牛诺如病毒实时荧光定量PCR检测方法的建立及应用

doi:10.11843/j.issn.0366-6964.2020.07.025

Abstract

Abstract: Bovine norovirus (BNoV) is an emerging causative agent of calf diarrhea in China, the aim of the study was to establish a real-time PCR assay for detecting BNoV. One pair of primers was designed based on RNA-dependent RNA polymerase (RdRp) gene of BNoV. The EvaGreen real-time PCR assay was successfully developed after the optimization of amplification conditions. The test results showed that the Ct value showed a good linear relationship with the standard in the range of 2.24×10²-2.24×10⁸copies·μL^-1 and the correlation coefficient R²=0.997, and the amplification efficiency was 98.44%. There is no specific amplification of other common calf diarrhea pathogens, only BNoV were positive. The detection limit of the method was 22.4 copies·μL^-1 for BNoV. The inter-assay and the intra-assay coefficient of variation were both less than 2%, indicating a good repeatability. 221 clinical samples that collected from the diarrheic calves in Henan Province during September 2017 to May 2019 were detected using this real-time PCR assay, and the BNoV detection rate was 11.31% (25/221), the farms positive rate was 92.86% (13/14). These results indicated that the real-time PCR assay has good sensitivity, specificity and repeatability, which can be provide an effective means for detection and epidemiological investigation of BNoV.

Key words: bovine norovirus, real-time PCR, calf diarrhea, detection

CLC Number:

S852.659.6

SHI Zhihai, WANG Wenjia, LAN Yali, ZHANG Bin, MENG Hongli, WANG Yazhou, HUA Liushuai, XU Zhaoxue. Establishment and Application of a Real-time PCR Assay for Detecting Bovine Norovirus[J]. Acta Veterinaria et Zootechnica Sinica, 2020, 51(7): 1728-1736.

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Establishment and Application of a Real-time PCR Assay for Detecting Bovine Norovirus

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