检测牛5种腹泻病毒的多重RT-PCR方法的建立及应用

doi:10.11843/j.issn.0366-6964.2022.01.021

Abstract

Abstract: Bovine group A rotavirus (BRVA), bovine coronavirus (BCoV), bovine viral diarrhea virus (BVDV), bovine norovirus (BNoV) and bovine nebovirus (BNeV) are common viruses that cause calf diarrhea, and there are mixed infections in clinic. The purpose of this experiment was to establish a multiplex PCR method that can simultaneously detect the above five viruses. The multiplex PCR method for simultaneous detection of BRVA, BCoV, BVDV, BNoV and BNeV was successfully established by designing primers, optimizing reaction system and conditions. This method only specifically detected the target virus, but did not amplify the diarrhea pathogens such as bovine astrovirus, bovine kobuvirus, bovine parvovirus, enterotoxigenic Escherichia coli, Salmonella and Campylobacter jejuni, which had good specificity and stability. The lowest detection limits for each virus were as follows:BRVA 1.63×10⁵copies·μL^-1, BCoV 2.0×10⁶copies·μL^-1, BVDV 1.9×10⁵copies·μL^-1, BNoV 1.96×10⁵copies·μL^-1 and BNeV 2.0×10⁵copies·μL^-1. The comparison experiment showed that the coincidence rate between the multiplex RT-PCR method and the RT-PCR method for detecting single virus was 100%. The detection results of 220 yak diarrhea samples collected from northwest Sichuan grassland from 2019 to 2020 showed that the detection rates of BRVA, BNoV and BNeV were 40.5%, 5.9% and 4.5%, respectively, while BVDV and BCoV were not detected. The multiplex RT-PCR method established in this study can simultaneously detect five common diarrhea viruses in fecal samples of calf diarrhea, providing technical support for the rapid diagnosis of bovine diarrhea.

Key words: bovine group A rotavirus, bovine coronavirus, bovine viral diarrhea virus, Norvirus, nebovirus, multiple RT-PCR

CLC Number:

S855.3

SUN Ji, YUE Hua, TANG Cheng. Establishment and Application of Multiplex RT-PCR for Detection of Five Bovine Diarrhea Viruses[J]. Acta Veterinaria et Zootechnica Sinica, 2022, 53(1): 209-218.

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Establishment and Application of Multiplex RT-PCR for Detection of Five Bovine Diarrhea Viruses

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