水禽圆环病毒巢式PCR检测方法的建立与应用  

Establishment and application of nested PCR detection method for waterfowl circovirus

作　　者：姚鑫炎 张玉倩 袁生 杨惠湖 李文俊 黄惠兰 梁昭平[2] 黄淑坚 张雪莲 YAO Xinyan;ZHANG Yuqian;YUAN Sheng;YANG Huihu;LI Wenjun;HUANG Huilan;LIANG Zhaoping;HUANG Shujian;ZHANG Xuelian(School of Life Science and Engineering,Foshan University,Foshan 528231,China;College of Veterinary Medicime,South China Agricultural University,Guangzhou 511300,China)

机构地区：[1]佛山科学技术学院生命科学与工程学院,广东佛山528231 [2]华南农业大学兽医学院,广州511300

出　　处：《黑龙江畜牧兽医》2022年第13期84-88,94,共6页Heilongjiang Animal Science And veterinary Medicine

基　　金：广东省现代农业产业技术体系创新团队项目(2019KJ137);广东省基础与应用基础研究区域联合基金-青年基金项目(2019A1515110157);广东省教育厅普通高校重点领域专项(乡村振兴)(2020ZDZX1004);广东省教育厅青年创新人才项目(2018KQNCX277)。

摘　　要：为了建立一种快速、灵敏、特异的水禽圆环病毒检测方法,试验根据GenBank中已公布的鸭圆环病毒(DuCV)和鹅圆环病毒(GoCV)的Rep基因序列设计3条特异性引物,对扩增条件进行优化,并检验优化后方法的特异性和灵敏性,最后用建立的水禽圆环病毒巢式PCR检测方法对40份疑似水禽圆环病毒感染的病料进行检测。结果表明:水禽圆环病毒巢式PCR方法可成功扩增出719 bp和543 bp的目的片段;优化后的一轮、二轮PCR中退火温度分别为50,56℃,引物加入量均为0.6μL,二轮PCR的模板稀释倍数为1×10^(3)倍。优化后的方法对水禽圆环病毒的检测具有较好的特异性,与鸭甲肝病毒1型(DHAV-1)、鸭甲肝病毒3型(DHAV-3)、鸭坦布苏病毒(DTMUV)、H5N6亚型禽流感病毒(AIV)、H9N2 AIV、新城疫病毒(NDV)、鸭瘟病毒(DEV)、鹅细小病毒(GPV)及鹅星状病毒(GoAstV)均无交叉反应;该方法的最低检出限达4.04×10^(2)拷贝/μL,分别是一轮、二轮PCR最低检出限的1×10^(4),1×10^(3)倍;利用该方法对肝脏病料进行检测,GoCV的阳性率为60%(12/20),DuCV的阳性率为40%(8/20)。说明试验建立的巢式PCR检测方法可用于水禽圆环病毒的检测。In order to establish a rapid, sensitive and specific detection method for waterfowl circovirus, three specific primers were designed according to the Rep gene sequences of Duck circovirus(DuCV) and Goose circovirus(GoCV) published in GenBank. Through optimization of amplification conditions and specificity and sensitivity tests, 40 diseased samples suspected of waterfowl circovirus infection were detected by the established nested PCR detection method for waterfowl circovirus. The results showed that the nested PCR method of waterfowl circovirus could successfully amplify the target fragments of 719 bp and 543 bp. The annealing temperature in the optimized first and second rounds of PCR was 50 ℃ and 56 ℃, respectively;the amount of primers added was 0.6 μL;the template dilution ratio of the second round of PCR was 1×10^(3) times. The optimized method had good specificity for the detection of waterfowl circovirus;there was no cross-reaction with Duck hepatitis A virus type 1(DHAV-1), Duck hepatitis A virus type 3(DHAV-3), Duck Tembusu virus(DTMUV), H5 N6 subtype Influenza A virus(AIV), H9 N2 AIV, Newcastle disease virus(NDV), Anatid alphaherpesvirus 1(DEV), Goose parvovirus(GPV) and Goose astrovirus(GoAstV). The detection limit of this method was 4.04×10^(2) copies/μL, which were 1×10^(4) and 1×10^(3) times the detection limit of the first and second rounds of PCR respectively. By using this method to detect diseased samples, the positive rate of GoCV was 60%(12/20), and the positive rate of DuCV was 40%(8/20). The results suggested that the nested PCR detection method established in the experiment could be used for the detection of waterfowl circovirus.

关 键 词：水禽圆环病毒 巢式PCR Rep基因 条件优化 特异性 灵敏性 应用 

分 类 号：S852.65[农业科学—基础兽医学]