三重PCR快速检测H7N9亚型流感病毒方法的建立

摘要

To develop a simple and rapid method for detection of influenza A virus subtype H7N9,three pairs of specific primers were designed according to the conserved regions on the HA sequences of influenza A virus subtype H7,the NA sequences of influenza A virus subtype N9 and the M sequences of all influenza A viruses in GenBank,respectively. A triple polymerase chain reaction (PCR) method was developed for detection of influenza A virus subtype H7N9 through optimization of triple PCR conditions,specificity test and sensitivity test. In this triple PCR,three specific fragments of the 304 bp of HA gene,the 160 bp of NA gene and the 458 bp of M gene could be amplified on the templates as influenza A virus subtype H7N9,but just one specific fragment of the 458 bp of M gene was amplifiedon the templates asthe other influenza A viruses,andthe triple PCR was negative for other viruses. Sensitivity test results showed that the detection limitof the triple PCR method was 0.6 pg of templates as H7N9 virus. Four hundred and seventy clinical samples were detected in the triple PCR,no influenza A virus subtype H7N9 was found. It was shown that the triple PCR method was rapid,sensitiveand specific,and it provided technical support for clinical test,effective prevention and control for influenza A virus subtype H7N9.%为建立简便快速检测 H7N9 亚型流感病毒的方法,根椐 GenBank 中 H7亚型流感病毒的HA基因、N9亚型流感病毒的 NA 基因和所有亚型流感病毒M基因的保守序列,分别设计了3对特异性引物,优化反应条件,并通过特异性试验、敏感性试验,建立了H7N9亚型流感病毒三重PCR检测方法.利用该法对H7N9亚型流感病毒进行扩增,可得到3条特异性条带(HA基因304 bp,NA基因160 bp,M基因458 bp),其他亚型流感病毒只出现1条特异性条带,大小为458 bp,对其他病原体的检测均无扩增条带.敏感性试验结果表明,该法最低能检测到0.6 pg H7N9 亚型流感病毒.用该法对470个样品进行检测,未发现H7N9 亚型流感病毒.该法具有快速、敏感和特异等优点,可为临床检测和疾控防控提供技术支持.