Primer, kit and method for conducting genetic typing on hantavirus by means of PCR direct sequencing method

Abstract

The invention discloses a primer, kit and method for conducting genetic typing on the hantavirus by means of a PCR direct sequencing method. The primer comprises an upstream primer: ATTAGCCCWGTCATGAGTGT, and a downstream primer: CTTTGACTCYTTTGKYTCCA, wherein the Y is a C or a T, the W is an A or a T, and the K is a G or a T. The method comprises the steps of extracting a total RNA of a virus sample to be tested, conducting reverse transcription to obtain a cDNA, using the cDNA as a template, utilizing the primer for conducting PCR amplification, conducting sequencing on an amplified target fragment, constructing a phylogenetic tree by using corresponding fragments of known genotype representative strains as a reference on the basis of sequence information of the target fragment, and conducting the genetic typing on the virus sample to be tested according to the phylogenetic tree. When the primer is used, a specificity sequence of an S gene of the hantavirus can be obtained by only conducting one-time PCR amplification, and the genetic typing can be easily, conveniently and rapidly carried out on the hantavirus by utilizing the specificity sequence.

Description

technical field [0001] The invention relates to the technical field of gene diagnosis, in particular to a primer, a kit and a method for genotyping hantavirus by PCR direct sequencing. Background technique [0002] Hantavirus (HV) belongs to the genus Hantavirus of the Bunyaviridae family, and is a segmented, enveloped, negative-strand RNA virus, including Hantaviruses that cause hemorrhagic fever with renal syndrome (HFRS). Tan type, Seoul type, Puumala type, etc., as well as New York type and unknown type that cause Hantavirus pulmonary syndrome (HPS), Hantaan type and Seoul type are the main epidemic strains in my country. The natural host of hantavirus is mainly small rodents, and it is transmitted to humans through rodents, causing HFRS or HPS. [0003] Hantavirus infection, also known as hemorrhagic fever, is an important severe zoonotic disease, and is listed as a second-class harmful pathogen by the Ministry of Health and the Ministry of Agriculture. Experimental an...

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Problems solved by technology

[0007] Previous studies have shown that by constructing a phylogenetic tree based on the full-length nucleotide sequences of the M gene segment and the S gene segment, the results of genotyping Hantavirus are consistent with the results of serological typing, but the whole gene needs to be fully nuclear Nucleic acid sequencing analysis, there is no literature report to achieve genotyping of different strains of Hantavirus with only one direct sequencing result of PCR products

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