水禽3种免疫抑制性病毒多重PCR检测方法的建立与应用  

Establishment and application of multiplex PCR for detection of three immunosuppressive viruses in waterfowl

作　　者：戴银[1,2] 杨雪 童雨芹[1] 吴希 陈瑝儿 仲月怡 胡晓苗 王洁茹[1,2] 殷冬冬 尹磊 沈学怀 潘孝成 赵瑞宏[1,2] DAI Yin;YANG Xue;TONG Yuqin;WU Xi;CHEN Huanger;ZHONG Yueyi;HU Xiaomiao;WANG Jjeru;YIN Dongdong;YIN Lei;SHEN Xuehuai;PAN Xiaocheng;HAO Ruihong(Livestock and Poultry Epidemic Diseases Research Center of Anhui Province,Institute of Animal Husbandry and Veterinary Science,Anhui Academy of A gricultural Science,Hefei 230031,China;Anhui Province Key Laboratory of Livestock and Poultry Product Safety Engineering,Hefei 230031,China)

机构地区：[1]安徽省农业科学院畜牧兽医研究所,畜禽疫病研究中心,安徽合肥230031 [2]畜禽产品安全工程安徽省重点实验室,安徽合肥230031

出　　处：《中国兽医科学》2024年第3期330-336,共7页Chinese Veterinary Science

基　　金：安徽省科技重大专项(202003a06020012);安徽省家禽产业技术体系项目(AHCYJSTX-06)。

摘　　要：禽流感病毒 H9 亚型(AIV-H9)、鹅圆环病毒(GoCV)和番鸭呼肠孤病毒(MDRV)是水禽常见病原,常发生混合感染且易导致水禽的免疫抑制性疾病。 本研究针对 AIV-H9、GoCV 和 MDRV 的基因序列,分别设计特异性引物,通过优化多重 PCR 的反应条件,建立了这 3 种病毒的多重 PCR 检测方法,并进行特异性、灵敏度和可重复性验证。 结果显示,建立的三重 PCR 检测方法能特异性扩增 AIV-H9、GoCV 和 MDRV的目的片段,与新型鹅星状病毒、鸭瘟病毒、鹅细小病毒、禽腺病毒 4 型无交叉反应;最低检出浓度分别为2.93×10^(4)copies/ μL、2.5×10^(2)copies/ μL、3.0×10^(6)copies/ μL;利用该方法对 30 份临床样品进行检测,MDRV检出率为 6.7%,GoCV 和 AIV-H9 的检出率较高,分别为 36.7%和 26.7%。 三重 PCR 与单重 PCR 的符合率为 100%。 结果表明,该三重 PCR 检测方法特异、灵敏、稳定性好,能够用于这 3 种病毒的流行病学调查和临床检测。Avian influenza virus H9 subtype(AIV-H9),goose circovirus (Go CV) and muscovy duck reovirus (MDRV) are common waterfowl pathogens.These viruses can easily cause immunosuppressive diseases in waterfowl,and mixed infections often occur.The specific primers were designed for the gene sequences of AIV-H9,Go CV and MDRV,respectively.After optimizing the reaction conditions of multiplex PCR,the method for three viruses was established.Then,the specificity,sensitivity and repeatability were verified.The results showed that the established triple PCR assay could specifically amplify the target fragments of AIV-H9,Go CV and MDRV,and it had no cross-reaction with novel-goose astrovirus,duck enteritis virus,goose parvovirus and fowl adenovirus serotype 4.Sensitivity assay showed that the minimum concentrations were 2.93 ×10^(4)copies/μL,2.5 ×10^(2)copies/μL and 3.0 ×10^(6) copies/μL,respectively.The triple PCR was used to detect 30 clinical samples,the positive rates of MDRV was 6.7% ,Go CV and AIV-H9 were 36.7% and 26.7%,respectively.The coincidence rate of triple PCR and single PCR was 100% .The results showed that the triple PCR method was specific,sensitive and reproducible,it could be used for epidemiological investigation and clinical detection of the three viruses.

关 键 词：禽流感病毒H9亚型 鹅圆环病毒 番鸭呼肠孤病毒 三重PCR 

分 类 号：S852.659.5[农业科学—基础兽医学] S852.659.2[农业科学—兽医学]