Multiplex RT-PCR method for diagnosing equine influenza virus and identifying subtype thereof

Abstract

The invention discloses a method for diagnosticating an EIV and evaluating a multiple RT-PCR of the EIV subtype. The method comprises the following steps that: a RNA of a sample to be detected is taken as a template, two groups of ribonucleotide sequences of SEQ ID NO:1 and SEQ ID NO:2 as well as SEQ ID NO:3 and SEQ ID NO:4 are taken as primers to perform the RT-PCR reaction; if only the product of 227bp is amplified in the sample, the sample is H7N7 subtype of the EIV; if the products of both 227bp and 595bp are amplified simultaneously, the sample is H3N3 subtype of the EIV; if the product of 227bp is not amplified, the sample does not contain the EIV. According to the RT-PCR technical theory, the multiple RT-PCR detection method is established according to the characteristic that the EIV only has two HA subtypes. The method has strong sensitivity and high specificity and can be applied to the rapid diagnosis and the subtype verification of the EIV.

Description

technical field [0001] The invention relates to a method for diagnosing and identifying a virus and its subtype, in particular to a multiple RT-PCR method for diagnosing equine influenza virus and identifying its subtype, and belongs to the field of virus molecular biology. Background technique [0002] Equine influenza virus (equine influenza virus, EIV) belongs to type A influenza virus, causes equine animal influenza, and can also infect dogs and other animals (Crawford PC, Edward J D, William L C, et al. Transmission of equine influenza virus to dogs [J].Science.2005.310:482-85; Kuiken T, Edwed CH,John M, et al.Host species barriers to influenza virus infections.Science[J].2006.312:394-97.). The influenza virus genome consists of 8 negative-sense single-stranded RNA segments including M gene, HA gene and NA gene. Among them, the M genome sequence is highly conserved, while the HA and NA genomes are highly variable, and the variation of certain sites can lead to the emer...

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Problems solved by technology

[0004] Virus isolation and serological tests are still commonly used methods for diagnosing equine influenza and typing EIV, but these methods are relatively complicated to operate and cannot quickly diagnose the virus, which is not conducive to the timely and effective prevention and control of equine influenza (Quinlivan M, Cullinane A, Nelly M, et al.Comparison of sensitivities of virusisolation,antigen detectoion,and nucleic acid amplification for detection of equineinfluenza virus[J].J.Cli.Micro.2004.Vol.42,No.2:759-63 .)

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