為建立可以區(qū)分H7N9流感病毒強毒株和弱毒株的方法，通過比對GenBank和GISAID中H7N9流感病毒的HA基因序列以及本實驗室保存的病毒序列，設(shè)計1對特異性引物和2條探針，建立了區(qū)分H7N9流感病毒強弱毒株的實時熒光RT-PCR方法，同時對該方法的反應(yīng)體系和反應(yīng)參數(shù)進(jìn)行優(yōu)化，并開展特異性和敏感性試驗以及臨床應(yīng)用試驗。特異性試驗顯示，該方法具有良好的特異性，對其他常見亞型的禽流感病毒和其他禽病病毒檢測均為陰性。敏感性試驗顯示，該方法對H7N9流感強毒和弱毒HA基因的檢測下限分別為0.004 fg 和0.1 fg RNA模板，高于兩種商品化試劑盒的靈敏度。利用該方法對40株經(jīng)實驗室分離鑒定的H7N9流感病毒進(jìn)行對比驗證，發(fā)現(xiàn)檢出率和符合率均為100%。結(jié)果表明，該方法具有特異、敏感、準(zhǔn)確的優(yōu)點，可用于H7N9流感病毒的快速檢測，同時還可區(qū)分強弱毒株，對H7N9流感病毒，尤其是高致病性流感病毒的早期診斷和有效防控具有重要意義。
Development of a Real-time RT-PCR Method
for Identification of Virulent and Avirulent Strains of H7N9Influenza Virus
In order to develop a method for identifying virulent andavirulent strains of H7N9 influenza virus，a pair ofspecific primers and probes were designed by comparing the HA gene sequences ofH7N9 influenza virus in Genbank and GISAID and the sequence of which wasisolated and sequenced by our laboratory，a real-timeRT-PCR method was developed，meantime，the reaction system and parameters of the method were optimized，and the specificity and sensitivity as well as its clinicalapplication were tested. The specificity test showed that，the development method didn't react with other common subtypes ofavian influenza virus and other avian virus，showing agood specificity. Sensitivity test showed that，by theRT-PCR method，the lower detection limits of HA genes ofvirulent and attenuated H7N9 strains were 0.004 fg and 0.1 fg respectively，which were all lower than the detection limits of the two kinds ofcommercial kits. By comparing and verifying 40 strains of H7N9 influenza virusisolated and identified by the laboratory，it was foundthat both the detection rate and coincidence rate were 100%. Therefore, it wasconcluded that the method could be used for rapid detection of H7N9 virus andidentifying the virulent and avirulent strains due to its advantages of goodspecificity，sensitivity and high accuracy，which was of great significance for early diagnosis and effectiveprevention and control of H7N9 virus，especially thehighly pathogenic virus.
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