WO2024055627A1 - Primer-molecular beacon combination for molecular typing of wild type and mutant type of monkey poxvirus and use thereof - Google Patents

Abstract

The present application provides a primer-molecular beacon combination for molecular typing of a wild type and a mutant type of a monkey poxvirus and use thereof. The primer-molecular beacon combination comprises a primer pair and a molecular beacon that specifically amplify and detect a monkey poxvirus F3L gene, respectively. Nucleotide sequences of the primer pair that specifically amplifies the monkey poxvirus F3L gene are set forth in SEQ ID No. 1 and SEQ ID No. 2. The primer-molecular beacon combination of the present application features good specificity and high detection sensitivity, and the detection lower limit reaches 1 copy. Rapid, simple, convenient, accurate, efficient, practical, and economical detection of a wild type of monkey poxvirus and variants thereof can be realized by using the kit of the present application, and the wild type of the monkey poxvirus and variants thereof can also be effectively distinguished by means of a molecular beacon melting curve method, which can meet the requirements of related pathogen detection in actual clinical examination.

Description

A primer-molecular beacon combination for molecular typing of wild-type and mutant monkeypox virus and its application Technical field

This application belongs to the field of biological detection technology, and specifically relates to a primer-molecule beacon combination for molecular typing of wild-type and mutant monkeypox virus and its application.

Background technique

At present, the probes used in fluorescence quantitative PCR are mainly 5'-end reporter fluorophores and 3'-end quencher fluorophores. This method achieves the effect of quenching fluorescence through random curls between probes. However, Due to the spatial distance, this labeling method cannot achieve zero-distance contact between the reporter fluorophore and the quencher fluorophore, making it impossible to achieve zero-background fluorescence.

Although, super quenching probes have been developed (such as Shanghai Bailig Biotechnology Co., Ltd.), which add BHQ quenching group in the middle based on the original 5'-reporter fluorophore and 3'-quenching fluorophore. Killing groups, but still cannot completely achieve the goal of zero background fluorescence. Bailig's comparative study shows that the super quenching probe can completely quench the background fluorescence, which can not only increase the fluorescence value Rn value of the detection system, but also significantly reduce the Ct value of the same template, thereby significantly improving molecular detection. sensitivity.

However, adding two fluorescent quenching groups will not only increase the load of the detection system, but also increase the production cost of the kit. At the same time, the design of double quenching fluorescent groups has certain difficulties and risks of failure, thus increasing the research and development costs. Difficulty, period and cost.

Monkeypox is a zoonotic disease caused by infection with monkeypox virus, which belongs to a subset of the Poxviridae family, known as the genus Orthopoxvirus, and is a series of viruses that infect mammals and cause pustules. Monkeypox is mainly spread through close contact and respiratory tract. Items contaminated with the virus may also be contagious. According to past news reports, human cases of monkeypox infection are mainly transmitted through contact with animals carrying the virus. Skin wounds or mucous membranes may also be transmitted through contact with the blood and body fluids of sick animals. For example, people may be infected through being in the same room for a long time. Respiratory tract infections.

Through sequence comparison, it was found that the monkeypox virus mutant strain contains 56 SNPs and 3 deletions. These mutation sites can be used as molecular markers of the wild type or mutant type of monkeypox virus. Through traceability analysis, it was found that this mutant strain has been raging around the world since 2018 and has become one of the hidden public health safety risks that endanger society.

Therefore, accurate genotyping detection of monkeypox plays a vital role in controlling the monkeypox epidemic. Although there are reports and patents on the use of molecular beacons for multiple fungal detection, there is no use of molecular beacons to detect monkeypox. Reports labeled for genotyping. Therefore, the development and design of a highly sensitive molecular beacon has important application prospects in genotyping detection of monkeypox virus.

Contents of the invention

This application provides a primer molecular beacon combination for molecular typing of monkeypox virus wild type and mutant types and its application. This application uses fluorescence quantitative PCR technology to quickly, accurately and more sensitively detect and classify the wild type and mutant types of monkeypox virus. It has high detection sensitivity and good specificity, and the lower detection limit can reach 1 copy. It has the characteristics of high throughput and low cost.

In a first aspect, the present application provides a primer molecular beacon combination for molecular typing of monkeypox virus wild type and mutant types. The primer molecular beacon combination includes specific amplification and detection of monkeypox virus F3L genes respectively. Primer pairs and molecular beacons;

Wherein, the nucleotide sequence of the primer pair that specifically amplifies the monkeypox virus F3L gene is shown in SEQ ID No. 1 and SEQ ID No. 2.

Preferably, the nucleotide sequence of the molecular beacon for detecting wild-type monkeypox virus is shown in SEQ ID No. 3, and the nucleotide sequence of the molecular beacon for detecting mutant monkeypox virus is shown in SEQ ID No. 4.

In this application, without changing the original probe, a certain number of reverse complementary sequences at the 3' end are added to the 5' end of the probe so that the dissociated probe forms a stem-loop structure similar to single-stranded RNA, thereby making The reporter fluorophore and the quencher fluorophore are infinitely close in spatial structure, thus achieving the purpose of completely quenching the background fluorescence.

Preferably, the primer molecular beacon combination also includes a primer pair and a molecular beacon that respectively specifically amplify and detect an internal standard gene, and the internal standard gene is the RNase P gene.

Preferably, the nucleotide sequence of the primer pair that specifically amplifies the internal standard gene is as shown in SEQ ID No. 7 and SEQ ID No. 8.

Preferably, the nucleotide sequence of the molecular beacon for detecting the internal standard gene is shown in SEQ ID No. 9.

Preferably, the molecular beacons are equipped with fluorescent groups and quenching groups, and are used to detect wild-type monkeypox virus, to detect mutant monkeypox virus, and to detect internal standard genes. The fluorophores are all different.

Preferably, the fluorescent group includes any one or a combination of at least two of FAM, HEX, VIC, ROX, TAMRA, JOE, TET, CY3, CY5, Texas Red or LC RED460.

Preferably, the quenching fluorescent group includes TAMRA, BHQ1, BHQ2, MGB, DABCYL, Any one or a combination of at least two of BHQ3.

In a second aspect, the present application provides a detection kit for detecting monkeypox virus wild type and mutant types. The detection kit includes the primers for molecular typing of monkeypox virus wild type and mutant types described in the first aspect. Molecular beacon combination.

In this application, the detection kit also includes detection reagents, and the detection reagents include PCR buffer, enzyme mixture and water.

In a third aspect, this application provides a method of using the detection kit for detecting monkeypox virus wild type and mutant types described in the second aspect for the purpose of non-disease diagnosis and/or treatment, and the method of use includes the following steps:

(1) Collect samples, extract DNA, and prepare a PCR amplification system;

(2) Perform amplification detection on the PCR system in step (1); and

(3) Detect and interpret the detection results of monkeypox virus and its mutant strains based on the amplification curve obtained in step (2) to determine the pathogen infection in the sample.

Preferably, the PCR amplification system includes an enzyme mixture, upstream primers, downstream primers, wild-type molecular beacons, mutant molecular beacons and templates.

In this application, the PCR amplification system includes a thermostable DNA polymerase with or without 5'-3' exonuclease activity.

Preferably, the final concentrations of the upstream primer and the downstream primer are each independently 100-400 nM, for example, they can be 100 nM, 150 nM, 200 nM, 250 nM, 300 nM, 350 nM or 400 nM, etc.

Preferably, the final concentrations of the wild-type molecular beacon and the mutant molecular beacon are each independently 100-400 nM, for example, they can be 100 nM, 150 nM, 200 nM, 250 nM, 300 nM, 350 nM or 400 nM, etc.

Preferably, the final concentration of the template is 10 ⁰ -10 8 copies, for example, it can be 10 ⁰ copies, 10 ¹ copies, 10 ² copies, 10 ³ copies, 10 ⁴ copies, 10 ⁵ copies, 10 ⁶ copies, 10 ⁷ copies Or 10 ⁸ copies etc.

Preferably, the reaction conditions for the amplification detection are: pre-denaturation: 94-97°C, 4-5min; denaturation: 94-97°C, 8-12s, extension 58-62°C, 28-32s, 40-45 cycle;

The melting curve program is: denaturation 94-97°C, 0.8-1.2min; annealing 44-46°C, 115-125s; heating to 94-97°C at a heating rate of 0.01-0.3°C/s.

In this application, the reaction conditions for the amplification detection are:

Pre-denaturation: 94-97°C, 4-5 minutes. The pre-denaturation temperature can be, for example, 94°C, 95°C, 96°C or 97℃, etc., the pre-denaturation time can be, for example, 4min, 4.5min or 5min, etc.;

Denaturation: 94-97°C, 8-12s. The denaturation temperature can be, for example, 94°C, 95°C, 96°C or 97°C, etc. The denaturation time can be, for example, 8s, 9s, 10s, 11s or 12s, etc.;

Extension: 58-62°C, 28-32s, 40-45 cycles. The extension temperature can be, for example, 58°C, 59°C, 60°C, 61°C or 62°C, etc. The extension time can be, for example, 28s, 29s, 30s. , 31s or 32s, etc., the number of cycles can be, for example, 40, 41, 42, 43, 44 or 45.

Melting curve program: denaturation 94-97°C, 0.8-1.2min. The denaturation temperature can be, for example, 94°C, 95°C, 96°C or 97°C, etc. The denaturation time can be, for example, 0.8min, 0.9min, 1.0min, 1.1 min or 1.2min, etc.;

Annealing 44-46°C, 115-125s, the annealing temperature can be, for example, 44°C, 45°C, or 46°C, etc., and the annealing time can be, for example, 115s, 117s, 119s, 120s, 122s, or 125s, etc.;

Raise the temperature to 94-97°C at a heating rate of 0.01-0.3°C/s. The heating rate is 0.01°C/s, 0.03°C/s, 0.1°C/s, 0.2°C/s or 0.3°C/s, etc.

Preferably, during the amplification and detection process, the PCR amplification system is provided with at least one double-labeled oligonucleotide wild-type molecular beacon whose Tm value is greater than the Tm value of the two matching primers and corresponds to the same target. The real-time amplified fluorescent signal of the double-labeled oligonucleotide wild-type molecular beacon of the sequence can be detected normally.

In this application, the Tm value of the molecular beacon is higher than that of the two matching primers, mainly because it binds to the template DNA before the primers during the annealing process, thereby further ensuring the accuracy of the reporter fluorescence cleavage through the specificity of the probe sequence.

As the preferred technical solution of this application, the usage method includes the following steps:

(1) Extraction of nucleic acid samples: Collect samples and extract DNA, and transfer the extracted DNA to the refrigerator for storage until later use.

(2) Fluorescent quantitative PCR reaction:

Prepare a PCR amplification system, mix the prepared PCR amplification system with the DNA extracted in step (1) and add it to a 96-well sample plate for PCR reaction; PCR reaction conditions: 95°C, 5 min pre-denaturation; then sequentially 95°C, 10s, 60°C, 30s for 40-45 cycles; the melting curve program is: denaturation 94-97°C, 0.8-1.2min; annealing 44-46°C, 115-125s; 0.01-0.3°C/s The heating rate is increased to 94-97℃.

The PCR amplification system in step (2) also includes a thermostable amplification system with or without 5'-3' exonucleation Enzymatically active DNA polymerase.

The PCR amplification system of step (2) is provided with at least one dual-labeled oligonucleotide wild-type molecular beacon whose Tm value is greater than the Tm value of the two matching primers and corresponds to the dual-labeled oligonucleotide of the same target sequence. The real-time amplified fluorescent signal of the glycolic acid wild-type molecular beacon can be detected normally.

(3) PCR result analysis: Detect and interpret the detection results of monkeypox virus and its mutant strains based on the amplification curve obtained in step (2) to determine whether the sample contains the pathogen infection to be detected.

In the fourth aspect, this application provides a system for detecting monkeypox virus wild type and mutant types, the system includes:

(1) Sample preparation module: collect samples, extract DNA, and prepare a PCR amplification system; and

(2) Amplification detection module: perform amplification detection on the PCR system in the sample preparation module;

(3) Analysis module: Detect and interpret the detection results of monkeypox virus and its mutant strains based on the amplification curve obtained in the amplification detection module to determine the pathogen infection in the sample.

In the fifth aspect, this application provides the primer-molecule beacon combination for molecular typing of monkeypox virus wild type and mutant types described in the first aspect, and the detection method for detecting the wild type and mutant types of monkeypox virus described in the second aspect. The application of any one or a combination of at least two of the kits or the system for detecting monkeypox virus wild type and mutant type described in the fourth aspect in preparing a product for detecting monkeypox virus.

The numerical range described in this application not only includes the point values listed above, but also includes any point value between the above numerical ranges that are not listed. Due to space limitations and for the sake of simplicity, this application will not exhaustively list the ranges. Specific point values included.

Compared with the existing technology, this application has the following beneficial effects:

(1) This application can quickly, accurately and more sensitively detect and classify the wild type and mutant types of monkeypox virus by applying fluorescence quantitative PCR technology, with high detection sensitivity (the lower detection limit can reach 1 copy), It has good specificity, high throughput and low cost.

(2) The fluorescence quantitative PCR reaction procedure is completed in one step, without the need for secondary processing such as product purification and sequencing. The operation is extremely simple and the required sample amount is small.

(3) This application can use the asymmetric + melting curve analysis method, which breaks through the limitation of the traditional detection system that one channel can only detect one target. One fluorescence channel in this application can detect at least two targets.

(4) The spatial structure of the molecular beacon used in this application can bring the reporter fluorescence and the quenching fluorescence group to an infinitely close level, thus making the amplified fluorescence value significantly higher than that of ordinary TaqMan probes.

Description of drawings

Figure 1 is the beacon method amplification chart in Example 5. The amplification curve in Figure 1 is the ROX channel, which is the internal standard.

Figure 2 is the beacon method amplification chart in Example 5. The amplification curve in Figure 2 is the FAM channel, which is the wild type.

Figure 3 is the beacon method amplification chart in Example 5. The amplification curve in Figure 3 is the VIC channel, which is the mutant type.

Figure 4 is the TaqMan probe method amplification chart in Example 5. The amplification curve in Figure 4 is the ROX channel, which is the internal standard.

Figure 5 is the TaqMan probe method amplification chart in Example 5. The amplification curve in Figure 5 is the FAM channel, which is the wild type.

Figure 6 is the TaqMan probe method amplification chart in Example 5. The amplification curve in Figure 6 is the VIC channel, which is the mutant type.

Figure 7 is a melting curve of the molecular beacon in Example 6.

Detailed ways

The technical solutions of the present application will be further described below through specific implementations. Those skilled in the art should understand that the embodiments are only to help understand the present application and should not be regarded as specific limitations of the present application.

If specific techniques or conditions are not specified in the examples, the techniques or conditions described in literature in the field shall be followed, or the product instructions shall be followed. If the manufacturer of the reagents or instruments used is not indicated, they are all conventional products that can be purchased through regular channels.

Example 1

This embodiment provides a primer molecular beacon combination for molecular typing of monkeypox virus wild type and mutant types. The primer molecular beacon combination includes a primer pair that specifically amplifies and detects monkeypox virus F3L gene and Molecular beacon; the nucleotide sequence of the primer pair that specifically amplifies the monkeypox virus F3L gene is shown in SEQ ID No. 1 and SEQ ID No. 2; the nucleoside of the molecular beacon for detecting wild-type monkeypox virus The acid sequence is shown in SEQ ID No. 3, and the nucleotide sequence of the molecular beacon for detecting mutant monkeypox virus is shown in SEQ ID No. 4.

The primer molecular beacon combination also includes a primer pair and a molecular beacon that specifically amplify and detect the internal standard gene respectively; the nucleotide sequence of the primer pair that specifically amplifies the internal standard gene is such as SEQ ID No. 7 and SEQ ID No. 8; the nucleotide sequence of the molecular beacon for detecting the internal standard gene is shown in SEQ ID No. 9.

MonkeyPox-F is the upstream primer for amplifying the F3L gene;

MonkeyPox-R is the downstream primer for amplifying the F3L gene;

MonkeyPox-P-MB-Wt is a molecular beacon for detecting wild-type monkeypox virus. The fluorescent group of the molecular beacon is FAM and the quenching fluorescent group is BHQ1;

MonkeyPox-P-MB-Mt is a molecular beacon for detecting mutant monkeypox virus. The fluorescent group of the molecular beacon is VIC and the quenching fluorescent group is BHQ1;

RNase PF is the upstream primer for amplifying the internal standard gene (RNase P gene);

Rnase PR is the downstream primer for amplifying the internal standard gene (RNase P gene);

RNase P-MB-R is a molecular beacon for detecting the internal standard gene (RNase P gene). The fluorescent group of the molecular beacon is ROX and the quenching fluorescent group is BHQ2;

Comparative example 1

This comparative example provides a primer molecular beacon combination for molecular typing of wild type and mutant monkeypox virus. The only difference between the primer molecular beacon combination and Example 1 is that it specifically amplifies the monkeypox virus. The nucleotide sequence of the primer pair of the poxvirus F3L gene is shown in SEQ ID No. 1 and SEQ ID No. 10. The molecular beacon for detecting monkeypox virus is consistent with Example 1, and the internal standard gene is specifically amplified and detected. The primer pairs and molecular beacons are consistent with Example 1.

Comparative example 2

This comparative example provides a primer molecular beacon combination for molecular typing of wild type and mutant monkeypox virus. The only difference between the primer molecular beacon combination and Example 1 is that it specifically amplifies the monkeypox virus. The nucleotide sequence of the primer pair of the poxvirus F3L gene is shown in SEQ ID No. 11 and SEQ ID No. 12. The molecular beacon for detecting monkeypox virus is consistent with Example 1, and the internal standard gene is specifically amplified and detected. The primer pairs and molecular beacons are consistent with Example 1.

Comparative Example 3

This comparative example provides a primer molecular beacon combination for molecular typing of wild type and mutant monkeypox virus. The only difference between the primer molecular beacon combination and Example 1 is that it specifically amplifies the monkeypox virus. The nucleotide sequence of the primer pair of the poxvirus F3L gene is shown in SEQ ID No. 13 and SEQ ID No. 14. The molecular beacon for detecting monkeypox virus is consistent with Example 1, and the internal standard gene is specifically amplified and detected. The primer pairs and molecular beacons are consistent with Example 1.

The primers in Comparative Examples 1-3 are as follows:

MonkeyPOX virus-F1：SEQ ID No.1：

MonkeyPOX virus-R1：SEQ ID No.10：

MonkeyPOX virus-F2：SEQ ID No.11：

MonkeyPOX virus-R2：SEQ ID No.12：

MonkeyPOX virus-F3：SEQ ID No.13：

MonkeyPOX virus-R3：SEQ ID No.14：

Example 2

This embodiment provides a detection kit for detecting monkeypox virus wild type and mutant type. The detection kit includes the primer molecular beacon for molecular typing of monkeypox virus wild type and mutant type described in Example 1. Combination; the detection kit also includes detection reagents, and the detection reagents include PCR buffer, enzyme mixture and water.

Example 3

This example takes the primer-molecule beacon combination for molecular typing of monkeypox virus wild type and mutant types described in Example 1 as an example, and uses the beacon method to simultaneously detect and distinguish monkeypox virus wild type and mutant types; TaqMan probes were used as controls to perform synchronized experiments. The primer and molecular beacon combinations for molecular typing of wild-type and mutant monkeypox virus are shown in Table 1.

Table 1

MonkeyPox-P-TaqMan-Wt is a TaqMan probe (SEQ ID No. 5) for detecting wild-type monkeypox virus. The fluorescent group of the probe is FAM and the quenching fluorescent group is BHQ1.

MonkeyPox-P-TaqMan-Mt is a TaqMan probe for detecting mutant monkeypox virus (SEQ ID No. 6). The fluorophore of the probe is VIC and the quenching fluorophore is BHQ1.

The detection steps are as follows:

1. Extraction of nucleic acid samples. The kit used for extraction is independently developed by Baoteng Biomedical Technology Co., Ltd. (Cat. No.: BT-02-11).

1.1 The preparation and inspection of reagent materials before the experiment are as follows:

(1) Check the shelf life of the kit and ensure that ethanol has been added to Wash Buffer 1 and 2, and tick the corresponding mark on the bottle;

(2) Isopropyl alcohol (if not available, absolute ethanol can be used instead) and 75% ethanol;

(3) 1.5mL Eppendorf tubes and various pipette tips within the validity period of autoclaving.

1.2 Take out the EDTA anticoagulant tube containing whole blood from the 4℃ refrigerator, and mix it by turning it upside down several times.

1.3 Mark the 1.5mL Eppendorf tube corresponding to the unique identification of the specimen.

1.4 Pipette 900 μL of Cell Lysis Solution into sterile 1.5 mL Eppendorf tubes.

1.5 Carefully transfer 300μL of whole blood to the 1.5mL EP tube containing Cell Lysis Solution.

1.6 Cover the Eppendorf tube and incubate at room temperature for 10 minutes.

1.7 Centrifuge at room temperature at 13000 rpm for 20 seconds.

1.8 Take out the Eppendorf tube and observe the white precipitate.

1.9 Open the cap of the Eppendorf tube, hold the bottom of the tube, tilt the EP tube opening to discard part of the red supernatant, and try to absorb as much red supernatant as possible.

1.10 Cover the Eppendorf tube and tap the bottom of the Eppendorf tube with your finger to resuspend the white precipitate.

1.11 Pipette 300 μL of Nuclei Lysis Solution into the above-mentioned Eppendorf tube, cap the tube, and mix by inverting several times.

1.12 Open the Eppendorf tube, transfer 100 μL of the protein precipitation solution (Protein Precipitation Solution) into the above Eppendorf tube, cover the tube cap, and shake vigorously on the oscillator for 20 seconds; centrifuge at 13,000 rpm at room temperature for 3 minutes.

1.13 Transfer the supernatant to a new sterilized 1.5mL Eppendorf tube.

1.14 Pipette 300 μL of isopropyl alcohol into an Eppendorf tube, cap the tube, and mix by inverting several times. White flocculent gDNA can be seen precipitating.

1.15 Centrifuge at room temperature for 1 minute at 13000 rpm.

1.16 Open the Eppendorf tube, pinch the bottom of the tube, tilt the tube opening and discard the supernatant.

1.17 Pipette 300 μL of 75% ethanol into the Eppendorf tube, cover the tube, and gently invert the tube upside down to wash the precipitate.

1.18 Centrifuge at room temperature at 13000 rpm for 1 min.

1.19 Open the Eppendorf tube, hold the bottom of the tube, tilt the tube opening and discard the supernatant.

1.20 Place new filter paper on the experimental bench, turn the Eppendorf tube upside down, absorb the liquid, and open the Eppendorf tube and place it on its side to air dry.

1.21 Visually check the size of the precipitate and add 50 to 100 μL DNA Rehydration Solution. to sedimentation.

1.22 After dissolving overnight, use a NanoDrop UV spectrophotometer to measure the nucleic acid concentration. The nucleic acid concentration is greater than or equal to 20ng/μL and the OD ₂₆₀ /OD ₂₈₀ is 1.9±0.2. It is considered qualified. If the concentration is not enough, add ethanol to precipitate the DNA again, and then add an appropriate amount again. DNA Rehydration Solution dissolves nucleic acids.

1.23 Mark the unique sample number again on the tube wall and tube cap, and wrap it with transparent tape for protection.

1.24 Store nucleic acid samples in a 4°C refrigerator.

2. Fluorescent quantitative PCR reaction. The kit used in the PCR reaction is independently developed by Baoteng Biomedical Technology Co., Ltd. (Cat. No.: BT-20-15).

2.1 Prepare a 25 μL PCR amplification system (except for template addition) in the reagent preparation area. The components and addition amounts are as shown in Table 2.

Table 2

2.2 In the specimen preparation area, briefly centrifuge the gDNA template containing it and add 2.0 μL to the amplification system. Mark the unique identification of the specimen on the wall of the PCR tube and the detection project code on the tube cover; shake and mix the PCR tube, and put it on the desktop centrifuge Centrifuge briefly.

2.3 After setting the program, put the PCR tube into the adapter and install it into the amplification instrument, and set the reaction program according to Table 3; the selected channels for the reaction program include FAM/VIC/ROX.

table 3

2.4 Click "start" to start the instrument operation.

3. Interpretation of results: Check whether there are specific amplification curves in the corresponding FAM, VIC and ROX channels. In this embodiment, a wild-type S-shaped amplification curve will appear in the FAM channel, a mutated S-shaped amplification curve will appear in the VIC channel, and an internal standard amplification curve will appear in the ROX channel. The amplification results were consistent with expectations. A wild-type S-shaped amplification curve appeared in the FAM channel, a mutated S-shaped amplification curve appeared in the VIC channel, and an internal standard amplification curve appeared in the ROX channel. The results showed that MonkeyPOX virus-F, MonkeyPOX virus- R and the corresponding molecular beacon can detect monkeypox and have good discrimination ability between wild type and mutant type.

4. Analysis of first-generation sequencing results

Double-click the mouse in the "experiment" folder to open the above run file, select "gene scanning", and click the "Calculate" button to perform genotype analysis on all test specimens.

Comparative example 4

This comparative example uses the primer SEQ ID No. 10-15 in Comparative Example 1-3 for amplification, and the amplification results are shown in Table 4.

Table 4

It can be seen from the results in Table 4 that compared with the combination of MonkeyPOX virus-F1 and MonkeyPOX virus-R1, the combination of MonkeyPOX virus-F2 and MonkeyPOX virus-R2, and the combination of MonkeyPOX virus-F3 and MonkeyPOX virus-R3, MonkeyPOX virus-F The combination with MonkeyPOX virus-R can specifically amplify the F3L gene. In the combination of MonkeyPOX virus-F1 and MonkeyPOX virus-R1, although the sequences of MonkeyPOX virus-F1 and MonkeyPOX virus-F are consistent, due to changes in downstream primers, non-specific amplification eventually occurred, indicating that MonkeyPOX virus-F and MonkeyPOX The combination of virus-R is the optimal primer for detecting monkeypox.

Example 4

In this example, 12 samples to be tested (pseudoviruses synthesized by Fubaiao (Suzhou) Biotechnology Co., Ltd.) were subjected to fluorescence quantitative PCR detection, and the experiment was performed with reference to the steps shown in Example 3. A comparative experiment was conducted between fluorescence quantitative PCR detection and first-generation sequencing of 12 samples.

The time consumption of fluorescence quantitative PCR detection and first-generation sequencing was calculated. Among them, the fluorescence quantitative PCR detection of 12 samples took 1.5 hours + the result analysis took 0.5 hours, totaling 2 hours. The first-generation sequencing test takes 8 hours + result analysis takes 1 hour, totaling 9 hours. Furthermore, fluorescence quantitative PCR detection is a closed-tube operation and does not require secondary processing such as product purification and sequencing, so there is no risk of amplification product contamination. The comparison results of fluorescence quantitative PCR and first-generation sequencing of 12 samples are shown in Table 5.

table 5

As can be seen from Table 5, the positive detection rate (positive coincidence rate, sensitivity) of the self-developed qPCR is significantly higher than that of first-generation sequencing. Compared with first-generation sequencing, this detection system has the advantages of short detection time, simple operation and closed-tube operation.

Example 5

This embodiment performs sensitivity detection.

The wild-type and mutant amplicon regions to be detected were cloned into two plasmids respectively. The synthesis was completed by Shanghai Fubaiao Biotechnology Co., Ltd. and the plasmids were quantified. The plasmid distribution was diluted 10 times according to the wild type, mutant type and hybrid type to 10000copies/μL, 1000copies/μL, 10copies/μL, and 1copies/μL, and fluorescent quantitative PCR reaction was performed on the viruses at the above dilution ratios. The steps of the fluorescence quantitative PCR reaction are as described in Example 3. The amounts of each component added in the PCR system are as shown in Table 6. Prepare the amplification system with the listed molecular beacons and TaqMan probes respectively, and proceed according to the reaction conditions in Example 3. Amplification and melting curve experiments.

Table 6

The amplification detection results are shown in Figure 1-6. Figure 1, Figure 2 and Figure 3 are the beacon method amplification maps, where, In the figure, the amplification curve in Figure 2 is the FAM channel, which is the wild type, the amplification curve in Figure 3 is the VIC channel, which is the mutant type, and the amplification curve in Figure 1 is the ROX channel, which is the internal standard.

Figure 4, Figure 5, and Figure 6 are TaqMan probe method amplification charts. The amplification curve in Figure 5 is the FAM channel, which is the wild type, the amplification curve in Figure 6 is the VIC channel, which is the mutant type, and the amplification curve in Figure 4 is ROX. The channel is the internal standard.

The sensitivity test results show that the lower detection limit of wild-type monkeypox virus, mutant monkeypox virus and hybrid monkeypox virus using the beacon method is 1 copy; the detection sensitivity of the beacon method is higher than that of the TaqMan probe method. The effect is better.

Example 6

In this example, a melting curve experiment was performed to verify the ability of the molecular beacon to distinguish between wild-type and mutant monkeypox viruses. The PCR system and reaction procedures are shown in Tables 7 and 8.

Table 7

Reaction program: (Channel selection FAM).

Table 8

Prepare an amplification system with the listed molecular beacons and perform amplification according to the reaction conditions. The melting curve results are shown in Figure 7. The melting peak corresponding to around 70°C is the wild-type melting peak, and the melting peak corresponding to around 65°C is the mutant melting peak. The results show that the melting curve obtained by the beacon method can significantly distinguish between wild type and mutant transform.

In summary, the primers designed in this application can specifically amplify the target sequence, and the designed molecular beacons can efficiently distinguish between wild-type and mutant monkeypox viruses. The application of the kit of the present application can realize rapid, simple, accurate, efficient, practical and economical detection of monkeypox virus wild type and its mutant strains. The detection sensitivity is high and the specificity is good. The lower detection limit can reach 1 copy. It has the characteristics of high throughput and low cost; it can also effectively distinguish monkeypox virus wild type and its mutant strains through the molecular beacon melting curve method, which can meet the requirements of related pathogen detection in practical clinical testing.

The applicant declares that the above are only specific implementation modes of the present application, but the protection scope of the present application is not limited thereto. Persons skilled in the technical field should understand that any person skilled in the technical field will not disclose any information disclosed in this application. Within the technical scope, changes or substitutions that can be easily imagined fall within the protection scope and disclosure scope of this application.

Claims (10)

A primer-molecular beacon combination for molecular typing of wild-type and mutant monkeypox virus, which includes a pair of primers and a molecular beacon that specifically amplify and detect the F3L gene of monkeypox virus respectively; Wherein, the nucleotide sequence of the primer pair that specifically amplifies the monkeypox virus F3L gene is shown in SEQ ID No. 1 and SEQ ID No. 2. The primer molecular beacon combination for monkeypox virus wild type and mutant molecular typing according to claim 1, wherein the nucleotide sequence of the molecular beacon for detecting wild type monkeypox virus is as shown in SEQ ID No. 3 As shown, the nucleotide sequence of the molecular beacon for detecting mutant monkeypox virus is shown in SEQ ID No. 4. The primer molecular beacon combination for molecular typing of wild type and mutant monkeypox virus according to claim 1 or 2, wherein the primer molecular beacon combination further includes specifically amplifying and detecting internal standard genes respectively Primer pair and molecular beacon, the internal standard gene is the RNase P gene; Preferably, the nucleotide sequence of the primer pair that specifically amplifies the internal standard gene is as shown in SEQ ID No. 7 and SEQ ID No. 8; Preferably, the nucleotide sequence of the molecular beacon for detecting the internal standard gene is shown in SEQ ID No. 9. The primer molecular beacon combination for molecular typing of wild type and mutant monkeypox virus according to any one of claims 1-3, wherein the molecular beacons are equipped with fluorescent groups and quenching groups. The fluorescent groups of the molecular beacon that detects wild-type monkeypox virus, the molecular beacon that detects mutant monkeypox virus, and the molecular beacon that detects the internal standard gene are all different; Preferably, the fluorescent group includes any one or a combination of at least two of FAM, HEX, VIC, ROX, TAMRA, JOE, TET, CY3, CY5, Texas Red or LC RED460; Preferably, the quenching fluorescent group includes any one or a combination of at least two of TAMRA, BHQ1, BHQ2, MGB, DABCYL, and BHQ3. A detection kit for detecting monkeypox virus wild type and mutant type, which includes the primer-molecule beacon combination for molecular typing of monkeypox virus wild type and mutant type according to any one of claims 1-4. A method of using the detection kit for detecting monkeypox virus wild type and mutant type according to claim 5 for the purpose of non-disease diagnosis and/or treatment, which includes the following steps: (1) Collect samples, extract DNA, and prepare a PCR amplification system; (2) Perform amplification detection on the PCR system in step (1); and (3) Detect and interpret the detection results of monkeypox virus and its mutant strains based on the amplification curve obtained in step (2) to determine the pathogen infection in the sample. The detection kit for detecting monkeypox virus wild type and mutant type according to claim 6 is based on non- A method of use for the purpose of disease diagnosis and/or treatment, wherein the PCR amplification system includes an enzyme mixture, upstream primers, downstream primers, wild-type molecular beacons, mutant molecular beacons and templates; Preferably, the final concentrations of the upstream primer and downstream primer are each independently 100-400 nM; Preferably, the final concentrations of the wild-type molecular beacon and the mutant molecular beacon are each independently 100-400 nM; Preferably, the final concentration of the template is 10 ⁰ -10 ⁸ copies; Preferably, the reaction conditions for the amplification detection are: pre-denaturation: 94-97°C, 4-5min; denaturation: 94-97°C, 8-12s, extension 58-62°C, 28-32s, 40-45 cycle; The melting curve program is: denaturation 94-97°C, 0.8-1.2min; annealing 44-46°C, 115-125s; heating to 94-97°C at a heating rate of 0.01-0.3°C/s. The method of using the detection kit for detecting monkeypox virus wild type and mutant type according to claim 6 or 7 for the purpose of non-disease diagnosis and/or treatment, wherein during the amplification and detection process, the PCR amplification The system is provided with at least one dual-labeled oligonucleotide wild-type molecular beacon whose Tm value is greater than the Tm value of the two matching primers and corresponds to the real-time amplification of the dual-labeled oligonucleotide wild-type molecular beacon with the same target sequence. The increased fluorescence signal can be detected normally. A system for detecting wild type and mutant forms of monkeypox virus, comprising: (1) Sample preparation module: collect samples, extract DNA, and prepare a PCR amplification system; (2) Amplification detection module: perform amplification detection on the PCR system in the sample preparation module; and (3) Analysis module: Detect and interpret the detection results of monkeypox virus and its mutant strains based on the amplification curve and melting curve obtained in the amplification detection module to determine the pathogenic infection in the sample. The primer molecular beacon combination for molecular typing of monkeypox virus wild type and mutant types according to any one of claims 1 to 4, and the detection reagent for detecting monkeypox virus wild type and mutant types according to claim 5 The application of any one or a combination of at least two of the kit or the system for detecting monkeypox virus wild type and mutant type according to claim 9 in the preparation of a product for detecting monkeypox virus.