereto.

The biological sample may include, but is not limited to, saliva sample, sputum sample, nasal swab sample, throat swab sample, nasopharyngeal sample, urine sample, feces sample, rectal swab sample, cerebrospinal fluid sample, body fluid sample, etc. In one embodiment, the biological sample may be a saliva sample.

In addition, in the above-mentioned method for detecting GAPDH nucleic acid disclosed herein, the above-mentioned constant temperature circular nucleic acid amplification method may include a standard constant temperature circular nucleic acid amplification method, a reverse transcription constant temperature circular nucleic acid amplification method, etc., but is not limited thereto.

In one embodiment, the constant temperature circular nucleic acid amplification method in the method for detecting GAPDH nucleic acid disclosed above can be a reverse transcription constant temperature circular nucleic acid amplification method. In a specific embodiment, in the reverse transcription constant temperature circular amplification method, the reverse transcription process and the constant temperature circular amplification process are performed in one step, and in the reverse transcription constant temperature circular amplification method, a polymerase having the function of a reverse transcriptase can be used, for example, a Bst DNA polymerase, but not limited thereto. Examples of the above Bst DNA polymerase can include, but are not limited to, a Bst DNA polymerase whose sequence can include the amino acid sequence of sequence identification number: 10. In another specific embodiment, in the above-mentioned reverse transcription constant temperature cyclic amplification method, a constant temperature cyclic amplification process is performed after a reverse transcription process. In this specific embodiment, the reverse transcription process can be performed by a reverse transcriptase having a ribonuclease (RNase) function, but is not limited to this. The above-mentioned ribonuclease (RNase) can include, but is not limited to, ribonuclease H (RNase H) and the like.

Furthermore, according to the above, the present disclosure may also provide a method for detecting a target nucleic acid.

The method for detecting a target nucleic acid disclosed herein may include, but is not limited to, the following steps.

First, a sample to be tested is provided. The relevant description of the source of the sample to be tested described herein can be related to the relevant description of the source of the sample to be tested described in the above paragraph about the kit of detection targets disclosed in the present invention, and therefore will not be repeated here. In one embodiment, the source of the sample to be tested can be a saliva sample.

Next, the above-mentioned sample to be tested is subjected to the above-mentioned first constant temperature circular nucleic acid amplification method and the above-mentioned second constant temperature circular nucleic acid amplification method respectively using the primer set for detecting GAPDH nucleic acid and the primer set for detecting target nucleic acid in any of the above-mentioned GAPDH nucleic acid detection kits disclosed herein. If the above-mentioned sample to be tested contains the above-mentioned target nucleic acid, an amplification product of a GAPDH nucleic acid as an internal control group is obtained from the above-mentioned first constant temperature circular nucleic acid amplification method, and an amplification product of a target nucleic acid is obtained from the above-mentioned second constant temperature circular nucleic acid amplification method.

In one embodiment, in the method for detecting the target nucleic acid disclosed above, the method for confirming the presence of the amplification product of the GAPDH nucleic acid or the amplification product of the target nucleic acid is not particularly limited, as long as the presence of the amplification product of the GAPDH nucleic acid or the amplification product of the target nucleic acid can be confirmed. The method for confirming the presence of the amplification product of the GAPDH nucleic acid or the amplification product of the target nucleic acid may include, but is not limited to, a gel electrophoresis analysis, a lateral flow immunoassay, etc.

In the method for detecting target nucleic acid disclosed above, the first constant temperature circular nucleic acid amplification method and the second constant temperature circular nucleic acid amplification method can use the single-stranded RNA or the first-stranded cDNA in the sample to be tested as the starting template, but is not limited thereto.

In one embodiment, in the method for detecting target nucleic acid disclosed above, the sample to be tested may be a sample that has not been subjected to any purification treatment, such as nucleic acid purification treatment. That is, the method for detecting target nucleic acid disclosed above may directly perform the first constant temperature cycle nucleic acid amplification method and the second constant temperature cycle nucleic acid amplification method on a biological sample that has not been subjected to any purification treatment, and obtain accurate target nucleic acid detection results, thereby achieving the effect of reducing or eliminating the need to process the sample to be tested. In a specific embodiment, the sample to be tested may be an original saliva sample.

In one embodiment, the method for detecting a target nucleic acid disclosed herein may further include pre-treating a biological sample to obtain the sample before providing the sample. The above-mentioned pre-treatment may include, but is not limited to, one of the following steps: (i) subjecting the biological sample to a heat treatment step; (ii) subjecting the above-mentioned biological sample to a nucleic acid purification step; (iii) subjecting the above-mentioned biological sample to a reverse transcription step; (iv) subjecting the above-mentioned biological sample to a nucleic acid purification step and then to a reverse transcription step; (v) subjecting the above-mentioned biological sample to a reverse transcription step and then to an RNA removal step; and (vi) subjecting the above-mentioned biological sample to a nucleic acid purification step and then to a reverse transcription step and then to an RNA removal step.

The temperature of the heat treatment step may be about 45-100°C, such as about 50-95°C, about 55-90°C, about 60°C, about 70°C, about 80°C, about 90°C, about 95°C, etc., but is not limited thereto.

The biological sample may include, but is not limited to, saliva sample, sputum sample, nasal swab sample, throat swab sample, nasopharyngeal sample, urine sample, feces sample, rectal swab sample, cerebrospinal fluid sample, body fluid sample, etc. In one embodiment, the biological sample may be a saliva sample.

In addition, in the method for detecting target nucleic acid disclosed above, the first constant temperature circular nucleic acid amplification method or the second constant temperature circular nucleic acid amplification method may include a standard constant temperature circular nucleic acid amplification method, a reverse transcription constant temperature circular nucleic acid amplification method, etc., but is not limited thereto.

In one embodiment, the first constant temperature circular nucleic acid amplification method or the second constant temperature circular nucleic acid amplification method in the method for detecting the target nucleic acid disclosed above can be a reverse transcription constant temperature circular amplification method. In a specific embodiment, in the reverse transcription constant temperature circular amplification method, the reverse transcription process and the constant temperature circular amplification process are performed in one step, and in the reverse transcription constant temperature circular amplification method, a polymerase having the function of a reverse transcriptase can be used, for example, a Bst DNA polymerase, but not limited thereto. Examples of the above Bst DNA polymerase can include, but are not limited to, a Bst DNA polymerase whose sequence can include the amino acid sequence of sequence identification number: 10. In another specific embodiment, in the above-mentioned reverse transcription constant temperature cyclic amplification method, a constant temperature cyclic amplification process is performed after a reverse transcription process. In this specific embodiment, the reverse transcription process can be performed by a reverse transcriptase having the function of a ribonuclease (RNase), but is not limited to this. The above-mentioned ribonuclease (RNase) can include, but is not limited to, ribonuclease (RNase) H, etc.

In one embodiment, the target of the method for detecting target nucleic acid disclosed herein may be a nucleic acid of an RNA virus. Examples of the above RNA viruses may include coronavirus, influenza virus, human immunodeficiency virus, Ebola virus, hepatitis C virus, but are not limited thereto.

The above-mentioned coronaviruses may include, but are not limited to, severe acute respiratory syndrome coronavirus, severe acute respiratory syndrome coronavirus type 2 (novel coronavirus), Middle East respiratory syndrome coronavirus, etc.

In addition, the nucleic acid of the above-mentioned severe acute respiratory syndrome coronavirus type 2 (novel coronavirus) may include, but is not limited to, nucleic acid within the range of ORF1ab (such as nucleic acid of RdRp gene, but not limited to this), nucleic acid of spike protein (S) gene, nucleic acid of envelope (E) gene, nucleic acid of membrane protein (M) gene, nucleic acid of nucleoprotein (N) gene, etc.

In one embodiment, the detection target of the method for detecting target nucleic acid disclosed herein may be the nucleic acid of the RdRp gene of severe acute respiratory syndrome coronavirus type 2 (novel coronavirus).

In the embodiment in which the detection target of the method for detecting target nucleic acid disclosed herein may be the nucleic acid of the RdRp gene of severe acute respiratory syndrome coronavirus type 2 (novel coronavirus), the primer set for detecting target nucleic acid in the target nucleic acid detection kit disclosed herein may be any primer set for detecting target nucleic acid mentioned in the paragraph of the relevant embodiment when the detection target of the primer set for detecting target nucleic acid in the target nucleic acid detection kit disclosed herein is the nucleic acid of the RdRp gene of severe acute respiratory syndrome coronavirus type 2 (novel coronavirus), but is not limited thereto.

In addition, based on the above, the present disclosure can also provide a method for detecting the novel coronavirus.

The method for detecting the novel coronavirus disclosed herein may include, but is not limited to, the following steps.

First, a sample to be tested is provided. The relevant description of the source of the sample to be tested described herein can be related to the relevant description of the source of the sample to be tested described in the above paragraph about the kit of detection targets disclosed in the present invention, and therefore will not be repeated here. In one embodiment, the source of the sample to be tested can be a saliva sample.

Next, the sample to be tested is subjected to a constant temperature circular nucleic acid amplification method using any of the primer sets for detecting the nucleic acid of the novel coronavirus in the novel coronavirus detection kit disclosed herein. If the sample to be tested contains the novel coronavirus, an amplification product of the novel coronavirus nucleic acid is obtained from the constant temperature circular nucleic acid amplification method.

In one embodiment, in the method for detecting novel coronavirus disclosed above, the method for confirming the amplification product of the novel coronavirus nucleic acid is not particularly limited, as long as the presence of the amplification product of the novel coronavirus nucleic acid can be confirmed. The method for confirming the presence of the amplification product of the novel coronavirus nucleic acid may include, but is not limited to, a colloid electrophoresis analysis, a lateral flow immunoassay, etc.

In the method for detecting the novel coronavirus disclosed above, the constant temperature circular nucleic acid amplification method can use the single-stranded RNA or the first-stranded cDNA in the above-mentioned sample to be tested as the starting template, but is not limited thereto.

In one embodiment, in the method for detecting the novel coronavirus disclosed above, the sample to be tested may be a sample that has not been subjected to any purification treatment, such as a sample that has not been subjected to nucleic acid purification treatment. That is, in the method for detecting the novel coronavirus disclosed above, the constant temperature cycle nucleic acid amplification method may be directly performed on a biological sample that has not been subjected to any purification treatment to obtain an accurate novel coronavirus detection result, thereby achieving the effect of reducing or eliminating the need to process the sample to be tested. In a specific embodiment, the sample to be tested may be an original saliva sample.

In one embodiment, the method for detecting the novel coronavirus disclosed above may further include pre-processing a biological sample to obtain the above-mentioned sample before providing the above-mentioned sample to be tested. The above-mentioned pre-treatment may include, but is not limited to, one of the following steps: (i) subjecting the biological sample to a heat treatment step; (ii) subjecting the above-mentioned biological sample to a nucleic acid purification step; (iii) subjecting the above-mentioned biological sample to a reverse transcription step; (iv) subjecting the above-mentioned biological sample to a nucleic acid purification step and then to a reverse transcription step; (v) subjecting the above-mentioned biological sample to a reverse transcription step and then to an RNA removal step; and (vi) subjecting the above-mentioned biological sample to a nucleic acid purification step and then to a reverse transcription step and then to an RNA removal step.

The temperature of the heat treatment step may be about 45-100°C, such as about 50-95°C, about 55-90°C, about 60°C, about 70°C, about 80°C, about 90°C, about 95°C, etc., but is not limited thereto.

The biological sample may include, but is not limited to, saliva sample, sputum sample, nasal swab sample, throat swab sample, nasopharyngeal sample, urine sample, feces sample, rectal swab sample, cerebrospinal fluid sample, body fluid sample, etc. In one embodiment, the biological sample may be a saliva sample.

In addition, in the method for detecting the novel coronavirus disclosed above, the above-mentioned constant temperature circular nucleic acid amplification method may include a standard constant temperature circular nucleic acid amplification method, a reverse transcription constant temperature circular nucleic acid amplification method, etc., but is not limited thereto.

In one embodiment, the first constant temperature circular nucleic acid amplification method or the second constant temperature circular nucleic acid amplification method in the method for detecting the target nucleic acid disclosed above can be a reverse transcription constant temperature circular nucleic acid amplification method. In a specific embodiment, in the reverse transcription constant temperature circular nucleic acid amplification method, the reverse transcription process and the constant temperature circular nucleic acid amplification process are performed in one step, and in the reverse transcription constant temperature circular nucleic acid amplification method, a polymerase having the function of a reverse transcriptase can be used, for example, a Bst DNA polymerase, but not limited thereto. Examples of the above Bst DNA polymerase can include, but are not limited to, a Bst DNA polymerase whose sequence can include the amino acid sequence of sequence identification number: 10. In another specific embodiment, in the above-mentioned reverse transcription constant temperature circular nucleic acid amplification method, a constant temperature circular nucleic acid amplification process is performed after a reverse transcription process. In this specific embodiment, the reverse transcription process can be performed by a reverse transcriptase having a ribonuclease (RNase) function, but is not limited to this. The above-mentioned ribonuclease (RNase) can include, but is not limited to, ribonuclease (RNase) H, etc.

In addition, the nucleic acid of the above-mentioned novel coronavirus may include, but is not limited to, nucleic acid within the range of ORF1ab (such as nucleic acid of RdRp gene, but not limited to this), nucleic acid of spike protein (S) gene, nucleic acid of envelope (E) gene, nucleic acid of membrane protein (M) gene, nucleic acid of nucleoprotein (N) gene, etc.

In one embodiment, the detection target of the method for detecting the novel coronavirus disclosed herein may be the nucleic acid of the RdRp gene of the novel coronavirus.

Embodiment

A. Materials and Methods

A-1. Deactivated severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) (novel coronavirus)

Deactivated SARS-CoV-2 virus was purchased from BEI Resources (Catalog No. NR-52286; Lot: 70034991).

A-2. Preparation of synthetic SARS-CoV-2 RNA control group

Synthetic SARS-CoV-2 RNA (GenBank ID: MT007544.1; Model No.: 102019) was purchased from Twist Bioscience.

The synthesized SARS-CoV-2 RNA was diluted to a final concentration of 2.5x10 ⁴ copies/mL with sterile 1x RNAsecure™ RNase Inactivation Reagent (Brand: Thermo Fisher Scientific Inc.; Model: AM7006) to serve as a positive control for each RT-LAMP assay.

A-3. Primers used in reverse transcription constant temperature circular nucleic acid amplification method:

1. Primer set for GAPDH

A primer set targeting GAPDH was designed based on a portion of the GAPDH mRNA sequence (NCBI Accession No. NM_001256799) (SEQ ID No.: 1).

The five primer sets designed for reverse transcription constant temperature circular nucleic acid amplification are shown in Table 1 below.

Table 1. Six selected regions and designed primer sets for the nucleotide sequence of SEQ ID No. 1 Primer set design based on nucleotide sequence of sequence identification number: 1 Primer group GAPDH-103 Selected area and designed primer 5' end site 3' end site Length sequence F3 region (forward outer primer) 52 69 18 GATGCTGGCGCTGAGTAC (SEQ ID NO: 3) F2 area 87 105 19 CGTCTTCACCACCATGGAG (Seq ID: 19) F1c area 144 165 twenty two AGCAGAGGGGGCAGAGATGATG (Seq ID: 18) B1c area 176 197 twenty two TGTTCGTCATGGGTGTGAACCA (Serial ID: 20) B2 Area 221 240 20 GGAGGCATTGCTGATGATCT (Seq ID: 21) B3 region (reverse primer) 248 265 18 GGGGTGCTAAGCAGTTGG (SEQ ID NO: 5) FIP (Front Introducer) 47 AGCAGAGGGGGCAGAGATGATGTTTTTTCGTCTTCACCACCATGGAG (Serial ID: 2) BIP (Backward Introducing Primer) 48 TGTTCGTCATGGGTGTGAACCATTTTTTGGAGGCATTGCTGATGATCT (Sequence ID: 4) Primer set GAPDH-103-B3-I Selected area and designed primer 5' end site 3' end site Length sequence F3 region (forward outer primer) 52 69 18 GATGCTGGCGCTGAGTAC (SEQ ID NO: 3) F2 area 87 105 19 CGTCTTCACCACCATGGAG (Seq ID: 19) F1c area 144 165 twenty two AGCAGAGGGGGCAGAGATGATG (Seq ID: 18) B1c area 176 197 twenty two TGTTCGTCATGGGTGTGAACCA (Serial ID: 20) B2 Area 221 240 20 GGAGGCATTGCTGATGATCT (Seq ID: 21) B3 region (reverse primer) 248 265 18 GGGGTGCIIIIIAGTTGG (Seq ID: 9) FIP (Front Introducer) 47 AGCAGAGGGGGCAGAGATGATGTTTTTTCGTCTTCACCACCATGGAG (Serial ID: 2) BIP (Backward Introducing Primer) 48 TGTTCGTCATGGGTGTGAACCATTTTTTGGAGGCATTGCTGATGATCT (Sequence ID: 4) Primer set GAPDH-103-BIP-3 Selected area and designed primer 5' end site 3' end site Length sequence F3 region (forward outer primer) 52 69 18 GATGCTGGCGCTGAGTAC (SEQ ID NO: 3) F2 area 87 105 19 CGTCTTCACCACCATGGAG (Seq ID: 19) F1c area 144 165 twenty two AGCAGAGGGGGCAGAGATGATG (Seq ID: 18) B1c area 176 197 twenty two TGTTCGTCATGGGTGTGAACCA (Serial ID: 20) B2 Area 229 246 18 GGTGCAGGAGGCATTGCT (SEQ ID NO: 22) B3 region (reverse primer) 248 265 18 GGGGTGCTAAGCAGTTGG (SEQ ID NO: 5) FIP (Front Introducer) 47 AGCAGAGGGGGCAGAGATGATGTTTTTTCGTCTTCACCACCATGGAG (Serial ID: 2) BIP (Backward Introducing Primer) 46 TGTTCGTCATGGGTGTGAACCATTTTTTGGTGCAGGAGGCATTGCT (Sequence ID: 6) Primer set GAPDH-103-B3/BIP-3-I Selected area and designed primer 5' end site 3' end site Length sequence F3 region (forward outer primer) 52 69 18 GATGCTGGCGCTGAGTAC (SEQ ID NO: 3) F2 area 87 105 19 CGTCTTCACCACCATGGAG (Seq ID: 19) F1c area 144 165 twenty two AGCAGAGGGGGCAGAGATGATG (Seq ID: 18) B1c area 176 197 twenty two TGTTCGTCATGGGTGTGAACCA (Serial ID: 20) B2 Area 229 246 18 GGTGCAGGAGGCATTGCT (SEQ ID NO: 22) B3 region (reverse primer) 248 265 18 GGGGTGCIIIIIAGTTGG (SEQ ID NO: 9) FIP (Front Introducer) 47 AGCAGAGGGGGCAGAGATGATGTTTTTTCGTCTTCACCACCATGGAG (Serial ID: 2) BIP (Backward Introducing Primer) 46 TGTTCGTCATGGGTGTGAACCATTTTTTGGTGCIIIIIGCATTGCT (SEQ ID NO: 8) Primer set GAPDH-103-BIP-4 Selected area and designed primer 5' end site 3' end site Length sequence F3 region (forward outer primer) 52 69 18 GATGCTGGCGCTGAGTAC (SEQ ID NO: 3) F2 area 87 105 19 CGTCTTCACCACCATGGAG (Seq ID: 19) F1c area 144 165 twenty two AGCAGAGGGGGCAGAGATGATG (Seq ID: 18) B1c area 176 197 twenty two TGTTCGTCATGGGTGTGAACCA (Serial ID: 20) B2 Area 228 245 18 GTGCAGGAGGCATTGCTG (SEQ ID NO: 23) B3 region (reverse primer) 248 265 18 GGGGTGCTAAGCAGTTGG (SEQ ID NO: 5) FIP (Front Introducer) 47 AGCAGAGGGGGCAGAGATGATGTTTTTTCGTCTTCACCACCATGGAG (Serial ID: 2) BIP (Backward Introducing Primer) 46 TGTTCGTCATGGGTGTGAACCATTTTTTGTGCAGGAGGCATTGCTG (Seq ID: 7) Note: I stands for inosine

2. Primer set for SARS-CoV-2 Primer set for RdRp gene nucleic acid

A primer set targeting the RdRp gene nucleic acid was designed using a portion of the ORF1ab nucleic acid of SARS-CoV-2 (NCBI Accession No. MN908947) (SEQ ID No. 11).

The primer set designed for the reverse transcription constant temperature circular nucleic acid amplification method is shown in Table 2 below.

Table 2. Six selected regions and designed primer sets for the nucleotide sequence of SEQ ID No. 11 Primer set design based on sequence identification number: 11 nucleotide sequence Primer group RdRp Selected area and designed primer 5' end site 3' end site Length sequence F3 region (forward outer primer) 37 54 18 ATGGCCTCACTTGTTCTT (Serial ID: 13) F2 area 55 72 18 GCTCGCAAACATACAACG (Seq ID: 25) F1c area 100 124 25 CTTGAGCACACTCATTAGCTAATCT (Seq ID: 24) B1c area 133 155 twenty three GAAATGGTCATGTGTGGCGGTTC (SEQ ID: 26) B2 Area 180 198 19 TGTGGCATCTCCTGATGAG (SEQ ID NO: 27) B3 region (reverse primer) 236 253 18 TAACATTGGCCGTGACAG (Seq ID: 15) FIP (Front Introducer) 49 CTTGAGCACACTCATTAGCTAATCTTTTTTTGCTCGCAAACATACAACG (SEQ ID: 12) BIP (Backward Introducing Primer) 48 GAAATGGTCATGTGTGGCGGTTCTTTTTTTGTGGCATCTCCTGATGAG (SEQ ID NO: 14) FLP (Forward Loop Primer) 73 94 twenty two AACGGTGTGACAAGCTACAACA (Seq ID: 16) BLP (reverse loop primer) 156 177 twenty two ACTATATGTTAAACCAGGTGGA (Seq ID: 17)

A-5. Reverse transcription constant temperature circular nucleic acid amplification method

1. Primer concentration:

The concentrations of the primers used in the reverse transcription constant temperature circular nucleic acid amplification method are as follows.

The final concentrations of primers used to amplify the human GAPDH gene were: 0.8 μM for FIP/BIP and 0.1 μM for F3/B3.

The final concentrations of primers used to amplify the SARS-CoV-2 RdRp nucleic acid sequence were: 0.8 μM for FIP/BIP; 0.1 μM for F3/B3; and 0.2 μM for FLP/BLP.

2. Reagents and conditions

(1) Use commercially available RT/ Bst mix reagent as polymerase

The reverse transcription constant temperature circular nucleic acid amplification method was performed using a commercially available RT/ Bst mix reagent (WarmStart® LAMP kit (Cat No. E1700L); brand: New England Biolabs).

The reaction temperature is 65°C and the reaction time is 60 minutes. The components and volumes required for the reaction are shown in Table 3:

Table 3 Element Experimental group Positive Quality Control Group Negative Quality Control Group RT/ Bst mix 10 μL 10 μL 10 μL Introduction set 1 μL 1 μL 1 μL Samples to be tested* 9 μL 0 μL 0 μL Synthetic SARS-CoV-2 RNA Control Set 0 μL 9 μL 0 μL 1x RNAsecure™ RNase Inactivation Reagent 0 μL 0 μL 9 μL Total volume 20 μL 20 μL 20 μL *The sample to be tested can be saliva containing SARS-CoV-2 virus or SARS-CoV-2 RNA control group, or genomic DNA or total RNA from Expi293 cells.

(2) Using recombinant Bst DNA polymerase large fragment as the polymerase

The recombinant Bst DNA polymerase large fragment was used as the polymerase, and its amino acid sequence (SEQ ID NO: 10) is shown below: MGSSHHHHHHSGGPEQKLISEEDLPGGSWSHPQFEKSGLVPRGSGR The histidine-signature (HHHHHH) (SEQ ID NO: 28), c-myc-signature (EQKLISEEDL) (SEQ ID NO: 29), Strep-signature II (WSHPQFEK) (SEQ ID NO: 30) and thrombin cleavage site (LVPRGS) (SEQ ID NO: 31) contained in the N-terminal sequence are shown in bold and underlined.

The reaction temperature is 65°C and the reaction time is 60 minutes. The components and volumes required for the reaction are shown in Table 4:

Table 4 Element BEI No template control (NTC) 10x Constant Temperature Expansion Buffer (pH 8.8) 2 μL 2 μL 100 mM MgSO ₄ 1.2 μL 1.2 μL RdRp primer set 1 μL 1 μL 10 mM dNTP 2.8 μL 2.8 μL Bst DNA polymerase large fragment 1 μL 1 μL RNA template 9 μL 0 μL _H2O 3 μL 12 μL Total volume 20 μL 20 μL The composition of 10x isothermal expansion buffer (pH 8.8) is as follows: 200 mM Tris-HCl 100 mM (NH ₄ ) ₂ SO ₄ 500 mM KCl 20 mM MgSO ₄ 1% Tween-20

A-6. Lateral flow immunoassay

Lateral flow immunoassay was performed using a commercially available nucleic acid lateral flow immunoassay (NALFIA) test strip PCRD FLEX Dipstick (model FG-FD51676, brand Abingdon Health).

The PCRD FLEX Dipstick has an analyte addition zone, a binding zone, a first test line (T1), a second test line (T2) and a control line (C) in order according to the flow direction of the analyte. When the analyte addition zone of the test strip is inserted into a reaction sample containing the product of the reverse transcription constant temperature cyclic nucleic acid amplification method, the carbon particles coated with NeutrAvidin on the surface of the binding zone of the test strip will bind to the biotin of the DNA amplification product labeled with digitalis glycosides (DIG)/biotin and the DNA amplification product labeled with fluorescein (FAM)/biotin in the reaction sample and move along the nitrocellulose membrane using the capillary phenomenon.

If the reaction sample contains DNA amplification products labeled with DIG/biotin, the DIG of the DNA amplification products labeled with DIG/biotin can bind to the anti-DIG antibody in the T1 test line to form a dark gray complex that can be identified by the naked eye; if the reaction sample contains DNA amplification products labeled with FAM/biotin, the FAM of the DNA amplification products labeled with FAM/biotin can bind to the anti-FAM antibody in the T2 test line to produce a dark gray line visible to the naked eye.

Finally, the carbon particles coated with mouse serum are captured at the control line to produce dark gray lines visible to the naked eye, serving as the quality control line for the nucleic acid lateral flow immunoassay test strip.

B. Results

Embodiment 1

Detection of SARS-CoV-2 RdRp and human GAPDH in saliva by reverse transcription isothermal circular nucleic acid amplification (one-step reaction)

Synthetic SARS-CoV-2 RNA control (Twist Bioscience) was added to the SARS-CoV-2 negative saliva samples at a final concentration of ^1x104 copies/mL.

Afterwards, the saliva samples containing the synthetic SARS-CoV-2 RNA control group were extracted using QIAamp Viral RNA Mini Kit (brand: Qiagen, model number: 52906) to obtain total RNA.

9 μL of total RNA was used as a nucleic acid template and mixed with the RdRp primer set (the 5' end of the forward inner primer was labeled with FAM, and the 5' end of the reverse inner primer was labeled with biotin) and RT/ Bst mix, and then a reverse transcription constant temperature circular nucleic acid amplification method was performed. In addition, 9 μL of total RNA was used as a nucleic acid template and mixed with the GAPDH primer set (the 5' end of the forward inner primer was labeled with DIG, and the 5' end of the reverse inner primer was labeled with biotin) and RT/ Bst mix, and then another reverse transcription constant temperature circular nucleic acid amplification method was performed. On the other hand, 9 µL of 1x RNAsecure™ RNase Inactivation Reagent was used to replace the total RNA template, and the RdRp primer set and GAPDH primer set were added to them respectively, and mixed with RT/ Bst mix and then subjected to reverse transcription constant temperature circular nucleic acid amplification method as a no template control (NTC). In addition, the synthetic SARS-CoV-2 RNA control group was directly mixed with the RdRp primer set (the 5' end of the forward inner primer was labeled with FAM, and the 5' end of the reverse inner primer was labeled with biotin) and RT/ Bst mix and subjected to a reverse transcription constant temperature circular nucleic acid amplification method as a positive control.

The saliva samples containing the synthetic SARS-CoV-2 RNA control group were subjected to reverse transcription constant temperature cyclic nucleic acid amplification method using RdRp primer set and GAPDH primer set, respectively. The two products obtained were analyzed by electrophoresis using 2% agar. The results are shown in Figures 3A and 3B, respectively.

The saliva samples containing the synthetic SARS-CoV-2 RNA control group were subjected to reverse transcription constant temperature cyclic nucleic acid amplification method using RdRp primer set and GAPDH primer set respectively, and the mixture obtained was mixed for lateral flow immunoassay. The results are shown in Figure 3C.

Figure 3A shows that the total RNA extracted from the saliva sample containing the synthetic SARS-CoV-2 RNA control group was subjected to reverse transcription constant temperature circular nucleic acid amplification method using the RdRp primer set to obtain a ladder-like amplification product. The positive control group also obtained a ladder-like amplification product.

FIG. 3B also shows that the total RNA extracted from the saliva sample containing the synthetic SARS-CoV-2 RNA control group was subjected to reverse transcription constant temperature circular nucleic acid amplification method using the GAPDH primer set to obtain a ladder-shaped amplification product.

Figure 3C shows that the products obtained by reverse transcription constant temperature cyclic nucleic acid amplification method using GAPDH primer set and RdRp primer set in the saliva sample containing the synthetic SARS-CoV-2 RNA control group can generate dark gray lines on the T1 test line and T2 test line of the lateral flow immunolayer test strip. The product of the positive control group only generates a dark gray line on the T2 test line, while the product of the no template control group does not show a dark gray line on both the T1 test line and the T2 test line.

From the above results, it can be seen that the RdRp primer set disclosed in the present invention can indeed detect SARS-CoV-2 in saliva samples, and the GAPDH primer set can indeed obtain a product in the reverse transcription constant temperature circular nucleic acid amplification method of total RNA in saliva samples, and this product can be used as an internal control group.

Embodiment 2

Detection of SARS-CoV-2 RdRp by reverse transcription isothermal circular nucleic acid amplification (two-step reaction)

The synthetic SARS-CoV-2 RNA control group (Twist Bioscience) (final concentration of 1x10 ⁴ copies/ml) (single-stranded RNA) was mixed with SuperScript IV reverse transcriptase (Invitrogen) as a template for reverse transcription (50°C for 15 minutes) to form an RNA-cDNA hybrid.

Next, the reverse transcription product was heated at 95°C for 3 minutes to degrade the RNA and obtain single-stranded cDNA. Afterwards, the cDNA was diluted 5-fold and 10-fold, respectively.

2 µL cDNA (undiluted, 5-fold diluted, or 10-fold diluted) was subjected to constant temperature circular nucleic acid amplification method. The constant temperature circular nucleic acid amplification method product was analyzed by electrophoresis using 2% agar. The results are shown in Figure 4.

FIG. 4 shows that the RdRp primer set disclosed herein can be used to perform constant temperature circular nucleic acid amplification using single-stranded cDNA as a template to obtain products.

Embodiment 3

Detection of SARS-CoV-2 viral RNA by reverse transcription isothermal circular nucleic acid amplification using recombinant Bst DNA polymerase large fragment as polymerase

Add 15,000 copies/mL of deactivated SARS-CoV-2 virus solution (BEI Resources, Catalog No. NR-52286; Lot: 70034991) to the SARS-CoV-2 negative saliva sample.

The saliva sample containing the deactivated SARS-CoV-2 virus fluid was purified to obtain total RNA.

9 µL of total RNA was used as template and the RdRp primer set and recombinant Bst DNA polymerase large fragment were subjected to constant temperature circular nucleic acid amplification method at 65°C for 1 hour. The obtained products were analyzed by 2% agar electrophoresis and lateral flow immunoassay. The results are shown in Figures 5A and 5B, respectively.

Figures 5A and 5B both show that the above constant temperature circular nucleic acid amplification method can indeed obtain amplified products. From this experimental result, it can be known that the recombinant Bst DNA polymerase large fragment has endogenous reverse transcriptase activity, and the RdRp primer set disclosed in the present disclosure can use single-stranded RNA as a template through the recombinant Bst DNA polymerase large fragment to perform reverse transcription constant temperature circular nucleic acid amplification method at 65°C.

Embodiment 4

Direct detection of SARS-CoV-2 virus in heat-treated saliva by reverse transcription constant temperature circular nucleic acid amplification

Inactivated SARS-CoV-2 virus was added to 50 µL of COVID-19 negative saliva samples at a final concentration of 10,000 copies/mL or 100,000 copies/mL. The samples and negative saliva samples without virus addition were treated in the following two ways: (1) 50 μL of DPBS and 2× RNAsecure were added to the samples and heated at 95°C for 30 minutes; (2) Proteinase K and 2x RNAsecure were added to the samples and heated at 95°C for 5 minutes.

After the above two treatments, 9 μL of the heated supernatant was used as a nucleic acid template and mixed with the RdRp primer set or the GAPDH-103 primer set to perform reverse transcription constant temperature cyclic nucleic acid amplification method, and the obtained products were analyzed by electrophoresis with 2% agar. The results are shown in Figure 6.

The products amplified by RdRp and GAPDH at the same viral concentration were mixed and subjected to lateral flow immunoassay. The results are shown in Figure 7.

According to the experimental results, by using the RdRp primer set or GAPDH primer set disclosed herein, a saliva sample can be directly subjected to the reverse transcription constant temperature circular nucleic acid amplification method using RT/ Bst mix as a polymerase without going through a nucleic acid purification step to obtain the amplified products of the SARS-CoV-2 virus RdRp RNA and the human GAPDH gene.

Embodiment 5

RT-LAMP reaction temperature test of GAPDH primer set

Expi293 cells were collected. Genomic DNA was extracted from one part of the cells using QIAamp genomic DNA kits (Qiagen) and then treated with RNase A/T1 (Thermo Scientific). Total RNA was extracted from another part of the cells using Direct-zol RNA Miniprep (Zymo Research) and digested with DNase I on a column.

Purified genomic DNA and total RNA were used as nucleic acid templates to test the GAPDH primer set. The reaction temperature of the reverse transcription constant temperature circular nucleic acid amplification method was set to 65℃, 68℃, and 70℃ for testing. When the reaction temperature was set at 65℃, the GAPDH-103 primer set could amplify the GAPDH gene in both genomic DNA and total RNA, while the GAPDH-103-BIP-3 primer set and the GAPDH-103-BIP-4 primer set could only amplify the GAPDH gene in total RNA (Figure 8A).

When the reaction temperature was raised to 68°C, the GAPDH-103-BIP-3 and GAPDH-103-BIP-4 primer sets still retained the property of amplifying only the GAPDH gene in total RNA. For the GAPDH-103 primer set, increasing the temperature increased the discrimination of this primer set between genomic DNA and total RNA (Figure 8B).

When the reaction temperature was raised to 70°C, the GAPDH-103 primer set only slightly amplified the GAPDH gene in the total RNA, while the GAPDH-103-BIP-3 primer set and the GAPDH-103-BIP-4 primer set still had the characteristic of only amplifying the GAPDH gene in the total RNA (Figure 8C).

The above experimental results show that compared with the GAPDH-103 primer set, the GAPDH-103-BIP-3 primer set and the GAPDH-103-BIP-4 primer set have a wider temperature tolerance range.

Embodiment 6

Substituted GAPDH primer set amplification assay:

Expi293 cells were collected. Genomic DNA was extracted from one part of the cells using QIAamp genomic DNA kits (Qiagen) and then treated with RNase A/T1 (Thermo Scientific). Total RNA was extracted from another part of the cells using Direct-zol RNA Miniprep (Zymo Research) and digested with DNase I on a column.

Purified genomic DNA and total RNA were used as nucleic acid templates for testing the GAPDH primer set. The RT-LAMP reaction temperature was set at 65°C.

By replacing the five nucleic acids on B3 in the GAPDH-103 primer set with inosine (labeled as GAPDH-103-B3-I), the primer set can amplify the GAPDH gene using only total RNA as a template (Figure 9). This result shows that after the primer set of the human GAPDH gene is modified with inosine, it can be unaffected by the DNA sequence of the genome and only target mRNA for reverse transcription homeostatic cyclic nucleic acid amplification.

In addition, the BIP sequence in the GAPDH-103 primer set was moved to form the GAPDH-103-BIP-3 and GAPDH-103-BIP-4 primer sets. The experimental results showed that the two primer sets only used total RNA as a template to specifically amplify the GAPDH gene.

Embodiment 7

Detection of RdRp and GAPDH RNA in heat-treated samples:

Inactivated SARS-CoV-2 virus was added to 50 µL of novel coronavirus negative saliva sample at a final concentration of 10,000 copies/mL or 100,000 copies/mL. The above sample and the negative saliva sample without virus addition were heated at 95℃ for 5 minutes to obtain a heat-treated sample.

9 µL of the heat-treated sample was used as a template and mixed with the RdRp primer set and the GAPDH primer set with inosine substitution (GAPDH-103-B3-I) and subjected to reverse transcription constant temperature circular nucleic acid amplification at 65°C. The obtained products were analyzed by electrophoresis with 2% agar. The results are shown in Figures 10A and 10B, respectively.

The heat-treated samples were subjected to reverse transcription constant temperature circular nucleic acid amplification using RdRp primer set and GAPDH primer set, respectively, and the mixture was mixed and subjected to lateral flow immunoassay. The results are shown in FIG10C .

The experimental results show that, by using the RdRp primer set or GAPDH primer set disclosed herein, a saliva sample can be directly subjected to a reverse transcription constant temperature cyclic nucleic acid amplification method using RT/ Bst mix as a polymerase without going through a nucleic acid purification step to obtain amplification products of SARS-CoV-2 virus RdRp RNA and human GAPDH RNA, and the amplification products are confirmed by electrophoresis analysis and lateral flow immunoassay. Both the RdRp primer set and the inosine-substituted GAPDH primer set can be applied to heat-treated samples, and the GAPDH amplification product can confirm that the sample RNA is released during the heat treatment process.

Embodiment 8

Reverse transcription isothermal circular nucleic acid amplification method using reverse transcriptase with RNase H activity:

The in vitro transcribed RdRp RNA was used as a template and mixed with RocketScript reverse transcriptase (Bioneer) with RNase H activity to reverse transcribe the RNA into cDNA, followed by constant temperature circular nucleic acid amplification.

In the two-step reverse transcription constant temperature circular nucleic acid amplification method, in vitro transcribed RdRp RNA was added at a final concentration of 6x10 ⁶ copies/mL, reacted at 50°C and 65°C for 15 minutes, and then heated at 95°C for 5 minutes. 2 μL of the reverse transcription product was mixed with recombinant Bst DNA polymerase large fragment and RdRp primer set, and constant temperature circular nucleic acid amplification method was performed at 65°C for 1 hour. The obtained products were analyzed by electrophoresis with 2% agar and lateral flow immunoassay. The results are shown in Figures 11A and 11B, respectively.

In the one-step reverse transcription constant temperature circular nucleic acid amplification method, in vitro transcribed RdRp RNA (1x10 ⁴ copies) was mixed with RdRp primer set, RocketScript Reverse Transcriptase and recombinant Bst DNA polymerase large fragment and reacted at 65°C for 1 hour. The obtained products were analyzed by electrophoresis with 2% agar and lateral flow immunoassay. The results are shown in Figures 12A and 12B, respectively.

The experimental results show that the RdRp primer set disclosed herein can be reverse transcribed using single-stranded RNA as a template by a reverse transcriptase with RNase H activity.

Embodiment 9

Comparison of primer sets

The primer set disclosed in the present invention, the GAPDH-103 primer set, and the primer set not designed in the design segment disclosed in the present invention, such as the GAPDH-5 primer set and the GAPDH-90 primer set, were subjected to the reverse transcription constant temperature circular nucleic acid amplification method. The design segments and sequences of the primer set GAPDH-5 and the primer set GAPDH-90 are shown in Table 5.

Expi293 cells were harvested and total RNA was extracted using Direct-zol RNA Miniprep (Zymo Research). The extracted total RNA was then digested on the column with DNase I. The SARS-CoV-2-negative saliva samples were extracted using the QIAamp Viral RNA Mini Kit to obtain total RNA from SARS-CoV-2-negative saliva.

9 μL of total RNA from Expi293 was taken as nucleic acid template, added to tubes containing GAPDH-5 primer set, GAPDH-90 primer set and GAPDH-103 primer set, mixed with RT/ Bst mix and placed at 65°C for 1 hour for reverse transcription constant temperature cycle nucleic acid amplification method. 9 μL of total RNA extracted from the above-mentioned novel coronavirus negative saliva was taken as nucleic acid template, added to tubes containing GAPDH-5 primer set, GAPDH-90 primer set and GAPDH-103 primer set, mixed with RT/ Bst mix and reacted at 65°C for 1 hour. The obtained products were analyzed by electrophoresis with 2% agar, and the results are shown in Figure 13.

Table 5 Primer set design based on sequence identification number: 11 nucleotide sequence Primer group GAPDH-5 Selected area and designed primer 5' end site 3' end site Length sequence F3 region (forward outer primer) 268 287 20 GCCAAGGTCATCCATGACAA (Seq ID: 33) F2 area 291 310 20 TGGTATCGTGGAAGGACTCA (SEQ ID NO: 37) F1c area 333 353 twenty one TCCACAGTCTTCTGGGTGGCA (SEQ ID NO: 36) B1c area 387 408 twenty two CGGGGCTCTCCAGAACATCATC (Seq ID: 38) B2 Area 433 450 18 GATGACCTTGCCCACAGC (Seq ID: 39) B3 region (reverse primer) 452 469 18 GCTTCCCGTTCAGCTCAG (Serial ID: 35) FIP (Front Introducer) 47 TCCACAGTCTTCTGGGTGGCATTTTTTTGGTATCGTGGAAGGACTCA (SEQ ID NO: 32) BIP (Backward Introducing Primer) 46 CGGGGCTCTCCAGAACATCATCTTTTTTGATGACCTTGCCCACAGC (SEQ ID: 34) Primer group GAPDH-90 Selected area and designed primer 5' end site 3' end site Length sequence F3 region (forward outer primer) 291 310 20 TGGTATCGTGGAAGGACTCA (SEQ ID NO: 37) F2 area 315 333 19 CACAGTCCATGCCATCACT (Serial ID: 43) F1c area 362 381 20 ATCACGCCACAGTTTCCCGG (Seq ID: 42) B1c area 387 408 twenty two CGGGGCTCTCCAGAACATCATC (Seq ID: 38) B2 Area 433 450 18 GATGACCTTGCCCACAGC (Seq ID: 39) B3 region (reverse primer) 456 473 18 GTGAGCTTCCCGTTCAGC (Seq ID: 41) FIP (Front Introducer) 45 ATCACGCCACAGTTTCCCGGTTTTTTCACAGTCCATGCCATCACT (Serial ID: 40) BIP (Backward Introducing Primer) 46 CGGGGCTCTCCAGAACATCATCTTTTTTGATGACCTTGCCCACAGC (SEQ ID: 34)

The result is shown in Figure 13.

The experimental results showed that compared with the GAPDH-103 primer set, the GAPDH-5 primer set and the GAPDH-90 primer set also produced amplified products in the no-template control group, and therefore could not be used as primer sets for detecting GAPDH.

Although the present invention has been disclosed as above with preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art may make some changes and modifications without departing from the spirit and scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the scope defined in the attached patent application.

100: target gene 101: forward inner primer (FIP) 103: forward outer primer (FIP) 105: backward inner primer (BIP) 107: backward outer primer (BLP) 109: forward loop primer (FLP) 111: backward loop primer (BLP) BIP-G: reverse inner primer for GAPDH nucleic acid FIP-G: forward inner primer for GAPDH nucleic acid BIP-T: reverse inner primer for target nucleic acid FIP-T: forward inner primer for target nucleic acid M1: first marker M2: second marker M3: third marker 200, 200' or 200'': Lateral flow immunoassay test strip 201: Analyte addition zone 203: Binding zone 203BP: First binding particle 203B: First binding molecule 203P: Particle connected to first binding molecule 203B 205: GAPDH detection zone 205B: Second binding molecule 206: Target nucleic acid detection zone 206B: Fourth binding molecule 207: Test strip control zone 207B: Third binding molecule

Figures 1A, 1B, and 1C show the design and operating principle of the primer set for detecting GAPDH nucleic acid in the GAPDH nucleic acid detection kit disclosed herein, and the design and operating principle of the primer set for detecting GAPDH nucleic acid and the primer set for detecting target nucleic acid in the target nucleic acid detection kit. Figure 2A shows a schematic diagram of the mechanism of action of a lateral flow immunoassay strip in one embodiment of the present disclosure. Figure 2B shows a schematic diagram of the mechanism of action of a lateral flow immunoassay strip in another embodiment of the present disclosure. Figure 2C shows a schematic diagram of the mechanism of action of a lateral flow immunoassay strip in yet another embodiment of the present disclosure. Figure 3A shows the electrophoresis analysis of the total RNA of the saliva sample containing the synthetic SARS-CoV-2 RNA control group using the RdRp primer set (the 5' end of the forward inner primer is labeled with FAM, and the 5' end of the reverse inner primer is labeled with biotin) through the reverse transcription constant temperature circular nucleic acid amplification method (one-step reaction). Positive control group (PC): The product produced by mixing the synthetic SARS-CoV-2 RNA control group directly with the RdRp primer set and RT/ Bst mix and performing a reverse transcription constant temperature circular nucleic acid amplification method. No template control (NTC): The product generated by mixing 1x RNAsecure™ RNase Inactivation Reagent (used to replace the total RNA template) with the RdRp primer set and RT/ Bst mix to perform a reverse transcription constant temperature circular nucleic acid amplification method. M: DNA molecular weight standard; PC: positive control group; NTC: no template control group; 10 ⁴ : The product generated by the total RNA of the saliva sample containing 10 ⁴ copies/ml of the synthetic SARS-CoV-2 RNA control group using the RdRp primer set through a reverse transcription constant temperature circular nucleic acid amplification method. Figure 3B shows the electrophoresis analysis of the total RNA from the saliva sample containing the synthetic SARS-CoV-2 RNA control group using the GAPDH-103 primer set (the 5' end of the forward inner primer is labeled with DIG, and the 5' end of the reverse inner primer is labeled with biotin) through the reverse transcription constant temperature circular nucleic acid amplification method (one-step reaction). No template control group (NTC): 1x RNAsecure™ RNase Inactivation Reagent (used to replace the total RNA template) was mixed with GAPDH-103 and RT/ Bst mix to perform a reverse transcription constant temperature circular nucleic acid amplification method. M: DNA molecular weight standard; NTC: no template control; 10 ⁴ : products generated by reverse transcription constant temperature circular nucleic acid amplification method using GAPDH-103 primer set from total RNA of saliva sample containing 10 ⁴ copies/ml synthetic SARS-CoV-2 RNA control group. Figure 3C shows the lateral flow immunoassay results of the mixture obtained by mixing equal amounts of two products obtained by reverse transcription constant temperature circular nucleic acid amplification method (one-step reaction) using RdRp primer set and GAPDH-103 primer set from saliva sample containing synthetic SARS-CoV-2 RNA control group. Positive control group: the product produced by mixing the synthetic SARS-CoV-2 RNA control group directly with the RdRp primer set and RT/ Bst mix and performing a reverse transcription constant temperature circular nucleic acid amplification method. No template control group (NTC): the mixture obtained by mixing the no template control group in Figure 3A and the no template control group in Figure 3B in equal amounts. 10 ⁴ copies/ml: the mixture obtained by mixing the two products obtained by performing a reverse transcription constant temperature circular nucleic acid amplification method with the RdRp primer set and the GAPDH-103 primer set in the saliva sample containing 10 ⁴ copies/ml of the synthetic SARS-CoV-2 RNA control group in equal amounts; NTC: no template control group; C: control line; T1: first test line; T2: second test line. Figure 4 shows the electrophoresis analysis of the products produced by the reverse transcription constant temperature circular nucleic acid amplification method (two-step reaction) of the synthetic SARS-CoV-2 RNA control group using the RdRp primer set. M: DNA molecular weight standard; N: no template control group. Figure 5A shows the electrophoresis analysis of the products produced by the reverse transcription constant temperature circular nucleic acid amplification method using the RdRp primer set and the recombinant Bst DNA polymerase large fragment as the polymerase. M: DNA molecular weight standard; BEI: deactivated SARS-CoV-2 virus liquid; NTC: no template control group. Figure 5B shows the results of lateral flow immunoassay of the products produced by the reverse transcription constant temperature circular nucleic acid amplification method using the RdRp primer set and the recombinant Bst DNA polymerase large fragment as the polymerase for SARS-CoV-2 viral RNA. BEI virus: deactivated SARS-CoV-2 virus liquid; NTC: no template control group; C: control line; T2: second test line. Figure 6 shows the results of the reverse transcription constant temperature circular nucleic acid amplification directly performed on the negative saliva specimen of the new coronavirus with the deactivated SARS-CoV-2 virus liquid added after heat treatment with the RdRp primer set and the GAPDH-103 primer set, and the electrophoresis analysis of the products obtained. M: DNA molecular weight standard; PC: positive control group; NTC-RdRp: RdRp primer set no template control group; NTC-GAPDH: GAPDH-103 primer set no template control group. Figure 7 shows that after the novel coronavirus negative saliva sample was added with deactivated SARS-CoV-2 virus solution and heat-treated, the above sample was directly subjected to reverse transcription constant temperature cycle nucleic acid amplification using RdRp primer set and GAPDH-103 primer set, and the mixture obtained by mixing the products obtained was subjected to lateral flow immunoassay. NTC: no template control group; C: control line; T1: first test line; T2: second test line. Figure 8A shows the electrophoresis analysis results of the products generated by the inversion transcription constant temperature circular nucleic acid amplification method at a reaction temperature of 65°C using different GAPDH primer sets. M: DNA molecular weight standard; D: Expi293 cell genomic DNA; R: Expi293 cell total RNA; N: no template control group. Figure 8B shows the electrophoresis analysis results of the products generated by the inversion transcription constant temperature circular nucleic acid amplification method at a reaction temperature of 68°C using different GAPDH primer sets. M: DNA molecular weight standard; D: Expi293 cell genomic DNA; R: Expi293 cell total RNA; N: no template control group. Figure 8C shows the electrophoresis analysis results of the products produced by the inversion transcription constant temperature circular nucleic acid amplification method at a reaction temperature of 70°C using different GAPDH primer sets. M: DNA molecular weight standard; D: Expi293 cell genomic DNA; R: Expi293 cell total RNA; N: no template control group. Figure 9 shows the electrophoresis analysis results of the products produced by the inversion transcription constant temperature circular nucleic acid amplification method at a reaction temperature of 65°C using different modified GAPDH primer sets. M: DNA molecular weight standard; D: Expi293 cell genomic DNA; R: Expi293 cell total RNA; N: no template control group. Figure 10A shows the results of heat-treating a novel coronavirus-negative saliva sample supplemented with deactivated SARS-CoV-2 virus solution, performing reverse transcription thermostatic cyclic nucleic acid amplification on the sample using the RdRp primer set, and analyzing the resulting products by electrophoresis. M: DNA molecular weight standard; NTC: no template control group. Figure 10B shows the results of heat-treating a novel coronavirus-negative saliva sample supplemented with deactivated SARS-CoV-2 virus solution, performing reverse transcription thermostatic cyclic nucleic acid amplification on the sample using the GAPDH-103-B3-I primer set, and analyzing the resulting products by electrophoresis. M: DNA molecular weight standard; NTC: no template control group. Figure 10C shows the results of heat treatment of a novel coronavirus-negative saliva sample supplemented with deactivated SARS-CoV-2 virus solution, and then performing reverse transcription constant temperature circular nucleic acid amplification on the sample using the RdRp primer set and the GAPDH-103-B3-I primer set, respectively, and mixing the obtained products to obtain a mixture for lateral flow immunoassay. NTC: no template control group; C: control line; T1: first test line; T2: second test line. Figure 11A shows the results of electrophoresis analysis of the products produced by reverse transcription reaction of in vitro transcribed RdRp RNA with reverse transcriptase with RNase H activity at different temperatures, followed by constant temperature circular nucleic acid amplification (two-step reaction) using recombinant Bst DNA polymerase. M: DNA molecular weight standard; NTC: no template control group. Figure 11B shows the results of lateral flow immunoassay of the products produced by reverse transcription of in vitro transcribed RdRp RNA using reverse transcriptase with RNase H activity at different temperatures, followed by constant temperature circular nucleic acid amplification method (two-step reaction) using recombinant Bst DNA polymerase. NTC: no template control group; C: control line; T2: second test line. Figure 12A shows the results of electrophoresis analysis of the products produced by reverse transcription of in vitro transcribed RdRp RNA using reverse transcriptase with RNase H activity and recombinant Bst DNA polymerase at 65°C using constant temperature circular nucleic acid amplification method (one-step reaction). M: DNA molecular weight standard; NTC: no template control group. FIG. 12B shows the results of lateral flow immunoassay of the products produced by reverse transcription constant temperature circular nucleic acid amplification method (one-step reaction) of in vitro transcribed RdRp RNA using reverse transcriptase with RNase H activity and recombinant Bst DNA polymerase at 65°C. NTC: no template control group; C: control line; T2: second test line. FIG. 13 shows the results of electrophoresis analysis of reverse transcription constant temperature circular nucleic acid amplification method using primer set GAPDH-103 disclosed in the present invention and primer set GAPDH-5 and primer set GAPDH-90 which are not designed in the design section disclosed in the present invention. M: DNA molecular weight standard; 293: total RNA of Expi293 cells; Sal: total RNA of saliva negative for novel coronavirus; NTC: no template control group.

Claims (52)

A GAPDH nucleic acid detection kit includes: a primer set for detecting GAPDH nucleic acid, including: a forward inner primer for GAPDH nucleic acid; a forward outer primer for GAPDH nucleic acid; a reverse inner primer for GAPDH nucleic acid; and a reverse outer primer for GAPDH nucleic acid, wherein the forward inner primer for GAPDH nucleic acid is composed of a first segment and a second segment, and the 3' end of the first segment is connected to the 5' end of the second segment, or the forward inner primer for GAPDH nucleic acid is composed of a first segment, a first linker and a second segment, and the 3' end of the first segment is connected to the 5' end of the second segment. The 5' end of the first linker is connected to the 5' end of the second fragment, wherein the first fragment has 10-30 nucleotides and is composed of a complementary strand of a first sequence segment, and the first sequence segment is located between the 134th position and the 175th position of the nucleotide sequence of sequence identification number: 1, and the second fragment has 10-30 nucleotides and is composed of a second sequence segment, and the second sequence segment is located between the 77th position and the 115th position of the nucleotide sequence of sequence identification number: 1, and the first linker is composed of 1-6 thymine or peptide nucleic acid (peptide nucleic acid) The forward outer primer for GAPDH nucleic acid has 10-30 nucleotides and is composed of a third sequence segment, and the third sequence segment is located between the 42nd position and the 79th position of the nucleotide sequence of sequence identification number: 1, the reverse inner primer for GAPDH nucleic acid is composed of a third segment and a fourth segment, and the 3' end of the third segment is connected to the 5' end of the fourth segment, or the reverse inner primer for GAPDH nucleic acid is composed of a third segment, a second linker and a fourth segment, and the 3' end of the third segment is connected to the 5' end of the second linker, and the 3' end of the second linker is connected to the 5' end of the fourth segment, wherein the third segment has 10-30 nucleotides and is composed of a fourth sequence segment, and the fourth segment The sequence segment is located between the 156th position and the 207th position of the nucleotide sequence of sequence identification number: 1, and the fourth segment has 10-30 nucleotides and is composed of a complementary strand of a fifth sequence segment, and the fifth sequence segment is located between the 211th position and the 250th position of the nucleotide sequence of sequence identification number: 1, and the second linker is composed of 1-6 thymines or peptide nucleic acid, and the reverse external primer for GAPDH nucleic acid has 10-30 nucleotides and is composed of a complementary strand of a sixth sequence segment, and the sixth sequence segment is located between the 238th position and the 275th position of the nucleotide sequence of sequence identification number: 1, wherein the primer set for detecting GAPDH nucleic acid is used in a loop-mediated nucleic acid amplification method (loop-mediated nucleic acid amplification method) isothermal amplification, LAMP), and the isothermal circular nucleic acid amplification method includes standard isothermal circular nucleic acid amplification method or reverse transcription loop-mediated isothermal amplification, RT-LAMP. The GAPDH nucleic acid detection kit as described in claim 1, wherein the sequence of the forward inner primer for GAPDH nucleic acid includes the nucleotide sequence of sequence identification number: 2; the sequence of the forward outer primer for GAPDH nucleic acid includes the nucleotide sequence of sequence identification number: 3; the sequence of the reverse inner primer for GAPDH nucleic acid includes the nucleotide sequence of sequence identification number: 4, the nucleotide sequence of sequence identification number: 6 or the nucleotide sequence of sequence identification number: 7, and the sequence of the reverse outer primer for GAPDH nucleic acid includes the nucleotide sequence of sequence identification number: 5. The GAPDH nucleic acid detection kit as described in claim 1, wherein at least one of the forward inner primer for GAPDH nucleic acid, the forward outer primer for GAPDH nucleic acid, the reverse inner primer for GAPDH nucleic acid and the reverse outer primer for GAPDH nucleic acid, starting from any one of the 4th to 14th positions from the 3' end thereof, is independently replaced by one of the following nucleotides: inosine (I), guanine (G); and uracil (U). The GAPDH nucleic acid detection kit as described in claim 3, wherein the sequence of the reverse inner primer for GAPDH nucleic acid includes the nucleotide sequence of sequence identification number: 8 and/or the sequence of the reverse outer primer for GAPDH nucleic acid includes the nucleotide sequence of sequence identification number: 9. The GAPDH nucleic acid detection kit as described in claim 3, wherein the sequence of the forward inner primer for GAPDH nucleic acid includes the nucleotide sequence of sequence identification number: 2; the sequence of the forward outer primer for GAPDH nucleic acid includes the nucleotide sequence of sequence identification number: 3; the sequence of the reverse inner primer for GAPDH nucleic acid includes the nucleotide sequence of sequence identification number: 4 or the nucleotide sequence of sequence identification number: 8, and the sequence of the reverse outer primer for GAPDH nucleic acid includes the nucleotide sequence of sequence identification number: 9. The GAPDH nucleic acid detection kit as described in claim 1, wherein the 5' end of the reverse inner primer for GAPDH nucleic acid is labeled with a first marker, and the 5' end of the forward inner primer for GAPDH nucleic acid is labeled with a second marker, and the first marker is different from the second marker, and wherein the type of the first marker includes biotin, avidin, streptavidin (SA), digoxigenin (DIG) or fluorescein (FAM), and the type of the second marker includes biotin, avidin, streptavidin, digoxigenin or fluorescein. The GAPDH nucleic acid detection kit as described in claim 6 further includes a lateral flow immunoassay test strip, which includes an analyte addition zone, a binding zone, a GAPDH detection zone and a test strip control zone in order according to the flow direction of the analyte, wherein the binding zone has a first binding particle, which has a first binding molecule and a particle connected to the first binding molecule, wherein the first binding molecule has the ability to bind to the first marker, the GAPDH detection zone is fixed with a second binding molecule, which has the ability to bind to the second marker, and the test strip control zone is fixed with a third binding molecule, which has the ability to bind to the first binding molecule in the first binding particle, wherein the third binding molecule is the same as or different from the first marker. The GAPDH nucleic acid detection kit as described in claim 1 further includes a polymerase and/or a nucleotide matrix, wherein the polymerase has the function of a reverse transcriptase, and the polymerase is a Bst DNA polymerase, and its sequence includes the amino acid sequence of sequence identification number: 10. A target nucleic acid detection kit includes: a primer set for detecting GAPDH nucleic acid, including: a forward inner primer for GAPDH nucleic acid; a forward outer primer for GAPDH nucleic acid; a reverse inner primer for GAPDH nucleic acid; and a reverse outer primer for GAPDH nucleic acid, wherein the forward inner primer for GAPDH nucleic acid is composed of a first segment and a second segment, and the 3' end of the first segment The 5' end of the second fragment is connected, or the forward inner primer for GAPDH nucleic acid is composed of a first fragment, a first linker and a second fragment, and the 3' end of the first fragment is connected to the 5' end of the first linker, and the 3' end of the first linker is connected to the 5' end of the second fragment, wherein the first fragment has 10-30 nucleotides and is composed of a complementary strand of a first sequence segment, and the first sequence segment is located at The second fragment has 10-30 nucleotides and is composed of a second sequence segment, and the second sequence segment is located between the 77th and 115th positions of the nucleotide sequence of sequence identification number: 1, and the first linker is composed of 1-6 thymines or peptide nucleic acids, and the forward outer primer for GAPDH nucleic acid The reverse inner primer for GAPDH nucleic acid comprises 10-30 nucleotides and is composed of a third sequence segment, and the third sequence segment is located between the 42nd position and the 79th position of the nucleotide sequence of sequence identification number: 1, the reverse inner primer for GAPDH nucleic acid comprises a third segment and a fourth segment, and the 3' end of the third segment is connected to the 5' end of the fourth segment, or the reverse inner primer for GAPDH nucleic acid comprises a third segment, a fourth segment, and a 5' end of the fourth segment. The third segment is composed of two linkers and a fourth segment, and the 3' end of the third segment is connected to the 5' end of the second linker, and the 3' end of the second linker is connected to the 5' end of the fourth segment, wherein the third segment has 10-30 nucleotides and is composed of a fourth sequence segment, and the fourth sequence segment is located between the 156th position and the 207th position of the nucleotide sequence of sequence identification number: 1, and the fourth segment has 10- 30 nucleotides, and is composed of a complementary strand of a fifth sequence segment, and the fifth sequence segment is located between the 211th position and the 250th position of the nucleotide sequence of sequence identification number: 1, and the second linker is composed of 1-6 thymines or peptide nucleic acid, and the reverse exon primer for GAPDH nucleic acid has 10-30 nucleotides and is composed of a complementary strand of a sixth sequence segment, and the sixth sequence segment Located between the 238th position and the 275th position of the nucleotide sequence of sequence identification number: 1; and a primer set for detecting a target nucleic acid, comprising: a forward inner primer for the target nucleic acid; a forward outer primer for the target nucleic acid; a reverse inner primer for the target nucleic acid; and a reverse outer primer for the target nucleic acid, wherein the detection target of the primer set for detecting the target nucleic acid is different from the detection target of the primer set for detecting GAPDH nucleic acid. Target, wherein the primer set for detecting GAPDH nucleic acid and the primer set for detecting target nucleic acid are used in a first constant temperature circular nucleic acid amplification method and a second constant temperature circular nucleic acid amplification method respectively, and the first constant temperature circular nucleic acid amplification method and the second constant temperature circular nucleic acid amplification method independently include a standard constant temperature circular nucleic acid amplification method or a reverse transcription constant temperature circular nucleic acid amplification method, and wherein the result of the first constant temperature circular nucleic acid amplification method is used as an internal control group. The target nucleic acid detection kit as described in claim 9, wherein the sequence of the forward inner primer for GAPDH nucleic acid includes the nucleotide sequence of sequence identification number: 2; the sequence of the forward outer primer for GAPDH nucleic acid includes the nucleotide sequence of sequence identification number: 3; the sequence of the reverse inner primer for GAPDH nucleic acid includes the nucleotide sequence of sequence identification number: 4, the nucleotide sequence of sequence identification number: 6 or the nucleotide sequence of sequence identification number: 7, and the sequence of the reverse outer primer for GAPDH nucleic acid includes the nucleotide sequence of sequence identification number: 5. The target nucleic acid detection kit as described in claim 9, wherein at least one of the forward inner primer for GAPDH nucleic acid, the forward outer primer for GAPDH nucleic acid, the reverse inner primer for GAPDH nucleic acid and the reverse outer primer for GAPDH nucleic acid, starting from any one of the 4th to 14th positions from the 3' end thereof, is independently replaced by one of the following nucleotides: inosine (I) guanine (G); and uracil (U). The target nucleic acid detection kit as described in claim 11, wherein the sequence of the reverse inner primer for GAPDH nucleic acid includes the nucleotide sequence of sequence identification number: 8 and/or the sequence of the reverse outer primer for GAPDH nucleic acid includes the nucleotide sequence of sequence identification number: 9. The target nucleic acid detection kit as described in claim 11, wherein the sequence of the forward inner primer for GAPDH nucleic acid includes the nucleotide sequence of sequence identification number: 2; the sequence of the forward outer primer for GAPDH nucleic acid includes the nucleotide sequence of sequence identification number: 3; the sequence of the reverse inner primer for GAPDH nucleic acid includes the nucleotide sequence of sequence identification number: 4 or the nucleotide sequence of sequence identification number: 8, and the sequence of the reverse outer primer for GAPDH nucleic acid includes the nucleotide sequence of sequence identification number: 9. The target nucleic acid detection kit as described in claim 9, wherein the detection target of the primer set of the detection target nucleic acid is a nucleic acid of an RNA virus, and the RNA virus includes a coronavirus (Coronavirus), an influenza virus (Influenza virus), a human immunodeficiency virus (HIV), an Ebola virus (Ebolavirus) or a hepatitis C virus (Hepatitis C virus, HCV). The target nucleic acid detection kit as described in claim 14, wherein the coronavirus includes severe acute respiratory syndrome coronavirus (SARS-CoV), severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) (novel coronavirus) or Middle East respiratory syndrome coronavirus (MERS-CoV). The target nucleic acid detection kit as described in claim 9, wherein the detection target of the primer set for detecting the target nucleic acid is the nucleic acid of severe acute respiratory syndrome coronavirus type 2 (novel coronavirus), and wherein the forward inner primer for the target nucleic acid is composed of a fifth segment and a sixth segment, and the 3' end of the fifth segment is connected to the 5' end of the sixth segment, or the forward inner primer for the target nucleic acid is composed of a fifth segment, a third linker and a sixth segment, and the 3' end of the fifth segment is connected to the 5' end of the third linker, and the 3' end of the third linker is connected to the 5' end of the sixth segment, wherein the fifth segment has 10-30 nucleotides, and is composed of a complementary strand of a seventh sequence segment, and the seventh sequence segment is located between the 90th position and the 134th position of the nucleotide sequence of sequence identification number: 11, and the sixth fragment has 10-30 nucleotides and is composed of an eighth sequence segment, and the eighth sequence segment is located between the 45th position and the 82nd position of the nucleotide sequence of sequence identification number: 11, and the third linker is composed of 1-6 thymines or peptide nucleic acid, and the forward outer primer for the target nucleic acid has 10-30 nucleotides and is composed of a ninth sequence segment, and the ninth sequence segment is located between the sequence identification number The reverse inner primer for the target nucleic acid is composed of a seventh segment and an eighth segment, and the 3' end of the seventh segment is connected to the 5' end of the eighth segment, or the reverse inner primer for the target nucleic acid is composed of a seventh segment, a fourth linker and an eighth segment, and the 3' end of the seventh segment is connected to the 5' end of the fourth linker, and the 3' end of the fourth linker is connected to the 5' end of the eighth segment, wherein the seventh segment has 10-30 nucleotides and is composed of a tenth sequence segment, and the tenth sequence segment is located in the nucleic acid of sequence identification number: 11. The eighth segment has 10-30 nucleotides and is composed of a complementary strand of an eleventh sequence segment, and the eleventh sequence segment is located between the 170th and 208th positions of the nucleotide sequence of sequence identification number: 11, and the fourth linker is composed of 1-6 thymines or peptide nucleic acid, and the reverse exon primer for the target nucleic acid has 10-30 nucleotides and is composed of a complementary strand of a twelfth sequence segment, and the twelfth sequence segment is located between the 226th and 263rd positions of the nucleotide sequence of sequence identification number: 11. The target nucleic acid detection kit as described in claim 16, wherein the primer set for detecting the target nucleic acid further includes: a forward loop primer for the target nucleic acid; and a reverse loop primer for the target nucleic acid, wherein the forward loop primer for the target nucleic acid has 10-30 nucleotides and is composed of a thirteenth sequence segment, and the thirteenth sequence segment is located between the 63rd position and the 104th position of the nucleotide sequence of sequence identification number: 11, and the reverse loop primer for the target nucleic acid has 10-30 nucleotides and is composed of a fourteenth sequence segment, and the fourteenth sequence segment is located between the 146th position and the 187th position of the nucleotide sequence of sequence identification number: 11. The target nucleic acid detection kit as described in claim 17, wherein the sequence of the forward inner primer for the target nucleic acid includes the nucleotide sequence of sequence identification number: 12; the sequence of the forward outer primer for the target nucleic acid includes the nucleotide sequence of sequence identification number: 13; the sequence of the forward loop primer for the target nucleic acid includes the nucleotide sequence of sequence identification number: 16; the sequence of the reverse inner primer for the target nucleic acid includes the nucleotide sequence of sequence identification number: 14, the sequence of the reverse outer primer for the target nucleic acid includes the nucleotide sequence of sequence identification number: 15, and the sequence of the reverse loop primer for the target nucleic acid includes the nucleotide sequence of sequence identification number: 17. The target nucleic acid detection kit as described in claim 9, wherein the 5' end of the reverse inner primer for GAPDH nucleic acid and the 5' end of the reverse inner primer for the target nucleic acid are marked with a first marker, the 5' end of the forward inner primer for GAPDH nucleic acid is marked with a second marker, the 5' end of the forward inner primer for the target nucleic acid is marked with a third marker, and the first marker, the second marker and the third marker are all different, and wherein the type of the first marker includes biotin, avidin, streptavidin, fociglobin or luciferin, the type of the second marker includes biotin, avidin, streptavidin, fociglobin or luciferin, and the type of the third marker includes biotin, avidin, streptavidin, fociglobin or luciferin. The target nucleic acid detection kit as described in claim 19 further includes a lateral flow immunoassay test strip, which includes an analyte addition zone, a binding zone, a GAPDH detection zone, a target nucleic acid detection zone and a test strip control zone in sequence according to the flow direction of the analyte, or includes an analyte addition zone, a binding zone, a target nucleic acid detection zone, a GAPDH detection zone and a test strip control zone in sequence, wherein the binding zone has a first binding particle, wherein the first binding particle has a first binding molecule and a linker A particle connected to the first binding molecule, and the first binding molecule has the ability to bind to the first marker, the GAPDH detection area is fixed with a second binding molecule, which has the ability to bind to the second marker, the test piece control area is fixed with a third binding molecule, which has the ability to bind to the first binding molecule in the first binding particle, wherein the third binding molecule is the same as or different from the first marker, and the target nucleic acid detection area is fixed with a fourth binding molecule, which has the ability to bind to the third marker. The target nucleic acid detection kit as described in claim 9 further includes a polymerase and/or a nucleotide matrix, wherein the polymerase has the function of a reverse transcriptase, and the polymerase is Bst DNA polymerase, and its sequence includes the amino acid sequence of sequence identification number: 10. A novel coronavirus detection kit includes: a primer set for detecting GAPDH nucleic acid, including: a forward inner primer for GAPDH nucleic acid; a forward outer primer for GAPDH nucleic acid; a reverse inner primer for GAPDH nucleic acid; and a reverse outer primer for GAPDH nucleic acid, wherein the forward inner primer for GAPDH nucleic acid is composed of a first segment and a second segment, and the 3' end of the first segment is connected to the 5' end of the second segment, or the forward inner primer for GAPDH nucleic acid is composed of a first segment, a first linker and a second segment, and the 3' end of the first segment is connected to the 5' end of the first linker, and the 3' end of the first linker is connected to the 5' end of the second segment. The first fragment has 10-30 nucleotides and is composed of a complementary strand of a first sequence segment, and the first sequence segment is located between the 134th position and the 175th position of the nucleotide sequence of sequence identification number: 1, and the second fragment has 10-30 nucleotides and is composed of a second sequence segment, and the second sequence segment is located between the 77th position and the 115th position of the nucleotide sequence of sequence identification number: 1, and the first linker is composed of 1-6 thymines or peptide nucleic acid, and the forward external primer for GAPDH nucleic acid has 10-30 nucleotides and is composed of a third sequence segment, and the third sequence segment is located between the 77th position and the 115th position of the nucleotide sequence of sequence identification number: 1. The reverse inner primer for GAPDH nucleic acid is composed of a third segment and a fourth segment, and the 3' end of the third segment is connected to the 5' end of the fourth segment, or the reverse inner primer for GAPDH nucleic acid is composed of a third segment, a second linker and a fourth segment, and the 3' end of the third segment is connected to the 5' end of the second linker, and the 3' end of the second linker is connected to the 5' end of the fourth segment, wherein the third segment has 10-30 nucleotides and is composed of a fourth sequence segment, and the fourth sequence segment is located between the 156th and 207th positions of the nucleotide sequence of sequence identification number: 1. , The fourth fragment has 10-30 nucleotides and is composed of a complementary strand of a fifth sequence segment, and the fifth sequence segment is located between the 211th position and the 250th position of the nucleotide sequence of sequence identification number: 1, and the second linker is composed of 1-6 thymines or peptide nucleic acids, the reverse outer primer for GAPDH nucleic acid has 10-30 nucleotides and is composed of a complementary strand of a sixth sequence segment, and the sixth sequence segment is located between the 238th position and the 275th position of the nucleotide sequence of sequence identification number: 1; and a primer set for detecting nucleic acid of the novel coronavirus, including: a forward inner primer for nucleic acid of the novel coronavirus; a forward inner primer for nucleic acid of the novel coronavirus; a forward outer primer for a nucleic acid of a virus; a reverse inner primer for a nucleic acid of a novel coronavirus; and a reverse outer primer for a nucleic acid of a novel coronavirus, wherein the forward inner primer for the nucleic acid of the novel coronavirus is composed of a fifth segment and a sixth segment, and the 3' end of the fifth segment is connected to the 5' end of the sixth segment, or the forward inner primer for the nucleic acid of the target is composed of a fifth segment, a third linker and a sixth segment, and the 3' end of the fifth segment is connected to the 5' end of the third linker, and the 3' end of the third linker is connected to the 5' end of the sixth segment, wherein the fifth segment has 10-30 nucleotides and is composed of a complementary strand of a seventh sequence segment, and the seventh sequence segment The sixth fragment has 10-30 nucleotides and is composed of an eighth sequence segment, and the eighth sequence segment is located between the 45th and 82nd positions of the nucleotide sequence of sequence identification number: 11, and the third linker is composed of 1-6 thymines or peptide nucleic acids, the forward outer primer for the nucleic acid of the novel coronavirus has 10-30 nucleotides and is composed of a ninth sequence segment, and the ninth sequence segment is located between the 27th and 64th positions of the nucleotide sequence of sequence identification number: 11, and the reverse inner primer for the nucleic acid of the novel coronavirus is composed of a seventh The reverse inner primer for the target nucleic acid is composed of a seventh fragment, a fourth linker and an eighth fragment, and the 3' end of the seventh fragment is connected to the 5' end of the fourth linker, and the 3' end of the fourth linker is connected to the 5' end of the eighth fragment, wherein the seventh fragment has 10-30 nucleotides and is composed of a tenth sequence segment, and the tenth sequence segment is located between the 123rd position and the 165th position of the nucleotide sequence of sequence identification number: 11, and the eighth fragment has 10-30 nucleotides and is composed of the complementary strand of an eleventh sequence segment, and the eleventh sequence segment The invention relates to a method for detecting a novel coronavirus nucleic acid, wherein the primer set for detecting a GAPDH nucleic acid and the primer set for detecting a novel coronavirus nucleic acid are respectively used in a constant temperature circular nucleic acid amplification method, and the constant temperature circular nucleic acid amplification method includes a standard constant temperature circular nucleic acid amplification method or a reverse transcription constant temperature circular nucleic acid amplification method. As claimed in claim 22, the novel coronavirus detection kit, wherein the primer set for detecting the target nucleic acid of the novel coronavirus further includes: a forward loop primer for the nucleic acid of the novel coronavirus; and a reverse loop primer for the nucleic acid of the novel coronavirus, wherein the forward loop primer for the nucleic acid of the novel coronavirus has 10-30 nucleotides and is composed of a thirteenth sequence segment, and the thirteenth sequence segment is located between the 63rd position and the 104th position of the nucleotide sequence of sequence identification number: 11, and the reverse loop primer for the nucleic acid of the novel coronavirus has 10-30 nucleotides and is composed of a fourteenth sequence segment, and the fourteenth sequence segment is located between the 146th position and the 187th position of the nucleotide sequence of sequence identification number: 11. As in claim 23, the novel coronavirus detection kit, wherein the sequence of the forward inner primer for the nucleic acid of the novel coronavirus includes the nucleotide sequence of sequence identification number: 12; the sequence of the forward outer primer for the nucleic acid of the novel coronavirus includes the nucleotide sequence of sequence identification number: 13; the sequence of the forward loop primer for the nucleic acid of the novel coronavirus includes the nucleotide sequence of sequence identification number: 16; the sequence of the reverse inner primer for the nucleic acid of the novel coronavirus includes the nucleotide sequence of sequence identification number: 14, the sequence of the reverse outer primer for the nucleic acid of the novel coronavirus includes the nucleotide sequence of sequence identification number: 15, and the sequence of the reverse loop primer for the nucleic acid of the novel coronavirus includes the nucleotide sequence of sequence identification number: 17. As in claim 22, the novel coronavirus detection kit, wherein the 5' end marker of the reverse inner primer for the novel coronavirus nucleic acid shows a first marker, and the 5' end marker of the forward inner primer for the novel coronavirus nucleic acid shows a second marker, and wherein the first marker is different from the second marker, and wherein the type of the first marker includes biotin, avidin, streptavidin, cytoglobin or luciferin, and the type of the second marker includes biotin, avidin, streptavidin, cytoglobin or luciferin. The novel coronavirus detection kit of claim 25 further includes a lateral flow immunoassay test strip, which includes an analyte addition zone, a binding zone, a novel coronavirus detection zone, and a test strip control zone in order according to the flow direction of the analyte, wherein the binding zone has a first binding particle, which has a first binding molecule and a particle connected to the first binding molecule, wherein the first binding molecule has the ability to bind to the first marker, the novel coronavirus detection zone is fixed with a second binding molecule, which has the ability to bind to the second marker, and the test strip control zone is fixed with a third binding molecule, which has the ability to bind to the first binding molecule in the first binding particle, wherein the third binding molecule is the same as or different from the first marker. The novel coronavirus detection kit of claim 22 further includes a polymerase and/or a nucleotide matrix, wherein the polymerase has the function of a reverse transcriptase, and the polymerase is a Bst DNA polymerase, and its sequence includes an amino acid sequence of sequence identification number: 10. A method for detecting GAPDH nucleic acid, comprising: (a) providing a sample to be tested; and (b) subjecting the sample to be tested to the constant temperature circular nucleic acid amplification method using the primer set for detecting GAPDH nucleic acid in the GAPDH nucleic acid detection kit of claim 1, wherein if the sample to be tested contains GAPDH nucleic acid, a GAPDH nucleic acid amplification product is obtained from the constant temperature circular nucleic acid amplification method, wherein the constant temperature circular nucleic acid amplification method includes a standard constant temperature circular nucleic acid amplification method or a reverse transcription constant temperature circular nucleic acid amplification method. A method for detecting GAPDH nucleic acid as described in claim 28, wherein the constant temperature circular nucleic acid amplification method uses the single strand RNA or first strand cDNA in the sample to be tested as the starting template. A method for detecting GAPDH nucleic acid as described in claim 28, wherein the source of the sample to be tested includes a saliva sample, a sputum sample, a nose swab sample, a throat swab sample, a nasopharyngeal sample, a urine sample, a stool sample, a rectal swab sample, a cerebrospinal fluid (CSF) sample, or a body fluid sample. The method for detecting GAPDH nucleic acid as described in claim 28 further comprises, before step (a), subjecting a biological sample to a pre-treatment to obtain the sample to be tested, wherein the pre-treatment comprises a step selected from the group consisting of the following steps: (i) subjecting the biological sample to a heat treatment step; (ii) subjecting the biological sample to a nucleic acid purification step; (iii) The biological sample is subjected to a reverse transcription step; (iv) the biological sample is subjected to a nucleic acid purification step, followed by a reverse transcription step; (v) the biological sample is subjected to a reverse transcription step, followed by an RNA removal step; and (vi) the biological sample is subjected to a nucleic acid purification step, followed by a reverse transcription step, followed by an RNA removal step. A method for detecting GAPDH nucleic acid as described in claim 31, wherein the biological sample is a saliva sample. A method for detecting GAPDH nucleic acid as described in claim 28, wherein the sample to be tested is an original saliva sample. A method for detecting GAPDH nucleic acid as described in claim 28, wherein the constant temperature circular nucleic acid amplification method is a reverse transcription constant temperature circular nucleic acid amplification method. A method for detecting GAPDH nucleic acid as described in claim 34, wherein in the reverse transcription constant temperature circular nucleic acid amplification method, the reverse transcription process and the constant temperature circular nucleic acid amplification process are performed in one step, and in the reverse transcription constant temperature circular nucleic acid amplification method, a polymerase with the function of a reverse transcriptase is used, and wherein the polymerase is a Bst DNA polymerase, and the sequence of the Bst DNA polymerase includes the amino acid sequence of sequence identification number: 10. A method for detecting GAPDH nucleic acid as described in claim 34, wherein in the reverse transcription constant temperature circular nucleic acid amplification method, a constant temperature circular amplification process is performed after a reverse transcription process. A method for detecting GAPDH nucleic acid as described in claim 28, wherein the sequence of the forward inner primer for GAPDH nucleic acid includes the nucleotide sequence of sequence identification number: 2; the sequence of the forward outer primer for GAPDH nucleic acid includes the nucleotide sequence of sequence identification number: 3; the sequence of the reverse inner primer for GAPDH nucleic acid includes the nucleotide sequence of sequence identification number: 4, the nucleotide sequence of sequence identification number: 6 or the nucleotide sequence of sequence identification number: 7, and the sequence of the reverse outer primer for GAPDH nucleic acid includes the nucleotide sequence of sequence identification number: 5. A method for detecting GAPDH nucleic acid as described in claim 28, wherein at least one of the forward inner primer for GAPDH nucleic acid, the forward outer primer for GAPDH nucleic acid, the reverse inner primer for GAPDH nucleic acid and the reverse outer primer for GAPDH nucleic acid, starting from any one of the 4th to 14th positions from the 3' end thereof, is independently replaced by one of the following nucleotides: inosine (I), guanine (G); and uracil (U). A method for detecting GAPDH nucleic acid as described in claim 38, wherein the sequence of the forward inner primer for GAPDH nucleic acid includes the nucleotide sequence of sequence identification number: 2; the sequence of the forward outer primer for GAPDH nucleic acid includes the nucleotide sequence of sequence identification number: 3; the sequence of the reverse inner primer for GAPDH nucleic acid includes the nucleotide sequence of sequence identification number: 4 or the nucleotide sequence of sequence identification number: 8, and the sequence of the reverse outer primer for GAPDH nucleic acid includes the nucleotide sequence of sequence identification number: 9. A method for detecting GAPDH nucleic acid as described in claim 28, wherein the presence of the amplified product of the GAPDH nucleic acid is confirmed by a gel electrophoresis analysis or a lateral flow immunoassay. A method for detecting a novel coronavirus, comprising: (a) providing a sample to be tested; and (b) subjecting the sample to be tested to a constant temperature cyclic nucleic acid amplification method using the primer set for detecting GAPDH nucleic acid and the primer set for detecting nucleic acid of the novel coronavirus in the novel coronavirus detection kit described in claim 22, wherein the primer set for detecting GAPDH nucleic acid is used to amplify the nucleic acid of the sample to be tested. The result of the constant temperature circular nucleic acid amplification method is used as an internal control group. If the sample to be tested contains the novel coronavirus, a nucleic acid amplification product of the novel coronavirus is obtained from the constant temperature circular nucleic acid amplification method performed using the primer set for detecting the nucleic acid of the novel coronavirus, wherein the constant temperature circular nucleic acid amplification method includes a standard constant temperature circular nucleic acid amplification method or a reverse transcription constant temperature circular nucleic acid amplification method. A method for detecting a novel coronavirus as described in claim 41, wherein the constant temperature circular nucleic acid amplification method uses a single-stranded RNA or first-stranded cDNA in the sample to be tested as a starting template. A method for detecting the novel coronavirus as described in claim 41, wherein the source of the sample to be tested includes a saliva sample, a sputum sample, a nasal swab sample, a throat swab sample, a nasopharyngeal sample, a urine sample, a stool sample, a rectal swab sample, a cerebrospinal fluid sample, or a body fluid sample. The method for detecting the novel coronavirus as described in claim 41 further includes, before step (a), subjecting a biological sample to a pre-treatment to obtain the sample to be tested, wherein the pre-treatment includes a step selected from the group consisting of the following steps: (i) subjecting the biological sample to a heat treatment step; (ii) subjecting the biological sample to a nucleic acid purification step; (iii) The biological sample is subjected to a reverse transcription step; (iv) the biological sample is subjected to a nucleic acid purification step, followed by a reverse transcription step; (v) the biological sample is subjected to a reverse transcription step, followed by an RNA removal step; and (vi) the biological sample is subjected to a nucleic acid purification step, followed by a reverse transcription step, followed by an RNA removal step. A method for detecting a novel coronavirus as described in claim 44, wherein the biological sample is a saliva sample. A method for detecting a novel coronavirus as described in claim 41, wherein the sample to be tested is a raw saliva sample. A method for detecting a novel coronavirus as described in claim 41, wherein the constant temperature circular nucleic acid amplification method is a reverse transcription constant temperature circular nucleic acid amplification method. A method for detecting the novel coronavirus as described in claim 47, wherein in the reverse transcription constant temperature circular nucleic acid amplification method, the reverse transcription process and the constant temperature circular nucleic acid amplification process are performed in one step, and in the reverse transcription constant temperature circular nucleic acid amplification method, a polymerase having the function of a reverse transcriptase is used, and wherein the polymerase is a Bst DNA polymerase, and the sequence of the Bst DNA polymerase includes the amino acid sequence of sequence identification number: 10. A method for detecting a novel coronavirus as described in claim 47, wherein in the reverse transcription constant temperature circular nucleic acid amplification method, a constant temperature circular nucleic acid amplification process is performed after a reverse transcription process. The method for detecting the novel coronavirus as described in claim 41, wherein the primer set for detecting the target nucleic acid of the novel coronavirus further includes: a forward loop primer for the nucleic acid of the novel coronavirus; and a reverse loop primer for the nucleic acid of the novel coronavirus, wherein the forward loop primer for the nucleic acid of the novel coronavirus has approximately 10-30 nucleotides and is composed of a thirteenth sequence segment, and the thirteenth sequence segment is located between the 63rd and 104th positions of the nucleotide sequence of sequence identification number: 11, and the reverse loop primer for the nucleic acid of the novel coronavirus has approximately 10-30 nucleotides and is composed of a fourteenth sequence segment, and the fourteenth sequence segment is located between the 146th and 187th positions of the nucleotide sequence of sequence identification number: 11. A method for detecting a novel coronavirus as described in claim 50, wherein the sequence of the forward inner primer for the nucleic acid of the novel coronavirus includes the nucleotide sequence of sequence identification number: 12; the sequence of the forward outer primer for the nucleic acid of the novel coronavirus includes the nucleotide sequence of sequence identification number: 13; the sequence of the forward loop primer for the nucleic acid of the novel coronavirus includes the nucleotide sequence of sequence identification number: 16; the sequence of the reverse inner primer for the nucleic acid of the novel coronavirus includes the nucleotide sequence of sequence identification number: 14, the sequence of the reverse outer primer for the nucleic acid of the novel coronavirus includes the nucleotide sequence of sequence identification number: 15, and the sequence of the reverse loop primer for the nucleic acid of the novel coronavirus includes the nucleotide sequence of sequence identification number: 17. A method for detecting a novel coronavirus as described in claim 41, wherein the presence of a nucleic acid amplification product of the novel coronavirus is confirmed by a gel electrophoresis analysis or a lateral flow immunoassay.