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根据GenBank中H9亚型禽流感病毒HA基因和禽肺病毒F基因的保守序列,通过Blast进行比对,在各自保守的基因序列中分别设计了两对特异性扩增引物,引物扩增的片段大小分别为569bp和424bp,应用这两对引物建立了H9亚型禽流感病毒和禽肺病毒的二重RT-PCR检测方法,对建立的方法进行了特异性试验、敏感性试验和临床样品验证。对检测为阳性的样品进行克隆和序列分析,以验证本方法的准确性。特异性试验结果表明,所有参试的H9亚型禽流感病毒都能扩增出大小为569bp的片段,参试的禽肺病毒毒株扩增出424bp的片段,没有其他条带出现,而对照的参试毒株在569bp和424bp处没有任何条带;敏感性试验结果表明,本试验建立的方法能检测到10pg的H9亚型禽流感病毒和禽肺病毒RNA模板。应用本试验建立的方法对广西采集的病鸡样品132份进行检测,H9亚型禽流感病毒阳性的样品有6份,禽肺病毒阳性的样品2份;随机各挑取2份H9亚型禽流感病毒和禽肺病毒阳性PCR产物进行克隆和序列分析,经比对分析,2份H9亚型禽流感病毒阳性PCR产物与H9亚型禽流感病毒(No.JF715045.1)100%同源,2份禽肺病毒阳性PCR产物与禽肺病毒株(No.AF187154)100%同源。本试验建立的H9亚型禽流感病毒和禽肺病毒二重RT-PCR检测方法具有特异、敏感、快速的特点,可以用于临床快速准确检测H9亚型禽流感病毒和禽肺病毒。
According to the conserved sequence of HA gene of H9 subtype avian influenza virus and F gene of avian pneumovirus in GenBank, two pairs of specific amplification primers were designed respectively by Blast, Size of 569bp and 424bp respectively. Two pairs of primers were used to establish a double RT-PCR method to detect H9 subtype avian influenza virus and avian pneumovirus. The established methods were tested by specificity, sensitivity and clinical samples . Samples that tested positive were cloned and sequenced to verify the accuracy of the method. Specificity test results showed that all the tested H9 subtype avian influenza viruses could amplify a 569 bp fragment, and the tested avian Pneumovirus strain amplified a 424 bp fragment with no other bands, whereas the control Of the tested strains did not have any band at 569bp and 424bp. The results of the sensitivity test showed that the method established in this experiment can detect 10pg of H9 subtype avian influenza virus and avian influenza virus RNA template. 132 samples of sick chickens collected in Guangxi were tested by the method established in this experiment. There were 6 samples positive for H9 subtype avian influenza virus and 2 samples positive for avian Pneumovirus. Two samples of H9 subtype avian were randomly selected Influenza virus and avian Pneumovirus positive PCR products were cloned and sequenced. After comparison and analysis, two H9 subtype avian influenza virus positive PCR products were 100% homologous with H9 subtype avian influenza virus (No. JF715045.1) Two avian pneumovirus positive PCR products were 100% homologous to the avian pneumovirus strain (No. AF187154). The double RT-PCR detection method of H9 subtype of avian influenza virus and avian pneumovirus established in this study is specific, sensitive and rapid and can be used for clinical rapid and accurate detection of H9 subtype avian influenza virus and avian pneumovirus.