H3、H4、H6和H9亚型禽流感病毒多重TaqMan荧光定量RT-PCR检测方法的建立及应用  

Establishment and application of multiplex TaqMan quantitative RT-PCR assay for avian influenza viruses of subtypes H3,H4,H6 and H9

作　　者：龙凤 熊陈勇 施开创[1] 郑敏[1] 林昌华 林宝江 杜忠 韦显凯[1] 冯淑萍[1] 屈素洁[1] 陆文俊[1] 李剑锋 周明旭 尹彦文[1] LONG Feng;XIONG Chen-yong;SHI Kai-chuang;ZHENG Min;LIN Chang-hua;LIN Bao-jiang;DU Zhong;WEI Xian-kai;FENG Shu-ping;QU Su-jie;LU Wen-jun;LI Jian-feng;ZHOU Ming-xu;YIN Yan-wen(Guangxi Center for Animal Disease Control and Prevention,Nanning 530001,China;Guangxi State Farms Yongxin Animal Husbandry Group Xijiang Co.,Ltd.,Guigang 537104,China;Chongzuo Center for Animal Disease Control and Prevention,Chongzuo 532200,China;Guangxi Vocational University of Agriculture,Nanning 530007,China)

机构地区：[1]广西动物疫病预防控制中心,广西南宁530001 [2]广西农垦永新畜牧集团西江有限公司,广西贵港537104 [3]崇左市动物疫病预防控制中心,广西崇左532200 [4]广西农业职业技术大学,广西南宁530007

出　　处：《中国预防兽医学报》2025年第1期31-38,共8页Chinese Journal of Preventive Veterinary Medicine

基　　金：广西科技重大专项(桂科AA23062050);广西自然科学基金项目(2020GXNSFBA297066)。

摘　　要：为同时鉴别检测H3、H4、H6和H9亚型禽流感病毒(AIV),本研究根据上述4种亚型AIV HA基因的保守区,采用primer6.0设计4对特异性引物和TaqMan探针,经PCR扩增上述4种亚型AIVHA基因后,分别克隆至pMD18-T载体中构建重组质粒p-H3、p-H4、p-H6和p-H9,并均经PCR与测序鉴定正确后作为标准品。通过优化各反应条件,初步建立了检测H3、H4、H6和H9亚型AIV的多重TaqMan荧光定量RT-PCR(RT-qPCR)方法。分别以H1、H3、H4、H5、H6、H7、H9亚型AIV、传染性喉气管炎病毒(ILTV)、传染性支气管炎病毒(IBV)、鸭坦布苏病毒(DTMUV)、鸭圆环病毒(DuCV)、番鸭细小病毒(MDPV)、番鸭呼肠孤病毒(MDRV)和新城疫病毒(NDV)的RNA或者DNA作为模板,采用本研究建立的多重RT-qPCR检测,评估该方法的特异性;将4个重组质粒标准品等体积混合物10倍倍比稀释后作为模板,采用本研究建立的多重RT-qPCR检测,评估该方法的敏感性;选择3个浓度的重组质粒标准品等体积混合物作为模板,采用本研究建立的多重RT-qPCR分别进行批内和批间重复性试验,评估该方法的重复性。结果显示,该方法仅H3、H4、H6和H9亚型AIV出现扩增曲线,其他相关禽病病原均为阴性,特异性较强;该方法对H3、H4、H6和H9亚型AIV的检测限均为1.0×101拷贝/µL,敏感性较高;批内和批间重复性试验的变异系数为0.06%~1.09%,重复性较好。利用该方法检测来自广西不同地区的3360份临床家禽咽喉、泄殖腔拭子样品,结果显示H3、H4、H6和H9亚型AIV的检出率分别为4.08%(137/3360)、0.21%(7/3360)、1.13%(38/3360)和3.51%(118/3360),与各亚型AIV单一RT-qPCR商品化试剂盒的检测结果均一致,符合率达100%。表明本研究建立的多重RT-qPCR准确性较好,可以用于临床各类样品的检测。本研究建立了一种同时鉴别检测H3、H4、H6和H9亚型AIV的多重RT-qPCR方法,为AIV基因分型和流行病学调查提供便捷的技术手段。To simultaneously detect and differentiate H3,H4,H6,and H9 subtypes of avian influenza viruses(AIVs),this study designed four sets of specific primers and TaqMan probes targeting the conserved regions of the HA genes of these four AIV subtypes using Primer 6.0 software.The HA genes of the four AIV subtypes were amplified by PCR and cloned into the pMD18-T vector to construct recombinant plasmids p-H3,p-H4,p-H6,and p-H9.These plasmids were confirmed as standards through PCR and sequencing.A multiplex TaqMan reverse transcription fluorescence quantitative PCR(RT-qPCR)method for detecting H3,H4,H6,and H9 subtypes of AIV was developed by optimizing the reaction conditions.To evaluate the specificity of this method,RNA or DNA from AIV subtypes H1,H3,H4,H5,H6,H7,and H9,as well as from infectious laryngotracheitis virus(ILTV),infectious bronchitis virus(IBV),duck Tembusu virus(DTMUV),duck circovirus(DuCV),Muscovy duck parvovirus(MDPV),Muscovy duck reovirus(MDRV),and Newcastle disease virus(NDV),were used as templates.The sensitivity of the method was assessed by using a 10-fold serial dilution of an equimolar mixture of the four recombinant plasmid standards.The repeatability of the method was evaluated by testing three different concentrations of the equimolar mixture of the recombinant plasmid standards.The results showed that the technique only produced amplification curves for the H3,H4,H6,and H9 subtypes of AIV,while all other related avian pathogens were tested negative,indicating high specificity.The detection limit for the H3,H4,H6,and H9 subtypes of AIV was 1.0×101 copies/μL,demonstrating high sensitivity.The intra-and inter-assay coefficients of variation ranged from 0.06%to 1.09%,in-dicating good reproducibility.Using this method,3360 clinical samples of oropharyngeal and cloacal swabs from poultry in different regions of Guangxi were tested.The positive rates for H3,H4,H6,and H9 subtypes of AIV were 4.08%(137/3360),0.21%(7/3360),1.13%(38/3360),and 3.51%(118/3360),respectively.These results were consistent

关 键 词：禽流感病毒 荧光定量RT-PCR TAQMAN 

分 类 号：S852.65[农业科学—基础兽医学]