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Multiple primers, kit and method for high-throughput sequencing of enterovirus

A technology of enteroviruses and kits, applied in the analysis of high-throughput sequencing data, and in the field of multiple primers, can solve the problems of systematic monitoring, early warning and prevention and control of enteroviruses, and the study of the rules of virus molecular variation, etc. problem, to achieve rapid identification, perfect detection and analysis technology, and high accuracy

Inactive Publication Date: 2019-10-29
广东省公共卫生研究院 +1
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  • Application Information

AI Technical Summary

Problems solved by technology

At present, the main virus isolation and culture, serological test and PT-PCR detection technology can only carry out simple serotype or genotype identification of a small number of enterovirus types, and cannot be systematically monitored, let alone researched by obtaining viral genome sequences The law of molecular variation of viruses makes it impossible to implement effective early warning and prevention and control of enteroviruses

Method used

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  • Multiple primers, kit and method for high-throughput sequencing of enterovirus
  • Multiple primers, kit and method for high-throughput sequencing of enterovirus
  • Multiple primers, kit and method for high-throughput sequencing of enterovirus

Examples

Experimental program
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Effect test

Embodiment 1

[0095] Embodiment 1 multiplex PCR primer design

[0096] The base sequences of various genotypes of enteroviruses were obtained by using the public database (NCBI) and the resources of the Guangdong Province bacterial and virus strain bank, and the conserved segments at the head, tail and middle of the genome were identified through bioinformatics analysis. Different from the previous method of designing multiple primer sets to complete genome amplification through first-generation sequencing, and also different from the recently published method of determining the viral genome through polyA enrichment combined with next-generation sequencing, this example focuses on enterovirus A / B / C Conserved segment of the three genomes, divide the whole enterovirus genome into two segments of 4kb and 3.5kb, and design universal primers for the three groups of human enterovirus A / B / C in the conserved segment, each group contains two For primers, a total of six pairs of primers cover the en...

Embodiment 2

[0097] Embodiment 2 kit preparation

[0098] An embodiment of the kit that can be used for the construction and detection of the whole genome library of enteroviruses in the present invention, which includes reverse transcription reagents (reverse transcription primers, reverse transcription reaction solution, reverse transcriptase mixture, dNTP), multiplex PCR amplification Reagents (multiple PCR reaction solution, multiple PCR enzyme, multiple PCR primer mix 1, multiple PCR primer mix 2), library construction reagents (end repair reaction solution, end repair enzyme, ligation buffer, DNA ligase, linker, Specific adapters 1-12, library amplification reaction solution, library amplification enzyme). Wherein, multiplex PCR primer mixture 1 is formed by mixing polynucleotides SEQ ID NO.1 and 2, SEQ ID NO.1 and 5, SEQ ID NO.1 and 8; multiplex PCR primer mixture 2 is composed of polynucleotide SEQ ID NO.3 and 4, SEQ ID NO.6 and 7, and SEQ ID NO.9 and 10 are mixed.

[0099] Accor...

Embodiment 32019

[0115] Example 3 Detection of Nosocomial Infection Epidemic Samples of Enterovirus in 2019

[0116] Compared with the current fluorescent quantitative PCR (RT-PCR) method, the present invention has the advantage of providing a universal sequencing method for the whole genome of enteroviruses based on a high-throughput detection platform, which overcomes the need to design specific primers for specific types in conventional RT-PCR Disadvantages of Probes. More importantly, the acquisition of the complete genome sequence of enteroviruses can not only accurately identify enterovirus types, but also further analyze the characteristics of the virus genome. In April 2019, a nosocomial infection of enterovirus broke out in Shunde, Guangdong Province, causing 4 newborn deaths and dozens of people showing symptoms of infection, causing serious social impact. Now take this epidemic as an example to illustrate how to use the kit in Example 2 to perform rapid sequence determination, typi...

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Abstract

The invention discloses multiple primers and a kit for high-throughput detecting of a whole genome of enterovirus, and a high-throughput sequencing analysis method. Compared with the prior art, specific primers do not need to be used for detecting a certain generic type one by one in a targeted mode any more, but multiple PCR is utilized to conduct genomic amplification on various enterovirus subtypes through an experiment, the virus subtypes are accurately distinguished and identified, and the accuracy rate is higher, and repeatability is better; high-depth sequencing is adopted, the enough detection depth is ensured, and thus false positive is lower; and through the detection method, the gene types of the enterovirus are rapidly identified, genome variable site, diversity and recombination analysis of viruses can be further provided, and the important scientific basis of prevention and control over the enterovirus is provided.

Description

technical field [0001] The invention relates to the technical field of high-throughput sequencing of viral genomes, in particular to a method for analyzing multiple primers, kits and high-throughput sequencing data for high-throughput determination of the whole genome of enteroviruses. Background technique [0002] Enterovirus is a single-stranded positive-sense RNA virus with a total length of about 7.2-8.4kb. It causes a wide range of diseases. In addition to common hand, foot and mouth disease, enterovirus infection can also cause encephalitis, angina, and myocarditis , Respiratory tract infection, etc. From 2008 to 2018, a total of about 20 million cases of hand, foot and mouth disease were reported in my country, and other diseases such as encephalitis caused by enteroviruses also continued to occur. Since April 2019, enterovirus Echo11 has been detected in many hospitals in Guangdong Province, and the nosocomial infection caused by enterovirus has caused many deaths o...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/6869C12N15/11
CPCC12Q1/701C12Q1/6869C12Q2537/143C12Q2531/113C12Q2535/122
Inventor 陆靖李晖郑焕英曾汉日易丽娜夏昊强姚启聪
Owner 广东省公共卫生研究院
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