H7和N9亚型及A型流感病毒多重荧光定量RT-RCR检测方法的建立  

Establishment of multiplex real-time RT-PCR for detecting H7, N9subtype and A type influenza virus

作　　者：徐晓龙[1] 包红梅[1] 马勇[1] 施建忠[1] 曾显营[1] 李雁冰[1] 王秀荣[1] 陈化兰[1] 

机构地区：[1]中国农业科学院哈尔滨兽医研究所兽医生物技术国家重点实验室/农业部动物流感重点开放实验室,黑龙江哈尔滨150001

出　　处：《中国预防兽医学报》2014年第12期939-942,共4页Chinese Journal of Preventive Veterinary Medicine

基　　金：十二五农村领域国家科技课题(2012AA101303);国际合作项目(2014DFR31260)

摘　　要：为同时检测H7亚型、N9亚型和A型流感病毒,本研究根据流感病毒H7 HA、N9 NA以及M基因序列分别合成3对引物和3种荧光素标记的探针,建立了多重Taq Man荧光定量RT-PCR检测方法。特异性试验结果显示,仅H7亚型和N9亚型病毒分别在FAM和JOE通道监测到荧光信号,而所有A型参考流感病毒株均可获得Cy5荧光信号,此外其他相关的禽源病毒检测结果均为阴性。同时,以H7N9亚型病毒RNA为模板建立了标准曲线,检测H7 HA、N9 NA以及M基因的R2值均为0.999,最低检出量均为35.4 EID50/m L。批内和批间试验的变异系数均小于1.48%。利用该方法对1 072份临床样品(咽喉和泄殖腔拭子)检测,H7N9亚型和A型流感病毒分别检出7份和23份。该方法可以有助于H7N9亚型和A型流感病毒的快速检测及鉴定。To establish a rapid detection method for detecting H7,N9 subtype and A type influenza virus,the multiplex TaqMan real-time RT-PCR assay was developed with three sets of primers （targeting H7 HA,N9 NA,and M gene） and probes labeled with FAM,JOE,and Cy5,respectively.The specificity results showed that the fluorescence signals from FAM and JOE were detected only in H7 and N9 subtype virus,and the Cy5 fluorescence signal was detected in all the A type influenza virus strains; Whereas,no signal was detected in other related avian respiratory viruses.In addition,standard curve established with H7N9 subtype virus displayed the R2 index was 0.999,and the detection limit was 35.4 EID50/mL,corresponding to H7 HA,N9 NA,and M gene.The intra-and inter-assay coefficients of variation were all less than 1.48％.Furthermore,a total of 1,072 clinical samples were tested,and 7 positive samples for H7N9 and 23 for A type influenza virus were identified by the established assay.These data demonstrated the detection assay could be applied for rapid identification of H7N9 subtype and A type influenza virus.

关 键 词：A型流感病毒 H7N9亚型流感病毒 多重TaqMan荧光定量RT-PCR 

分 类 号：S852.65[农业科学—基础兽医学]