检测山羊嵴病毒的实时荧光定量PCR方法的建立和应用

doi:10.11843/j.issn.0366-6964.2021.05.020

Abstract

Abstract: Caprine kobuvirus (CKoV) is an emerging virus in goats in China, this study aimed to establish a real-time fluorescent quantitative PCR assay for detecting CKoV. Through designing primers targeting the 3D gene of CKoV and optimizing the reaction conditions and system, a TB Green Fluorescent Quantitative PCR assay was successfully established. The assay had a good linear relationship with CKoV in the range of 2.23×10²-2.23×10⁸copies·μL^-1 with a correlation coefficient (R²) of 0. 999 2, and its amplification efficiency was 110%. Moreover, the assay has good specificity and stability, and the detection limit was 2.23×10¹copies·μL^-1. Furthermore, out of 79 diarrhea samples of lambs collected from Sichuan, Chongqing, and Yunnan regions from January 2020 to April 2020, 25.3%samples (20/79) were detected as CKoV positive, and the farms’ positive rate was 53.3% (8/15). In addition, 13 complete 3D genes of CKoV were obtained in this study, and evolution analysis showed that these 13 3D genes had unique evolutionary trends. In conclusion, this study provides a new molecular method for detecting CKoV and basic epidemiological data of domestic CKoV.

Key words: caprine kobuvirus, real time fluorescent quantitative PCR assay, goat, diarrhea

CLC Number:

S852.659.6

ABI-Kehamo, YU Zhonghua, JING Zhizhong, TANG Cheng. Establishment and Application of a Real Time Fluorescent Quantitative PCR Assay for Detecting Caprine Kobuvirus[J]. Acta Veterinaria et Zootechnica Sinica, 2021, 52(5): 1349-1358.

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Establishment and Application of a Real Time Fluorescent Quantitative PCR Assay for Detecting Caprine Kobuvirus

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