Detection method for identifying American classical PRRSV strain, HP-PRRSV strain and new-type viral NADC30 strain at the same time

Abstract

The invention discloses a detection method for identifying the American classical PRRSV strain, the HP-PRRSV strain and a new-type viral NADC30 strain at the same time. In the method, a multiplex real-time fluorescence RT-PCR for identifying three kinds of strains at the same time is established based on an ABI7500 fluorescent quantitative PCR instrument, wherein the strains include the classical PRRSV strain, the highly-pathogenic HP-PRRSV strain with the deletion of L-amino acid at the 481st locus of the Nsp2 gene and the continuous deletion of 29 amino acids at the 533ird-561st loci of the Nsp2 gene, and NADC30like; specific primers and specific probes for the three kinds of strains are designed respectively based on the Nsp2 gene, universal primers and universal probes for PRRSV are designed according to the relatively conserved N gene, missing detection caused by Nsp2 region mutation can be effectively prevented, and false negative can be avoided.

Description

technical field [0001] The invention relates to a detection method, in particular to a detection method for simultaneously identifying American-type PRRSV classic strains, variant strains and new virus-like NADC30 strains. The invention belongs to the technical field of molecular biology detection. Background technique [0002] Porcine reproductive and respiratory syndrome (Porcine reproductive and respiratory syndrome, PRRS) is an acute infectious disease caused by porcine reproductive and respiratory syndrome virus (Porcine reproductive and respiratory syndrome virus, PRRSV). The disease can cause reproductive disorders such as fever, miscarriage, premature birth, stillbirth, and mummified fetuses in sows, increased mortality of piglets before and after weaning, and respiratory disorders in pigs of various ages. The disease first occurred in the United States in 1987. At present, the two prevalent PRRSVs in the world are the European type (genotype I) and the American typ...

AI Technical Summary

Problems solved by technology

[0004] At present, PRRSV detection techniques include virus isolation method, neutralization test, immunoperoxidase monolayer test, immunofluorescence antibody test, immunohistochemical technology, enzyme-linked immunosorbent assay, colloidal gold immunochromatography technology, reverse transcription polymerization Enzyme chain reaction, etc., the above methods are general-purpose and can only identify whether they are infected with PRRSV, but cannot distinguish which type of PRRSV is infected. There are limitations such as low specificity, easy to cause false positives, and low accuracy.

The application numbers are CN201610192662.2 and CN 201610195105.6, which provide a detection method for the identification of NADC30 virus strains, but this method is a common PCR method, and the risk of PCR product contamination is likely to occur in the opening operation through agarose gel electrophoresis detection, resulting in inaccurate detection results. precise

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