Whole genome sequencing method of influenza B virus

Abstract

The invention discloses a whole genome sequencing method of an influenza B virus. By the whole genome sequencing method, in allusion to conserved regions of PB2, PB1, PA, HA, NP, NA, MP and NS genes of the influenza B virus, 14 pairs of oligonucleotide primer sequences covering the full gene length (an open reading frame), altogether 28 oligonucleotide primer sequences shown as SEQ ID No. 1 to SEQ ID No. 28, are designed, M13 forward and reverse primer sequences are added to both ends of forward and reverse amplification primers, and a sequencing reaction step after gene amplification is simplified. The invention also discloses treatment to a sample to be detected, an RT-PCR reaction system, an RT-PCR reaction condition, a sequencing reaction system and a sequencing reaction condition. According to the whole genome sequencing method, whole genome amplification of the influenza B virus which is epidemic in China mainland can be achieved so as to obtain whole genome sequencing information of the influenza B virus; the whole genome sequencing method is easy to operate and convenient to apply; and according to the whole genome sequencing method, a feasible technical method is provided for the etiological research and molecular evolution analysis of the influenza B virus in China.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to a RT-PCR amplification technology, in particular to an influenza virus RT-PCR whole genome sequencing method and application thereof. Background technique [0002] Influenza is an acute respiratory disease caused by influenza A, B or C viruses, which is characterized by seasonal epidemics or local outbreaks. Influenza virus belongs to the Orthomyxoviridae family and is a single-stranded negative-sense RNA virus with a segmented genome. Among them, type A influenza virus often causes influenza pandemics worldwide, while type B is a seasonal epidemic trend. Unlike influenza A, humans are the only host of influenza B viruses, and influenza B viruses are not divided into HA and NA subtypes, but are divided into two branches with different antigens, namely, the Victoria branch and the Yamagata branch. [0003] Each year, one or two branches of influenza B virus co-circulate with seasonal ...

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Problems solved by technology

[0004] Obtaining the genome information of the virus through sequencing is conducive to grasping the epidemic trend and mutation of the virus. Sequencing with the first-generation sequencing method based on the Sanger dideoxy chain termination method requires the design of amplification primers in the conserved region of the virus gene sequence. The disadvantage of the method of designing sequencing primers while amplifying is that gene amplification primers are only designed for certain strain sequences in certain regions, and the versatility is not strong, and amplification primers need to be redesigned continuously

There are multiple sequencing primers for one gene, and there are dozens of sequencing primers for an influenza virus genome, which is inconvenient to operate

Although the next-generation sequencing technology does not rely on primers, the cycle is relatively long, the cost of measuring the genome of pathogenic microorganisms is high, and specialized bioinformatics personnel are required to splice and analyze the sequences

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