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Primer probe set used for detecting coxsackie viruses, enterovirus 71-type and enterovirus universal type through multiple PCR

A Coxsackie virus and enterovirus technology, applied in the biological field, can solve the problems of epidemiological development that is difficult to meet the diversity of hand, foot and mouth disease virus typing, single detection target of detection and identification technology, etc., and achieve high specificity , highly conservative and specific, time-saving effect

Inactive Publication Date: 2018-05-15
北京卓诚惠生生物科技股份有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] The detection and identification technology of the existing HFMD detection kits has a single detection target or only joint detection for common Coxsackievirus A16 and enterovirus 71, which is difficult to meet the epidemiological development of HFMD virus typing diversity need
At present, there is no report on simultaneous identification of coxsackievirus types A6, A10, A16, enterovirus 71 and enterovirus universal type by multiplex real-time fluorescent PCR

Method used

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  • Primer probe set used for detecting coxsackie viruses, enterovirus 71-type and enterovirus universal type through multiple PCR
  • Primer probe set used for detecting coxsackie viruses, enterovirus 71-type and enterovirus universal type through multiple PCR
  • Primer probe set used for detecting coxsackie viruses, enterovirus 71-type and enterovirus universal type through multiple PCR

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0115] Coxsackievirus A6 / A10 / A16 / Enterovirus 71 / Enterovirus Universal Multiplex Fluorescent Quantitative PCR Detection Kit:

[0116] The composition of the primer probe:

[0117] Tube A primer probe: the Coxsackie virus A6 upstream primer of the nucleotide sequence shown in SEQ ID NO.1, the Coxsackie virus A6 downstream primer of the nucleotide sequence shown in SEQ ID NO.2, with SEQ ID The Coxsackie virus A6 type probe with the nucleotide sequence shown in NO.3, the 5' end of the Coxsackie virus A6 type probe is marked with a CY5 luminescent group, and the 3' end is marked with a fluorescent quenching group BHQ3;

[0118] The Coxsackie virus A10 type upstream primer of the nucleotide sequence shown in SEQ ID NO.4, the Coxsackie virus A10 type downstream primer of the nucleotide sequence shown in SEQ ID NO.5, has shown in SEQ ID NO.6 Coxsackievirus A10 type probe of nucleotide sequence, the 5' end of the Coxsackievirus A10 type probe is marked with a VIC luminescent group, a...

Embodiment 2

[0129] Operation and result judgment of embodiment 2 kit

[0130] 1. Genome extraction

[0131] Use a commercial virus genome kit to extract RNA from the sample to be tested as a test sample.

[0132] 2. Preparation of reaction system

[0133] Referring to the same preparation method as in Example 1, take 200 μL of PCR tubes and configure two 20 μL reaction systems of A and B, Coxsackie virus A6 type / A10 type / A16 type and positive internal quality control primer probe mixture, B The tube is a mixture of enterovirus universal type / enterovirus 71 and positive internal quality control primer probe plasmid template (pET28a 0.0003ng), and its configuration is as follows: 2×RT-PCR reaction solution 10 μL; 20×reverse transcriptase 1 μL; 2 μL of 10× primer-probe mixture; 5 μL of RNA template, 2 μL of nuclease-free water.

[0134] 3. PCR reaction

[0135] Put the PCR tube into the fluorescent quantitative PCR instrument, and carry out the PCR reaction according to the following pro...

Embodiment 3

[0140] The shelf life test of embodiment 3 kit

[0141] Take strong positive 10 respectively 5 PFU / mL and Weak Positive 10 3 PFU / mL pseudovirus dilutions of Coxsackievirus A6, A10, A16, and Enterovirus 71 are the test samples for evaluation. On day 0, divide into 9 parts and store in a -70°C refrigerator middle. The completed kits were stored at -20°C, and kits of 0, 10, 15, 30, 60, 90, 120, 150, 180, and 360 days were used for storage test. The test results of the shelf life are shown in Table 2:

[0142] Table 2 shelf life test results

[0143] shelf life

[0144] It can be seen from Table 2 that the kit is stored in a -20°C refrigerator, and it is effective in detecting the four target viruses and the common type of enterovirus at different storage periods. The experimental results show that the storage period of the kit is at least 6 months.

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Abstract

The invention relates to a primer probe set used for detecting coxsackie viruses A6 type / A10 type / A16 type / enterovirus 71-type / enterovirus universal type through multiple PCR. The invention also provides a kit used for detecting coxsackie viruses A6 type / A10 type / A16 type / enterovirus 71-type / enterovirus universal type through multiple quantitative fluorescence PCR. The kit comprises the primer probe set. The above technical scheme provides a complete solution scheme for rapidly screening coxsackie viruses A6 type / A10 type / A16 type / enterovirus 71-type / enterovirus universal type in food, can increase emergent processing for hand-foot-and-mouth disease and comprehensive control capability, provides the necessary technical guarantee for reducing large-scope spreading of hand-foot-and-mouth disease in population, and eliminates the social influence and economic loss due to outbreak of infectious diseases.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a set of primers and probes for multiplex PCR detection and detection of Coxsackievirus A6 / A10 / A16 / enterovirus 71 / enterovirus general type. Background technique [0002] The detection method of HFMD-related virus includes three aspects: virus isolation and culture, serological examination and virus nucleic acid detection. The first two methods are cumbersome to operate, the identification period is long, and the antiserum is easy to cross-react with other human enteroviruses. Molecular biology methods to identify human enterovirus typing methods are being used more and more. At present, the existing in vitro diagnostic kits for hand, foot and mouth disease include real-time fluorescent PCR method, isothermal amplification PCR method, liquid phase chip technology, etc. . Real-time fluorescent PCR technology based on fluorescently labeled probes is currently the most widely used in t...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/6851C12N15/11
CPCC12Q1/6851C12Q1/701C12Q2600/16C12Q2537/143C12Q2531/113C12Q2563/107
Inventor 王月王雷林笑冬张志强
Owner 北京卓诚惠生生物科技股份有限公司
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