Pyrophosphoric acid sequencing method for identifying and detecting Class II Newcastle disease virus virulent strains or attenuated strains

Abstract

The invention belongs to the technical field of animal pathogen detection, and relates to a pyrophosphoric acid sequencing method for identifying and detecting Class II Newcastle disease virus virulent strains or attenuated strains. The method comprises the following steps: designing specific RT-PCR (reverse transcription-polymerase chain reaction) amplification primers and pyrophosphoric acid sequencing primers capable of distinguishing all Class II Newcastle disease virus virulent strains and attenuated strains; extracting RNA of a sample to be detected, and carrying out amplification by using the RT-PCR amplification primers; detecting the amplification product by agarose gel electrophoresis; and if the length of the amplification product is 218bp, preparing a pyrophosphoric acid sequencing single-strand template, carrying out pyrophosphoric acid sequencing reaction, and finally, judging whether the sample contains the Class II Newcastle disease virus virulent strains or attenuated strains according to the RT-PCR amplification result and pyrophosphoric acid sequencing result. By using the pyrophosphoric acid sequencing technique, the specific sequences are utilized to identify the Newcastle disease virus virulent strains or attenuated strains, so the method has the characteristics of high flux, low cost and the like; and the method has the advantages of small required sample quantity and simple result judgment standard, is simple to operate, achieves the goal of early diagnosis, and thus, has wide application prospects in epidemic situation monitoring, rapid diagnosis and exit-entry quarantine inspection.

Description

Technical field [0001] The invention belongs to the technical field of animal pathogen detection, and relates to a pyrosequencing method for distinguishing and detecting the strong and weak virulence of Type II Newcastle disease virus. Background technique [0002] Newcastle disease (ND) is an acute, highly contagious infectious disease of poultry caused by virulent Newcastle disease virus (NDV) infection. After the virulent Newcastle disease virus infects poultry, it can cause typical Newcastle disease symptoms and pathological changes in poultry, with a mortality rate as high as 100%, causing huge economic losses to the poultry industry. The Ministry of Agriculture of my country lists Newcastle disease as a type of animal disease, and the World Organization for Animal Health (OIE) lists it as a statutory reported disease. In the "National Medium and Long-term Animal Disease Prevention and Control Plan (2012-2020)" promulgated by the State Council, Newcastle disease has been i...

AI Technical Summary

Problems solved by technology

[0005] Newcastle disease virus virulence identification methods mainly include two methods: traditional biological test and virus gene sequence determination. Index (ICPI) and 6-week-old Chicken Intravenous Pathogenicity Index (IVPI) for identification. These traditional virulence identification methods are time-consuming and laborious, and require a certain level of biosafety laboratories to operate, so they are not suitable for large-scale promotion Application: Viral gene sequence determination is based on RT-PCR combined with sequence determination to evaluate the pathogenic characteristics of the strain, which takes a relatively long time and cannot meet the needs of the first diagnosis

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