鸭常见RNA病毒的多重PCR检测方法的建立与初步应用  

Establishment and preliminary application of multiplex PCR for detection of common RNA viruses in ducks

作　　者：董亚青[1] 王永娟[1] 王豪伟 DONG Ya-qing;WANG Yong-juan;WANG Hao-wei(Jiangsu Agri-animal Husbandry Vocational College,Taizhou 225300,China)

机构地区：[1]江苏农牧科技职业学院,江苏泰州225300

出　　处：《中国预防兽医学报》2022年第11期1183-1188,共6页Chinese Journal of Preventive Veterinary Medicine

基　　金：江苏省2020年度政策引导类计划(苏北科技专项)先导性项目(XZSZ202021);江苏农牧科技职业学院科研课题(NSF2022ZR07);江苏省高等学校自然科学研究重大项目(19KJA520008)。

摘　　要：1型鸭肝炎病毒(DHAV-1)、番鸭呼肠孤病毒(MDRV)、鸭坦布苏病毒(DTMUV)和鸭低致病性H9N2禽流感病毒(AIV)是危害养鸭业的常见病毒性传染病病原。为建立快速鉴别检测以上4种病毒的方法,本研究根据GenBank登录的DHAV-1 vp1基因、MDRV c基因、DTMUV e基因和H9N2(AIV)HA基因序列,分别设计并合成4对特异性引物,经各反应条件的优化,结果显示,4对引物均能分别扩增出DHAV-1、DTMUV、MDRV、H9N2AIV的特异性片段,确定各病毒最佳上、下游引物终浓度均为0.2μmol/L,退火温度为52℃,25个循环,初步建立了能同时检测DHAV-1、DTMUV、MDRV、H9N2 AIV的多重PCR检测方法。以优化的多重PCR方法分别对DHAV-1 SH、DTMUV JS804、MDRV HNF、H9N2 AIV C/SH/F/98、鸭瘟病毒(DPV F34)、番鸭细小病毒(MDPV FJM3)、传染性法氏囊病病毒(IBDV B87)、新城疫病毒(NDV La Sota)株基因组进行特异性检测,结果显示,分别扩增出DHAV-1、DTMUV、MDRV、H9N2 AIV的特异性片段,与其他4种常见病原均无交叉扩增,特异性较强。将重组质粒标准品p MD-DHAV-1、pMD-DTMUV、pMD-MDRV和pMD-H9N2分别10倍倍比稀释并等体积混合后作为模板,利用本实验建立的多重PCR检测,结果显示该方法对各重组质粒标准品检测下限为102拷贝/μL及以下,敏感性较高。利用该方法 3次重复检测随机3批次的4种病毒cDNA混合模板,结果显示各结果均一致,重复性好。利用建立的该方法检测人工混合感染上述4种病毒的10个鸭胚和87份临床疑似病料样品,评估建立的多重PCR方法临床应用的可行性,结果显示该方法能够检出人工混合感染鸭胚样品中的4种病毒;该方法对87份临床疑似病料样品进行检测,检测结果与各病毒单一PCR检测结果均一致。本研究建立的多重PCR检测方法为DHAV-1、DTMUV、MDRV、H9N2 AIV 4种病毒的快速鉴别检测提供了新的技术手段。Duck hepatitis virus type 1(DHAV-1),muscovy duck reovirus(MDRV),duck Tembusu virus(DTMUV) and H9N2avian influenza virus are common viral infectious pathogens endangering duck industry.The ultimate aim of the this study was to establish a rapid method for identification and detection of the above mentioned four viruses.Thus,four pairs of specific primers were designed and synthesized,according to the vp1 gene of DHAV-1,c gene of MDRV,e gene of DTMUV and HA gene of H9N2AIV.After single PCR amplification and optimization of the amplification conditions,a multiplex PCR method was established for the simultaneous detection of DHAV-1,DTMUV,MDRV and H9N2 AIV,and its specificity,sensitivity and repeatability were evaluated.Subsequently,the established method was used to detect 10 artificially mixed infected duck embryos and 87 clinically suspected samples,to confirm the feasibility and clinical application of the assay.The results showed that the specific fragments of the corresponding virus could be amplified by mixed four pairs of primers,with no cross amplification with other four common viral pathogens including duck plague virus(DPV),muscovy duck parvovirus(MDPV),infectious bursal disease virus(IBDV),and Newcastle disease virus(NDV).Moreover,the established multiplex PCR method was able to detect recombinant plasmid standards as low as 102copies/μL,with good repeatability,and could also detect various viruses in artificially mixed infected duck embryos.The results of 87 suspected clinical samples were consistent with those of single PCR.Our findings demonstrate that a multiplex PCR method for a rapid and simultaneous detection of DHAV-1,DTMUV,MDRV,H9N2 AIV was successfully established.

关 键 词：鸭 RNA病毒 多重PCR 检测 

分 类 号：S834[农业科学—畜牧学]