摘要
为了建立一种能快速检测鹅星状病毒(GoAstV)、鹅圆环病毒(GoCV)、鹅细小病毒(GPV)、鸭坦布苏病毒(DTMUV)的多重PCR检测方法,本研究根据GenBank数据库已公布的GoAstV的ORF2基因、GoCV的Rep基因、GPV的VP3基因及DTMUV的NS5基因序列,分别设计4对特异性扩增引物,通过对扩增条件的优化及特异性、灵敏性的检验,建立了可同时检测以上4种病原的多重PCR检测方法,并对40份临床样品进行检测。结果显示,建立的多重PCR检测方法具有良好的特异性和较高的灵敏性,可同时扩增出大小为731 bp(GoAstV)、543 bp(GoCV)、392 bp(GPV)、200 bp(DTMUV)的特异性片段,且利用本试验建立的多重PCR检测方法对水禽其他病原体进行扩增均呈阴性。该方法对GoAstV、GoCV、GPV及DTMUV最低检出限度分别为4×10^(3),4.22×10^(3),4.43×10^(3),4.73×10^(3)copies/μL。单一PCR检测方法对临床病料样品检测结果与多重PCR检测结果一致,证明该方法可用于这4种病原临床病料样品的检测。
This study aims to establish a multiplex PCR method for rapid detection of goose astrovirus(GoAstV),goose circovirus(GoCV),goose parvovirus(GPV)and duck Tembusu virus(DTMUV).According to the published sequences of NS5 gene of DTMUV,VP3 gene of GPV,Rep gene of GoCV and ORF2 gene of GoAstv in GenBank database,4 pairs of specific primers were designed,respectively.Through the optimization of amplification conditions,validating specificity and sensitivity,the multiplex PCR method was established for simultaneous detection of the four pathogens.And 40 clinical samples were detected by the multiplex PCR method.The results showed that the assay had good specificity and high sensitivity,and could simultaneously amplify the specific fragments of GoAstV(731 bp),GoCV(543 bp),GPV(392 bp)and DTMUV(200 bp),respectively.The multiplex PCR method established in this study was negative for the amplification of other waterfowl pathogens.The minimum detection limits of GoAstV,GoCV,GPV and DTMUV were 4×10^(3),4.22×10^(3),4.43×10^(3)and 4.73×10^(3)copies/μL,respectively.The detection results of clinical samples by single PCR were consistent with those by multiple PCR,which proved that the method could be used for the detection of the four pathogenic clinical samples.
作者
姚鑫炎
孙静
刘宏
吕志航
车艺俊
黄淑坚
袁生
张雪莲
YAO Xinyan;SUN Jing;LIU Hong;LYU Zhihang;CHE Yijun;HUANG Shujian;YUAN Sheng;ZHANG Xuelian(College of Life Science and Engineering,Foshan University,Foshan,Guangzhou 528231,China;Guangdong GeFeng Animal Health Technology Co.,LTD,Zhaoqing,Guangdong 526238,China)
出处
《中国兽医学报》
CAS
CSCD
北大核心
2022年第11期2190-2194,2210,共6页
Chinese Journal of Veterinary Science
基金
广东省基础与应用基础研究区域联合基金-青年基金资助项目(2019A1515110157)
广东省教育厅普通高校重点领域专项(乡村振兴)资助项目(2020ZDZX1004)
广东省教育厅青年创新人才资助项目(2018KQNCX277)
广东省现代农业产业技术体系创新团队资助项目(2019KJ137)。
