Rapid detection method for monkey pox virus based on microdroplet digital PCR (Polymerase Chain Reaction) technology

Abstract

The invention discloses a monkey pox virus rapid detection method based on a microdroplet digital PCR technology. The monkey pox virus rapid detection method comprises the following steps: acquiring gene sequence data of monkey pox virus, analyzing a gene conserved region, and designing a specific primer and a probe based on the gene conserved region; establishing reaction conditions based on a fluorescent PCR detection system, and analyzing the sensitivity of the specific primer and the probe based on the reaction conditions to obtain an analysis result; selecting the same specific primer and probe based on the analysis result, and setting the same reaction condition to establish a digital PCR detection system; setting condition parameters, and verifying the sensitivity of the specific primer and the probe based on the condition parameters; adjusting condition parameters based on the verification result, generating an optimal reaction condition, detecting the detected sample based on the optimal reaction condition to obtain a detection result, and generating a monkey pox virus detection report; the monkey pox virus nucleic acid in an import and export sample is detected by using a digital PCR method, the sensitivity is higher, and the detection result is more accurate.

Description

Technical Field

[0001] The present invention relates to the technical field of monkeypox virus detection, and in particular to a method for rapid detection of monkeypox virus based on droplet digital PCR technology. Background Art

[0002] Monkeypox is a zoonosis caused by the monkeypox virus. Strengthening the detection and prevention of diseases at ports has become a top priority. At present, the diagnosis is confirmed by combining clinical and laboratory diagnosis internationally. my country's port quarantine mainly uses fluorescent PCR to confirm the diagnosis. However, due to the wide variety of samples, insufficient sampling volume, viral load, incubation period and method critical value determination and other problems, more sensitive, efficient and accurate detection methods are urgently needed as technical reserves. Specific primers were designed based on the conserved regions of the monkeypox virus gene, and fluorescent PCR and digital PCR detection methods for monkeypox virus were established. The monkeypox virus nucleic acid detection was absolutely quantitative, and the reaction system was optimized to enhance the sensitivity and specificity of the test method. Verification was carried out during the quarantine of susceptible animals and animal products entering and leaving the country. Summary of the invention

[0003] The purpose of the present invention is to propose a method for rapid detection of monkeypox virus based on droplet digital PCR technology to solve the problem of rapid detection of monkeypox virus.

[0004] In order to achieve the above-mentioned object of the invention, the technical solution adopted by the present invention is: a method for rapid detection of monkeypox virus based on droplet digital PCR technology, comprising the following steps: S1, obtain the gene sequence data of monkeypox virus, analyze the conserved regions of the gene, and design specific primers and probes based on the conserved regions of the gene; S2, establishing reaction conditions based on the fluorescent PCR detection system, analyzing the sensitivity of specific primers and probes based on the reaction conditions, and obtaining analysis results; S3, selecting the same specific primers and probes based on the analysis results, setting the same reaction conditions to establish a digital PCR detection system; S4, setting condition parameters, verifying the sensitivity of specific primers and probes based on the condition parameters, and obtaining verification results; S5, adjusting the condition parameters based on the verification results to generate the optimal reaction conditions, testing the sample to be tested based on the optimal reaction conditions, obtaining the test results, and generating a monkeypox virus test report.

[0005] Optionally, in the method for rapid detection of monkeypox virus based on droplet digital PCR technology described in the embodiment of the present application, in step S1, the genetic sequence data of the monkeypox virus includes the viral extracellular envelope protein gene (B6R), the DNA polymerase gene (E9L), the DNA-dependent RNA polymerase subunit 18 gene (rpo18) and the double-stranded RNA binding protein region F3L gene.

[0006] Optionally, in the method for rapid detection of monkeypox virus based on droplet digital PCR technology described in the embodiments of the present application, step S1 further includes: Analyze the conservative regions, select one of the conservative regions of the gene, design specific binding primers using PRIMER QUEST TOOL software, and compare and verify the specificity of primers and probes; The selected gene is artificially synthesized into a gene fragment and inserted into the plasmid pUC57 as a recombinant plasmid positive template.

[0007] Optionally, in the method for rapid detection of monkeypox virus based on droplet digital PCR technology described in the examples of the present application, the sensitivity verification of the probe in step S2 is verified using a positive control gradient dilution solution.

[0008] Optionally, in the method for rapid detection of monkeypox virus based on droplet digital PCR technology described in the embodiments of the present application, qPCR is used in step S4 to verify the sensitivity of primers and probes; Determine whether the sensitivity meets the set sensitivity threshold; If satisfied, the optimal reaction conditions are generated; If not, correction information is generated, and condition parameters are adjusted based on the correction information to achieve the optimal reaction condition.

[0009] Optionally, in the method for rapid detection of monkeypox virus based on droplet digital PCR technology described in the embodiments of the present application, the condition parameters include Taq enzyme, primer probe dosage, cycle parameters and cycle conditions.

[0010] Optionally, in the method for rapid detection of monkeypox virus based on droplet digital PCR technology described in the embodiments of the present application, step S5 further includes: Select the monkeypox fluorescent PCR detection kit to test the sample to be tested and obtain the kit test report; Compare the test report of the kit with the monkeypox virus test report to obtain a comparison result; Analyze similarity based on the comparison results; The monkeypox virus test report with a similarity greater than or equal to the set similarity threshold is used as the final test report; Monkeypox virus test reports with similarity less than the set similarity threshold will be subjected to secondary testing.

[0011] Optionally, in the method for rapid detection of monkeypox virus based on droplet digital PCR technology described in the embodiments of the present application, the sample to be detected in step S5 is a virus of the same genus as monkeypox or a virus with cross-reaction.

[0012] Due to the application of the above technical solution, the present invention has the following advantages compared with the prior art: This application uses a digital PCR method to detect monkeypox virus nucleic acid in samples suitable for entry and exit, which has higher sensitivity and more accurate detection results. Digital PCR can achieve absolute quantitative detection of the starting sample and can directly calculate the number of target gene DNA molecules. It is suitable for situations where absolute quantification is required and the Ct value that relies on fluorescent quantitative PCR cannot well distinguish the detection results. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 A flowchart of a method for rapid detection of monkeypox virus based on droplet digital PCR technology provided in an embodiment of the present application; Figure 2 This is a flow chart of the test report analysis of the method for rapid detection of monkeypox virus based on droplet digital PCR technology provided in the embodiments of the present application. DETAILED DESCRIPTION

[0014] In order to enable those skilled in the art to better understand the solution of the present application, the technical solution in the embodiments of the present application will be clearly and completely described below in conjunction with the drawings in the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, not all of the embodiments. Based on the embodiments in the present application, all other embodiments obtained by ordinary technicians in this field without creative work should fall within the scope of protection of the present application.

[0015] It should be noted that the terms "first", "second", etc. in the specification and claims of the present application and the above-mentioned drawings are used to distinguish similar objects, and are not necessarily used to describe a specific order or sequential order. It should be understood that the data used in this way can be interchanged where appropriate, so that the embodiments of the present application described here. In addition, the terms "including" and "having" and any of their variations are intended to cover non-exclusive inclusions, for example, a process, method, system, product or device comprising a series of steps or units is not necessarily limited to those steps or units clearly listed, but may include other steps or units that are not clearly listed or inherent to these processes, methods, products or devices.

[0016] It should be noted that, in the absence of conflict, the embodiments and features in the embodiments of the present application can be combined with each other. The present application will be described in detail below with reference to the accompanying drawings and in combination with the embodiments.

[0017] Please refer to Figure 1 , Figure 1 Flowchart of a method for rapid detection of monkeypox virus based on droplet digital PCR technology in some embodiments of the present application. The embodiment of the present invention provides a method for rapid detection of monkeypox virus based on droplet digital PCR technology, comprising the following steps: S1, obtain the gene sequence data of monkeypox virus, analyze the conserved regions of the gene, and design specific primers and probes based on the conserved regions of the gene; S2, establishing reaction conditions based on the fluorescent PCR detection system, analyzing the sensitivity of specific primers and probes based on the reaction conditions, and obtaining analysis results; S3, selecting the same specific primers and probes based on the analysis results, setting the same reaction conditions to establish a digital PCR detection system; S4, setting condition parameters, verifying the sensitivity of specific primers and probes based on the condition parameters, and obtaining verification results; S5, adjusting the condition parameters based on the verification results to generate the optimal reaction conditions, testing the sample to be tested based on the optimal reaction conditions, obtaining the test results, and generating a monkeypox virus test report.

[0018] It should be noted that the same pair of primers and probes were used to establish a monkeypox virus droplet digital PCR detection system, and the specificity of the detection system was verified using multiple control viruses of the same genus. Its sensitivity was verified using gradient dilutions of positive templates, and the concentrations of primers and probes in the digital PCR reaction system were optimized. The sensitivity and specificity of the analysis method were analyzed, and the established digital PCR method was compared with other commonly used monkeypox virus nucleic acid molecular biology detection methods, such as conventional PCR and fluorescent quantitative PCR methods, for analysis and optimization.

[0019] According to an embodiment of the present invention, in step S1, the gene sequence data of the monkeypox virus includes the viral extracellular envelope protein gene (B6R), the DNA polymerase gene (E9L), the DNA-dependent RNA polymerase subunit 18 gene (rpo18) and the double-stranded RNA binding protein region F3L gene.

[0020] According to an embodiment of the present invention, step S1 further includes: Analyze the conservative regions, select one of the conservative regions of the gene, design specific binding primers using PRIMER QUEST TOOL software, and compare and verify the specificity of primers and probes; The selected gene is artificially synthesized into a gene fragment and inserted into the plasmid pUC57 as a recombinant plasmid positive template.

[0021] According to an embodiment of the present invention, the sensitivity verification of the probe in step S2 is performed using a positive control gradient dilution solution.

[0022] According to an embodiment of the present invention, qPCR is used in step S4 to verify the sensitivity of the primers and probes; Determine whether the sensitivity meets the set sensitivity threshold; If satisfied, the optimal reaction conditions are generated; If not, correction information is generated, and condition parameters are adjusted based on the correction information to achieve the optimal reaction condition.

[0023] According to an embodiment of the present invention, the condition parameters include Taq enzyme, primer and probe dosage, cycle parameters and cycle conditions.

[0024] Please refer to Figure 2 , Figure 2 This is a flow chart of the analysis of a test report of a method for rapid detection of monkeypox virus based on droplet digital PCR technology in some embodiments of the present application. According to an embodiment of the present invention, step S5 also includes: S501, selecting a monkeypox fluorescent PCR detection kit to detect the sample to be tested, and obtaining a kit test report; S502, comparing the test kit test report with the monkeypox virus test report to obtain a comparison result; S503, analyzing similarity based on the comparison results; S504, taking the monkeypox virus test report with a similarity greater than or equal to a set similarity threshold as the final test report; S505, performing a secondary test on the monkeypox virus test reports whose similarity is less than the set similarity threshold.

[0025] According to an embodiment of the present invention, the sample detected in step S5 is a virus of the same genus as monkeypox or a virus with cross-reaction.

[0026] In summary, the present application uses a digital PCR method to detect monkeypox virus nucleic acid in samples suitable for entry and exit, which has higher sensitivity and more accurate detection results. Digital PCR can achieve absolute quantitative detection of the starting sample and can directly calculate the number of target gene DNA molecules. It is suitable for situations where absolute quantification is required and the Ct value used in fluorescent quantitative PCR cannot well distinguish the detection results.

[0027] In the above embodiments, all or part of the embodiments may be implemented by software, hardware, firmware or any combination thereof. When implemented by software, all or part of the embodiments may be implemented in the form of a program product of computer programming. The computer program product includes one or more computer instructions. When the computer program instructions are loaded and executed on a processor, all or part of the processes or functions according to the embodiments of the present application are generated. The computer may be a general-purpose computer, a computer network, or other programmable device.

[0028] The device embodiments described above are only illustrative. For example, the division of units is only a logical function division. There may be other division methods in actual implementation, such as: multiple units or components can be combined, or can be integrated into another system, or some features can be ignored or not executed. In addition, the coupling, direct coupling, or communication connection between the components shown or discussed can be through some interfaces, and the indirect coupling or communication connection of devices or units can be electrical, mechanical or other forms.

[0029] The units described above as separate components may or may not be physically separated, and the components displayed as units may or may not be physical units; they may be located in one place or distributed on multiple network units; some or all of the units may be selected according to actual needs to achieve the purpose of the present embodiment.

[0030] In addition, all functional units in the embodiments of the present invention may be integrated into one processing unit, or each unit may be separately used as a unit, or two or more units may be integrated into one unit; the above-mentioned integrated units may be implemented in the form of hardware or in the form of hardware plus software functional units.

[0031] Finally, it should be noted that the above are only preferred embodiments of the present invention and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the aforementioned embodiments, it is still possible for those skilled in the art to modify the technical solutions described in the aforementioned embodiments, or to make equivalent substitutions for some of the technical features therein. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for rapid detection of monkeypox virus based on droplet digital PCR technology, characterized in that: The following steps are involved: S1, obtain the gene sequence data of monkeypox virus, analyze the conserved regions of the gene, and design specific primers and probes based on the conserved regions of the gene; S2, establishing reaction conditions based on the fluorescent PCR detection system, analyzing the sensitivity of specific primers and probes based on the reaction conditions, and obtaining analysis results; S3, selecting the same specific primers and probes based on the analysis results, setting the same reaction conditions to establish a digital PCR detection system; S4, setting condition parameters, verifying the sensitivity of specific primers and probes based on the condition parameters, and obtaining verification results; S5, adjusting the condition parameters based on the verification results to generate the optimal reaction conditions, testing the sample to be tested based on the optimal reaction conditions, obtaining the test results, and generating a monkeypox virus test report.

2. The method for rapid detection of monkeypox virus based on droplet digital PCR technology according to claim 1, characterized in that: In step S1, the gene sequence data of the monkeypox virus includes the viral extracellular envelope protein gene (B6R), the DNA polymerase gene (E9L), the DNA-dependent RNA polymerase subunit 18 gene (rpo18) and the double-stranded RNA binding protein region F3L gene.

3. The method for rapid detection of monkeypox virus based on droplet digital PCR technology according to claim 2, characterized in that: Step S1 also includes: Analyze the conservative regions, select one of the conservative regions of the gene, design specific binding primers using PRIMER QUEST TOOL software, and compare and verify the specificity of primers and probes; The selected gene is artificially synthesized into a gene fragment and inserted into the plasmid pUC57 as a recombinant plasmid positive template.

4. The method for rapid detection of monkeypox virus based on droplet digital PCR technology according to claim 1, characterized in that: The sensitivity of the probe in step S2 is verified using a positive control gradient dilution.

5. The method for rapid detection of monkeypox virus based on droplet digital PCR technology according to claim 4, characterized in that: In step S4, the sensitivity of the primers and probes was verified using qPCR; Determine whether the sensitivity meets the set sensitivity threshold; If satisfied, the optimal reaction conditions are generated; If not, correction information is generated, and condition parameters are adjusted based on the correction information to achieve the optimal reaction condition.

6. The method for rapid detection of monkeypox virus based on droplet digital PCR technology according to claim 1, characterized in that: The condition parameters include Taq enzyme, primer and probe dosage, cycle parameters and cycle conditions.

7. The method for rapid detection of monkeypox virus based on droplet digital PCR technology according to claim 6, characterized in that: Step S5 also includes: Select the monkeypox fluorescent PCR detection kit to test the sample to be tested and obtain the kit test report; Compare the test report of the kit with the monkeypox virus test report to obtain a comparison result; Analyze similarity based on the comparison results; The monkeypox virus test report with a similarity greater than or equal to the set similarity threshold is used as the final test report; Monkeypox virus test reports with similarity less than the set similarity threshold will be subjected to secondary testing.

8. The method for rapid detection of monkeypox virus based on droplet digital PCR technology according to claim 1, characterized in that: The sample detected in step S5 is a virus of the same genus as monkeypox or a virus with cross-reaction.

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