WO2024050998A1 - Nucleic acid detection reagent for rapidly detecting pathogen of pets, and kit and use thereof - Google Patents

Abstract

Disclosed are a nucleic acid detection reagent for rapidly detecting a pathogen of pets, and a kit and a use thereof. Specifically, the method comprises: step T1, lysing, in a constant-temperature environment, a sample to be detected; step T2, performing TSA amplification on nucleic acids of the lysed sample; step T3, detecting the amplified nucleic acids by using a Cas protein; and step T4, reading the detection result by using a colloidal gold test strip. In T1, the nucleic acids in the sample are extracted by using a strong alkaline nucleic acid extraction liquid having a nuclease inhibitor; in T2, isothermal amplification of the nucleic acids is realized by using a UvsX protein, a UvsY protein, a single-stranded binding protein GP32, and a Bsu protein; in T3, a specific fragment is detected by using Cas protein technology; and in T4, the detection result is displayed by using a Reporter and a colloidal gold test strip.

Description

A reagent, kit and application for rapid nucleic acid detection of pet pathogens Technical field

The invention belongs to the field of biosafety technology. Specifically, it particularly relates to a reagent, a kit and an application thereof that can quickly detect pet infection virus nucleic acid at room temperature.

Background technique

With the rapid development of molecular biology technology, it not only involves biology, medicine, genetics, botany, zoology, etc., but also gradually becomes the underlying technology for practical applications. As a relatively mature technology, PCR has the characteristics of high specificity and high efficiency. However, it has been difficult to get rid of the constraints of laboratories with higher cleanliness levels and professional operators.

For a long time, the shortcomings of extracting nucleic acids (DNA and RNA) from biologically active samples and their purification steps are numerous and time-consuming, which have hindered the rapid development of nucleic acid detection. Especially when the titer of the pathogen in the test sample is low, whether the nucleic acid in the sample can be completely extracted and stored affects the subsequent test results. Therefore, solving the two problems of multiple nucleic acid extraction steps and long time is of great significance for nucleic acid detection.

For rough samples (swabs, blood, etc.), the method of adding nuclease inhibitors and heating in a water bath is generally used to lyse microorganisms or cells and release nucleic acids. The Hudson method involves adding EDTA and TCEP, first bathing in water at 60°C for 2 minutes to inactivate microorganisms, and then raising the temperature to 95°C for 10 minutes to obtain an extraction solution mixed with nucleic acids. Due to its simple operation, the next step of detection can be carried out directly without purification. It is low cost, easy to operate, and has low nucleic acid loss. It can be used as one of the rapid detection operations.

Nucleic acid extraction solutions (preservation solutions) available on the market usually add guanidinium isothiocyanate as a lysis component. Although it can destroy virus activity at room temperature and release nucleic acids, it usually requires more than ten minutes of lysis time to achieve the best results. Extraction effect.

In addition, another commonly used nucleic acid extraction method is alkaline lysis. For example, Chinese patent application CN201210242862.6 uses a strong alkaline solution to lyse cells in biological samples at high temperatures to release nucleic acids, and then adds acidic buffer for neutralization and centrifugation to obtain the extracted nucleic acid solution. Its high temperature and centrifugation steps also make rapid extraction difficult.

Regardless of whether the Hudson method or the alkaline lysis method is used, the extraction and detection of nucleic acids in samples using existing technologies will involve a heating step, and the waiting time of the heating process and the dangers caused by the use of electrical appliances and heating equipment are contrary to the original intention of rapid detection. .

In recent years, the combination of isothermal amplification technology and CRISPR gene editing technology has created a new platform for the development of nucleic acid isothermal amplification detection technology. The current steps for isothermal amplification and gene editing to detect pathogens include nucleic acid extraction, isothermal amplification, Cas protein editing, and product detection.

Nucleic acid extraction is a key step in nucleic acid amplification technology. However, because the methods for extracting nucleic acids in laboratories are usually cumbersome, a simple, fast, efficient, and low-energy-consuming nucleic acid extraction method is needed. Commonly used methods for rapid nucleic acid extraction include solid-phase extraction kit rapid extraction method, magnetic bead method, alumina membrane method and lysis solution treatment method. However, the solid-phase extraction method requires the operation of instruments such as centrifuges, the magnetic bead method has high processing costs, and the aluminum oxide membrane method has a complicated manufacturing process, which limits its more practical and effective application in rapid nucleic acid detection. The constant temperature rapid extraction method of lysate is simple to operate, cheap and easy to obtain, and is an ideal nucleic acid extraction method.

Contents of the invention

1. Problems to be solved

In view of the shortcomings of the existing technology, the present invention provides a reagent, a kit and its application that can quickly detect pet infection virus nucleic acid at room temperature. The reagent, kit and its application all use highly alkaline nucleic acid extraction solutions. The nucleic acid in the sample is extracted, and then the acidic stop solution is added to the extraction solution to neutralize it for the next step of amplification. The alkali concentration of the alkaline nucleic acid extraction solution is appropriately reduced, and EDTA is added to the extraction solution to inhibit nuclease activity; Tris is also added as a buffer system. After adding the acidic stop solution, there is a buffer system in the liquid, and errors caused by operation Little effect on pH. For complex samples such as serum, plasma, whole blood, urine, mouth, nose and eye secretions, reproductive tract secretions, anal swabs, feces and other complex samples, nucleic acids can also be extracted when using lysis buffer, but the lysis effect will be affected by the state of the sample. To obtain the best extraction results, the sample can be properly pretreated.

2. Technical solutions

In order to solve the above problems, the present invention adopts the following technical solutions.

A reagent for rapid nucleic acid detection of pet pathogens. The reagent includes an extraction solution, a stop solution, a TSA reaction solution, a Cas12a detection solution, and a colloidal gold test strip;

The extraction liquid includes strong alkali hydroxide and Tris-HCl;

The stop solution includes acetic acid and Tris acetic acid;

The TSA reaction solution includes UvsX protein, UvsY protein, GP32 protein, Bsu protein, dNTP, ATP, Tris buffer, creatine phosphate, creatine kinase, polyethylene glycol 35000 and MgOAc;

The Cas12a detection solution includes Cas12a protein, NaCl, Tris-HCl, MgCl ₂ , BSA, crRNA and FAM-Bio modified ssDNA;

The colloidal gold test strip includes a T band and a C band, where the T band is labeled with streptavidin and the C band is labeled with anti-anti-FAM antibody.

The above-mentioned reagents for rapid nucleic acid detection of pet pathogens,

In the extract:

The mass concentration of the strong alkali hydroxide is 25mM ~ 500mM, and the strong alkali hydroxide is NaOH,

The mass concentration of Tris-HCl is 10mM ~ 70mM,

The mass concentration of EDTA is 10mM ~ 50mM;

The pH value of the extraction liquid is 11.4-13.7.

The above-mentioned reagents for rapid nucleic acid detection of pet pathogens,

In the stop solution:

The mass concentration of the acetic acid is 1M ~ 5M,

The mass concentration of Tris-acetic acid is 1M;

The pH value of the stop solution is 2-2.5.

The above-mentioned reagents for rapid nucleic acid detection of pet pathogens,

In the TSA reaction solution:

The mass concentration of the UvsX protein is 100-300ng/μL,

The mass concentration of the UvsY protein is 50-100ng/μL,

The mass concentration of the GP32 protein is 100-500ng/μL,

The mass concentration of the Bsu protein is 50-200ng/μL,

The mass concentration of the dNTP is 150-300 μM,

The mass concentration of ATP is 2-10mM,

The mass concentration of the Tris buffer is 20-80mM,

The mass concentration of creatine phosphate is 20-80mM,

The mass concentration of creatine kinase is 30-100ng/μL,

The mass concentration of polyethylene glycol 35000 is 1-7% (w/v),

The mass concentration of MgOAc is 20mM.

The above-mentioned reagents for rapid nucleic acid detection of pet pathogens,

In the Cas12a detection solution:

The mass concentration of the Cas12a protein is 1-10uM,

The mass concentration of NaCl is 20-75mM,

The mass concentration of Tris-HCl is 5-15mM,

The mass concentration of MgCl ₂ is 1-20mM,

The mass concentration of the BSA is 50-150ug/ml,

The mass concentration of crRNA is 10-50nM,

The mass concentration of the FAM-Bio modified ssDNA is 1-100nM,

The pH value of the Cas12a detection solution is 7.9.

A kit containing the above-mentioned reagents.

A kit as described above is used in detecting nucleic acid of pet pathogens.

The applications mentioned above,

Includes the following steps:

(1) Extract the nucleic acid in the sample using the extraction solution to obtain a sample nucleic acid solution;

(2) Add all the stop solution to the sample nucleic acid solution obtained in step (1) to neutralize the pH of the sample nucleic acid solution and obtain a mixed solution; the mixed pH value is 6.5-8;

(3) Use the mixed solution to perform an amplification reaction and detect nucleic acids in the sample.

The pH of the mixed solution is 6.5-8.

3. Beneficial effects

Compared with the existing technology, the beneficial effects of the present invention are:

(1) Compared with the cumbersome and complicated nucleic acid extraction, it only takes 2 minutes and 2 steps to extract the sample nucleic acid;

(2) The extracted nucleic acid can be stored for a long time and can be used for various detection and amplification;

(3) The nucleic acid amplification method provided by the present invention has a wide amplification temperature range and can complete the amplification operation in a short time without the need for large-scale precision equipment or professionals;

(4) The combination of the first step of nucleic acid extraction and the second step of nucleic acid amplification can quickly complete the arrangement and detection at the sampling site.

Description of the drawings

In order to more clearly explain the specific embodiments of the present invention or the technical solutions in the prior art, the following will briefly introduce the drawings that need to be used in the description of the specific embodiments or the prior art. Obviously, the drawings in the following description are some embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained based on these drawings without exerting creative efforts.

Figure 1 is a diagram of the extraction effect (PCR verification) of the extraction solution under different extraction times of the present invention;

Figure 2 shows the effect of nucleic acid amplification at different temperatures;

Figure 3 shows the effect of nucleic acid amplification at different times;

Figure 4 is a schematic diagram of the principle of isothermal nucleic acid amplification;

Figure 5 shows the cleavage effect of Cas12a at different concentrations;

Figure 6 shows the test results of colloidal gold test strips.

Detailed ways

The present invention will be further described below with reference to specific embodiments.

The basic technical solution of the present invention is as follows:

A reagent for rapid nucleic acid detection of pet pathogens. The reagent includes an extraction solution, a stop solution, a TSA reaction solution, a Cas12a detection solution, and a colloidal gold test strip;

The extraction liquid includes strong alkali hydroxide and Tris-HCl;

The stop solution includes acetic acid and Tris acetic acid;

The TSA reaction solution includes UvsX protein, UvsY protein, GP32 protein, Bsu protein, dNTP, ATP, Tris buffer, creatine phosphate, creatine kinase, polyethylene glycol 35000 and MgOAc;

The Cas12a detection solution includes Cas12a protein, NaCl, Tris-HCl, MgCl ₂ , BSA, crRNA and FAM-Bio modified ssDNA;

The colloidal gold test strip includes a T band and a C band, where the T band is labeled with streptavidin and the C band is labeled with anti-anti-FAM antibody.

The above-mentioned reagents for rapid nucleic acid detection of pet pathogens,

In the extract:

The mass concentration of the strong alkali hydroxide is 25mM ~ 500mM, and the strong alkali hydroxide is NaOH,

The mass concentration of Tris-HCl is 10mM ~ 70mM,

The mass concentration of EDTA is 10mM ~ 50mM;

The pH value of the extraction liquid is 11.4-13.7.

The above-mentioned reagents for rapid nucleic acid detection of pet pathogens,

In the stop solution:

The mass concentration of the acetic acid is 1M ~ 5M,

The mass concentration of Tris-acetic acid is 1M;

The pH value of the stop solution is 2-2.5.

The above-mentioned reagents for rapid nucleic acid detection of pet pathogens,

In the TSA reaction solution:

The mass concentration of the UvsX protein is 100-300ng/μL,

The mass concentration of the UvsY protein is 50-100ng/μL,

The mass concentration of the GP32 protein is 100-500ng/μL,

The mass concentration of the Bsu protein is 50-200ng/μL,

The mass concentration of the dNTP is 150-300 μM,

The mass concentration of ATP is 2-10mM,

The mass concentration of the Tris buffer is 20-80mM,

The mass concentration of creatine phosphate is 20-80mM,

The mass concentration of creatine kinase is 30-100ng/μL,

The mass concentration of polyethylene glycol 35000 is 1-7% (w/v),

The mass concentration of MgOAc is 20mM.

The above-mentioned reagents for rapid nucleic acid detection of pet pathogens,

In the Cas12a detection solution:

The mass concentration of the Cas12a protein is 1-10uM,

The mass concentration of NaCl is 20-75mM,

The mass concentration of Tris-HCl is 5-15mM,

The mass concentration of MgCl ₂ is 1-20mM,

The mass concentration of the BSA is 50-150ug/ml,

The mass concentration of crRNA is 10-50nM,

The mass concentration of the FAM-Bio modified ssDNA is 1-100nM,

The pH value of the Cas12a detection solution is 7.9.

A kit containing the above-mentioned reagents.

A kit as described above is used in detecting nucleic acid of pet pathogens.

The applications mentioned above,

Includes the following steps:

(1) Extract the nucleic acid in the sample using the extraction solution to obtain a sample nucleic acid solution;

(2) Add all the stop solution to the sample nucleic acid solution obtained in step (1) to neutralize the pH of the sample nucleic acid solution and obtain a mixed solution; the mixed pH value is 6.5-8;

(3) Use the mixed solution to perform an amplification reaction and detect nucleic acids in the sample.

The pH of the mixed solution is 6.5-8.

It should be noted,

The present invention adopts the following nucleic acid extraction technical solution: a strong alkaline nucleic acid extraction method, including the following steps: putting the sample into an extraction solution (a strong alkali hydroxide solution with a concentration of 25mM to 500mM) to extract nucleic acids; Shake and mix well, let stand at room temperature for extraction for 2 minutes; add stop solution (HCl solution with a concentration of 0.1M-1.5M and a pH of 0.1-1) and shake to mix.

As a further improvement of this technical solution, the strong alkali hydroxide is NaOH or KOH;

As a further improvement of this technical solution, the volume of the extraction liquid is 200 μl;

As a further improvement of this technical solution, the volume of the stop solution is 200 μl;

As a further improvement of this technical solution, after the third step, the nucleic acid can be stored at -20°C for one month;

Compared with the existing technology, the present invention can meet the existing needs of various molecular biology detection and amplification by changing the concentration, pH value, extraction temperature and time of reagents and other parameters, and can use the same equipment at the same time. Automatic DNA and RNA extraction work is performed simultaneously under experimental conditions.

At the same time, the present invention adopts the following nucleic acid amplification system solution: including UvsX protein, UvsY protein, GP32 protein and Bsu protein:

The UvsX protein is any one of the following:

A 1) The amino acid sequence is shown in SEQ ID NO:1;

A 2) Any one or several amino acid residues of the amino acid sequence shown in SEQ ID NO:1 has been obtained by substitution and/or deletion and/or addition of one or several amino acid residues and has the same characteristics as the protein shown in A1) Proteins with more than 90% identity;

The UvsY protein is any one of the following:

B 1) The amino acid sequence is shown in SEQ ID NO:2;

B2) Any one or several amino acid residues of the amino acid sequence shown in SEQ ID NO:2 has been obtained by substituting and/or deleting and/or adding one or several amino acid residues and has 90% similarity with the protein shown in B1) Proteins with more than % identity;

The single-chain binding protein GP32 is any of the following:

C1) The amino acid sequence is shown in SEQ ID NO:3;

C2) Any one or several amino acid residues of the amino acid sequence shown in SEQ ID NO:3 has been obtained by substituting and/or deleting and/or adding one or several amino acid residues and has 90% similarity with the protein shown in C1) Proteins with more than % identity;

DNA polymerase Bsu is any one of the following:

D1) The amino acid sequence is shown in SEQ ID NO:4;

D2) Any one or several amino acid residues of the amino acid sequence shown in SEQ ID NO:4 has been obtained by substituting and/or deleting and/or adding one or several amino acid residues and has 90% similarity with the protein shown in D1) Proteins with more than % identity;

Optionally, the mass ratio of the UvsX protein, UvsY protein, single-chain binding protein GP32, and Bsu protein is 1: (0.1-4.5): (0.75-2.4): (0.17-0.82); it is recommended that the UvsX be preferred The mass ratio of protein, UvsY protein, single-chain binding protein GP32, and Bsu protein is 260:88:300:90.

Optionally, it also includes one or more of magnesium ion reaction initiator, dNTP, ATP, Tris-HCl buffer, creatine phosphate, creatine kinase (CK), and polyethylene glycol (PEG35000).

Optionally, the reaction starter is magnesium ion, and the concentration of the starter in the reaction system is 20mM.

Optionally, the reaction initiator magnesium ion is MgCl ₂ or MgOAc.

The reaction system contains the following materials: a pair of forward and reverse primers (concentration of each primer is 300-600nM), 100-300ng/μL UvsX protein, 50-100ng/μL UvsY protein, 100-500ng/μL GP32 protein, 50 -200ng/μL Bsu protein, 150-300μM dNTP, 2-10mM ATP, 20-80mM Tris buffer, 20-80mM creatine phosphate; 3 0-100ng/μL creatine kinase; 1-7% (w/v) Polyethylene glycol 35000, 20mM MgOAc.

A kit for isothermal nucleic acid detection, which includes the above system.

An isothermal nucleic acid detection method, the sample to be tested is added to the above screening system, and the reaction is carried out at a constant temperature for 15-30 minutes; at 30°C-47°C, the reaction is carried out at a constant temperature for 15-30 minutes; the method is a non-disease diagnosis and treatment method.

Optionally, the sample to be tested is DNA or RNA.

Optionally, when the sample to be tested is RNA, reverse transcriptase (MuLV) needs to be added to the reaction system.

Third, the present invention adopts the following nucleic acid detection method: including Cas12a protein, NaCl, Tris-HCl, MgCl ₂ , BSA, crRNA and FAM-Bio modified ssDNA.

Optional, the NaCl concentration is 5mM, the Tris-HCl concentration is 1mM, the MgCl2 concentration is 1mM, the BSA concentration is 10μg/ml, the Cas12a concentration is 10μM, the crRNA concentration is 50nM, and the ssDNA reporter concentration is 15nM.

Optionally, MgCl2 can be replaced with MgOAc.

Optionally, the ssDNA reporter concentration can be replaced with 10mM.

Optionally, the ssDNA reporter concentration can be replaced with 5mM.

It should be noted that in the following examples, specific concentrations of reagents are not mentioned, and experiments are conducted with the intermediate values as the most preferred values. At the same time, regarding primer I, Primer F: 5'-GTAGTTGTAAATAATATGGATAAAACTGCAG-3', Primer R: 5'-GGCTGAGTAGCAGATTCTGAAACAGTCTTT-3', as shown in SEQ ID NO: 5 and SEQ ID NO: 6 respectively.

Example 1

The steps for extracting feline parvovirus are as follows:

(1) Put the collected samples into the extraction solution (a strong alkali hydroxide solution with a concentration of 25mM to 500mM, it is recommended to choose 100mM) to extract nucleic acids; shake and mix the above solution, and let it stand at room temperature for extraction. 2 minutes; add stop solution (HCl solution with a concentration of 0.1M-1.5M and a pH of 0.1-1, it is recommended to choose a concentration of 0.8M and a pH of 0.5) and shake to mix.

(2) Carry out the following sets of reactions:

Add a blank swab to a-;

b-Add cat fine sample swab for 20s;

c-Add cat fine sample swab for 40s;

Add cat fine sample swab to d- for 60 seconds;

Add cat fine sample swab to e- for 90s;

Add cat fine sample swab to f- for 120s.

(3) Use specific primer I and PCR master mix for the above liquids, and perform PCR amplification on the Applied Biosystems 2720 P CR instrument. The program settings are as follows: predenaturation (94°C, 5min) - denaturation (94°C, 30s) - Annealing (55℃, 30s)-extension (72℃, 20s) go to denaturation, 28cycle-final extension (72℃, 10min).

(4)Results

The real-time results are shown in Figure 1. The results show that under the same conditions, taking both effect and efficiency into consideration, the extraction effect is best and the efficiency is highest at 2 minutes.

Example 2

Conduct amplification temperature test on the above extraction liquid e:

(1) Configure the reaction system (50μL system): 5μL template DNA e, primer I (420nM each primer), 1μl MuLV reverse transcriptase, 260ng/μL UvsX protein, 88ng/μL UvsY protein, 300ng/μL GP32 protein, 90ng/ μL Bsu protein, 240 μM dNTP, 5mM ATP, 100mM potassium acetate, 100mM Tris buffer, 40mM creatine phosphate; 90ng/μL creatine kinase; 6% polyethylene glycol 35000.

(2) Carry out the following sets of reactions:

g-Replace template DNA with DEPC water e;

Start by adding MgOAc with a final concentration of 20mM to h-, the reaction temperature is 10°C, and the reaction is terminated by purifying the product after 30 minutes of reaction;

MgOAc with a final concentration of 20mM was added to i-1 to start, the reaction temperature was 20°C, and the reaction was terminated by purifying the product after 30 minutes of reaction;

MgOAc with a final concentration of 20mM was added to j-1 to start, the reaction temperature was 30°C, and the reaction was terminated by purifying the product after 30 minutes of reaction;

Start by adding MgOAc with a final concentration of 20mM to k-, the reaction temperature is 35°C, and the reaction is terminated by purifying the product after 30 minutes of reaction;

MgOAc with a final concentration of 20mM was added to m- to start, the reaction temperature was 40°C, and the reaction was terminated by purifying the product after 30 minutes of reaction;

Start by adding MgOAc with a final concentration of 20mM to n-, the reaction temperature is 47°C, and the reaction is terminated by purifying the product after 30 minutes of reaction;

(3)Results

The real-time results are shown in Figure 2. The results show that the reaction can achieve amplification effects at 20-47°C.

Example 3

Perform amplification time test on the above extract e

(1) Configure the reaction system (50μL system): 5μL template DNA e, primer I (420nM each primer), 1μl MuLV reverse transcriptase, 260ng/μL UvsX protein, 88ng/μL UvsY protein, 300ng/μL GP32 protein, 90ng/ μL Bsu protein, 240 μM dNTP, 5mM ATP, 100mM potassium acetate, 100mM Tris buffer, 40mM creatine phosphate; 90ng/μL creatine kinase; 6% polyethylene glycol 35000.

(2) Carry out the following sets of reactions:

o-Replace template DNA e with DEPC water;

Start by adding MgOAc with a final concentration of 20mM to p-, the reaction temperature is 37°C, and the reaction is terminated by purifying the product after 5 minutes of reaction;

Start by adding MgOAc with a final concentration of 20mM to q-, the reaction temperature is 37°C, and the reaction is terminated by purifying the product after 10 minutes of reaction;

Start by adding MgOAc with a final concentration of 20mM to r-, the reaction temperature is 37°C, and the reaction is terminated by purifying the product after 15 minutes of reaction;

Start by adding MgOAc with a final concentration of 20mM to s-, the reaction temperature is 37°C, and the reaction is terminated by purifying the product after 20 minutes of reaction;

Start by adding MgOAc with a final concentration of 20mM to t-, the reaction temperature is 37°C, and the reaction is terminated by purifying the product after 25 minutes of reaction;

Start by adding MgOAc with a final concentration of 20mM to u-, the reaction temperature is 37°C, and the reaction is terminated by purifying the product after 30 minutes of reaction;

(3)Results

The real-time results are shown in Figure 3. The results show that the amplification effect becomes stronger with time in the first 30 minutes and reaches the maximum amount at 30 minutes.

Example 4

Carry out Cas12a concentration test on the above extract u.

(1) Configure the reaction system (20μl system): NaCl 5mM, Tris-HCl 1mM, MgCl ₂ 1mM, BSA 10μg/ml, crRNA 50nM, ssDNA reporter 15nM (FQ modification), Target (u) 2μl.

(2) Carry out the following sets of reactions:

Cas12a is not added to v-;

Cas12a was added to w- at a final concentration of 1 μM;

Add Cas12a to x- at a final concentration of 5 μM;

Add Cas12a to y- at a final concentration of 10 μM;

Add Cas12a to z- at a final concentration of 20 μM;

(3) The results were read using the ABI StepOne Plus qPCR instrument to read the fluorescence data.

The real-time results are shown in Figure 5. The results show that in the system of the present invention, the shearing efficiency is highest when the final concentration of Cas12a is 10 μM.

Example 5

Perform Cas12a cleavage on the above extract u and read the result using a test strip.

(1) Configure the reaction system (20μl system): NaCl 5mM, Tris-HCl 1mM, MgCl ₂ 1mM, BSA 10μg/ml, crRNA 50nM, ssDNA reporter 15nM (F-Bio modified).

(2) Carry out the following sets of reactions:

Add Target(u)2μl to pc-;

Add DEPC 2μl to nc-;

(3) Incubate at 37°C for 20 minutes;

(4) Insert the colloidal gold test strips respectively;

(5)Results

The results are shown in Figure 6. The results show that C has a band and T has no band, which is positive (pc); C has a band and T has a band, which is negative.

The above content is a further detailed description of the present invention in combination with specific implementation modes. It cannot be concluded that the specific implementation of the present invention is limited to these descriptions. For those of ordinary skill in the technical field to which the present invention belongs, without departing from the concept of the present invention, Several simple deductions or substitutions can also be made, which should be deemed to fall within the protection scope determined by the claims submitted for the present invention.

Claims (8)

A reagent for rapid nucleic acid detection of pet pathogens, which is characterized by: The reagents include extraction solution, stop solution, TSA reaction solution, Cas12a detection solution, and colloidal gold test strips; The extraction liquid includes strong alkali hydroxide and Tris-HCl; The stop solution includes acetic acid and Tris acetic acid; The TSA reaction solution includes UvsX protein, UvsY protein, GP32 protein, Bsu protein, dNTP, ATP, Tris buffer, creatine phosphate, creatine kinase, polyethylene glycol 35000 and MgOAc; The Cas12a detection solution includes Cas12a protein, NaCl, Tris-HCl, MgCl ₂ , BSA, crRNA and FAM-Bio modified ssDNA; The colloidal gold test strip includes a T band and a C band, where the T band is labeled with streptavidin and the C band is labeled with anti-anti-FAM antibody. The reagent for rapid nucleic acid detection of pet pathogens according to claim 1, characterized in that: In the extract: The mass concentration of the strong alkali hydroxide is 25mM ~ 500mM, and the strong alkali hydroxide is NaOH, The mass concentration of Tris-HCl is 10mM ~ 70mM, The mass concentration of EDTA is 10mM ~ 50mM; The pH value of the extraction liquid is 11.4-13.7. The reagent for rapid nucleic acid detection of pet pathogens according to claim 2, characterized in that: In the stop solution: The mass concentration of the acetic acid is 1M ~ 5M, The mass concentration of Tris-acetic acid is 1M; The pH value of the stop solution is 2-2.5. The reagent for rapid nucleic acid detection of pet pathogens according to claim 1, characterized in that: In the TSA reaction solution: The mass concentration of the UvsX protein is 100-300ng/μL, The mass concentration of the UvsY protein is 50-100ng/μL, The mass concentration of the GP32 protein is 100-500ng/μL, The mass concentration of the Bsu protein is 50-200ng/μL, The mass concentration of the dNTP is 150-300 μM, The mass concentration of ATP is 2-10mM, The mass concentration of the Tris buffer is 20-80mM, The mass concentration of creatine phosphate is 20-80mM, The mass concentration of creatine kinase is 30-100ng/μL, The mass concentration of polyethylene glycol 35000 is 1-7% (w/v), The mass concentration of MgOAc is 20mM. The reagent for rapid nucleic acid detection of pet pathogens according to claim 1, characterized in that: In the Cas12a detection solution: The mass concentration of the Cas12a protein is 1-10uM, The mass concentration of NaCl is 20-75mM, The mass concentration of Tris-HCl is 5-15mM, The mass concentration of MgCl ₂ is 1-20mM, The mass concentration of the BSA is 50-150ug/ml, The mass concentration of crRNA is 10-50nM, The mass concentration of the FAM-Bio modified ssDNA is 1-100nM, The pH value of the Cas12a detection solution is 7.9. A kit containing the reagent according to claim 1. A kit according to claim 6 is used in detecting nucleic acid of pet pathogens. The application according to claim 7, characterized in that: Includes the following steps: (1) Extract the nucleic acid in the sample using the extraction solution to obtain a sample nucleic acid solution; (2) Add all the stop solution to the sample nucleic acid solution obtained in step (1) to neutralize the pH of the sample nucleic acid solution and obtain a mixed solution; the mixed pH value is 6.5-8; (3) Use the mixed solution to perform an amplification reaction and detect nucleic acids in the sample. The pH of the mixed solution is 6.5-8.

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