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当前位置: 首页 > 文章 > 双重RT-PCR快速检测H7N9流感病毒方法的建立 中国动物检疫 2015,32 (5) 65-68+81
Position: Home > Articles > Development of a Duplex RT-PCR Method for Rapid Detection of H7N9 Subtype Influenza Virus China Animal Health Inspection 2015,32 (5) 65-68+81

双重RT-PCR快速检测H7N9流感病毒方法的建立

作  者：
蒋文明;李金平;于美芳;孙文博;王素春;彭程;侯广宇;刘朔;杜翔
单  位：
中国动物卫生与流行病学中心;山东省农业科学院畜牧兽医研究所
关键词：
流感病毒;H7N9;双重RT-PCR
摘  要：
为建立简便快速检测H7N9亚型流感病毒的方法,根椐Gen Bank中H7亚型流感病毒的HA基因序列和N9亚型流感病毒的NA基因序列,设计2对特异性引物,在建立H7亚型和N9亚型单项RT-PCR的基础上,优化双重RT-PCR反应条件,建立同时检测H7亚型和N9亚型的双重RT-PCR。对H7N9亚型流感病毒核酸模板进行双重RT-PCR扩增,结果可同时扩增HA基因263bp、NA基因520 bp的特异性片段,而对其他常见亚型的流感病毒的RT-PCR扩增结果均为阴性。敏感性试验结果表明,该双重RT-PCR方法的检测下限为3 pg的H7N9病毒模板。利用该双重RT-PCR方法对30份临床样品进行H7N9病毒进行检测,与病毒分离方法进行对比,结果显示,两者的符合率为100%。结果表明建立的双重RT-PCR检测方法,具有特异、快速、敏感、准确的特点,可用于H7N9流感病毒的同时检测和鉴别诊断,为H7N9亚型流感的有效防控提供技术支撑。
译  名：
Development of a Duplex RT-PCR Method for Rapid Detection of H7N9 Subtype Influenza Virus
作  者：
Jiang Wenming;Li Jinping;Yu Meifang;Sun Wenbo;Wang Suchun;Peng Cheng;Hou Guangyu;Liu Shuo;Du Xiang;Yu Jianmin;Chen Jiming;China Animal Health and Epidemiology Center;Institute of Animal Husbandry and Veterinary,Shandong Academy of Agricultural Sciences;
关键词：
influenza virus;;H7N9;;duplex RT-PCR
摘  要：
To establish a simple and rapid method for detection of H7N9 subtype influenza virus,two pairs of specific primers were designed according to the conserved regions on the HA sequences of H7 subtype influenza virus and the NA sequences of N9 subtype influenza virus in Gen Bank. A duplex reverse transcription-polymerase chain reaction(RT-PCR) method was developed for simultaneously detection of H7 subtype and N9 subtype influenza virus based on the single RT-PCR for H7 and N9 subtypes and on further optimization of duplex RT-PCR conditions. Amplification of H7N9 subtype influenza virus nucleic acid template by this method showed that specific fragments of the 263 bp of HA gene and 458 bp of NA gene could be amplified,and other common subtype influenza viruses in this duplex RTPCR were negative. Sensitivity test results showed that the detection limit of the duplex RT-PCR method was 3 pg H7N9 virus templates. Compared with the results of virus isolation methods for testing30 clinical samples,the duplex RTPCR method showed that the coincidence rate was 100%. It was shown that the established duplex RT-PCR method was specific,rapid,sensitive,and accurate and it can be used for simultaneously detection of H7N9 influenza virus and differential diagnosis,and it provide technical support for effective prevention and control for H7N9 subtype influenza.

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