绵羊痘病毒和山羊痘病毒通用RPA检测方法的建立与优化  

Establishment and optimization of a RPA diganostic method for sheep pox virus and goat pox virus

作　　者：周峰[1] 王志宇 张梅[1] 康新华[1] 高军军 豆玲[1] 王雪莹 康文彪[1] ZHOU Feng;WANG Zhi-yu;ZHANG Mei;KANG Xin-hua;GAO Jun-jun;DOU Ling;WANG Xue-ying;KANG Wen-biao(The Animal Pestilence Prevention and Control Center of Gansu Province,Lanzhou 730046,China;InTec Products Inc.(Suzhou),Suzhou 215000,China)

机构地区：[1]甘肃省动物疫病预防控制中心,甘肃兰州730046 [2]英科新创(苏州)生物科技有限公司,江苏苏州215000

出　　处：《中国兽医科学》2022年第7期830-836,共7页Chinese Veterinary Science

基　　金：甘肃省农业农村厅科技项目(GNKJ-2021-24)。

摘　　要：根据选择的靶基因RPO35基因序列,使用Oligo 7软件设计并筛选合适的引物与探针,在37℃下通过反应时间和反应体系的优化,结合胶体金试纸条检测,建立了一种检测绵羊痘病毒/山羊痘病毒的重组酶聚合酶扩增(recombinase polymerase amplification,RPA)方法,对其敏感性和重复性进行了检验,并将其用于临床样品的检测,将检测结果与普通PCR检测结果进行了比较。试验结果显示,病毒核酸阳性样品能够在试纸条上显示出阳性条带,阴性样品不显示,试验具有良好的重复性、敏感性(达到620 fg/μL),与普通PCR方法相当。使用本方法,对PCR方法确定的绵羊痘病毒核酸阳性的5份样品和随机抽取的5份临床样品进行了检测,结果显示,绵羊痘病毒核酸阳性的5份样品全部为阳性;5份临床样品中1份为阳性,4份为阴性,检测结果与普通PCR检测结果完全(100%)符合。上述结果表明,本研究建立的RPA技术适用于绵羊痘病毒/山羊痘病毒的快速检测,为绵羊痘和山羊痘的临床诊断提供了新的选择。To develop a recombinase polymerase amplification(RPA)method for rapid detection of sheep pox virus(SPPV)and goat pox virus(GTPV),specific primers were designed using Oligo 7 software based on the selected target RPO35 gene sequence and the probe was established by optimizing the reaction time and reaction system at 37℃.Combined with the detection of colloidal gold test strip,the sensitivity and repeatability of this method were tested,and it was preliminarily used in the detection of clinical samples.The results showed that the bands of positive samples on the strip,but no bands for the negative samples.Compared with PCR method,the RPA in our study indicating an acceptable good repeatability and sensitivity(620 fg/μL).Using the RPA to detect 5 positive samples which were detected by PCR method and 5 random clinic samples.Five positive samples is confirmed and 1 of the 5 clinic samples were positive,4 of the 5 clinic samples were negative and the detection results were 100%consistent with the results by PCR.The RPA will apply to rapid detection and will be a new selection for epidemiological investigation of SPPV and GTPV.

关 键 词：重组酶聚合酶扩增技术(RPA) 试纸条 绵羊痘病毒 山羊痘病毒 

分 类 号：S852.659.1[农业科学—基础兽医学]