检测山羊嵴病毒的实时荧光定量PCR方法的建立和应用

山羊嵴病毒（caprine kobuvirus，CKoV）是国内山羊新发病毒，本试验目的是建立检测CKoV的实时荧光定量PCR方法，并对西南三省腹泻山羊粪样进行该病毒的检测。选择CKoV最保守的3D基因序列，设计3D基因引物，初步设计实时荧光定量PCR，经优化反应条件和反应体系，成功建立了检测CKoV的TB Green染料法实时荧光定量PCR方法。该方法在病毒浓度2.23×10²～2.23×10⁸copies·μL^-1范围内线性关系良好，相关系数为0.999 2，扩增效率为110%；此方法具有良好的特异性和稳定性，最低检测限为2.23×10¹copies·μL^-1。采用所建立的方法对2020年1—4月采集自四川、云南和重庆的共15个场79份山羊腹泻粪样本进行检测，结果CKoV的平均检出率为25.3%，场阳性率为53.3%。并且本试验获得了13个完整的CKoV 3D基因，遗传进化分析表明这13个3D基因具有独特的进化趋势。本研究为CKoV的分子检测提供了一种新的技术手段和基础流行病学数据。

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

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.