摘要
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。
A rapid and specific method for detection of avian influenza virus H9 subtype （AIV H9） and avian pneumovirus（APV） by duplex RT-PCR was developed. Two pairs of primers were designed and synthesized base on HA gene of AIV H9 and the F gene of APV after comparisons by Blast. Duplex RT-PCR was set to detect AIV H9 and APV with two pair of primers,569 bp in length of AIV H9,424 bp in length of APV were amplified. Specificity test results showed that all test strains from AI H9, and APV were positive. Other pathogen strains as contrasts were negative. This duplex RT-PCR was able to detect at least 10 pg template each of AI H9 and APV. 132 clinical samples collected from different chicken farms were detected by this RT-PCR. 6 of 132 samples were positive for AI H9,2 of 132 samples were positive for APV. 2 AIV H9 positive samples and 2 APV positive samples were used for PCR amplification and sequencing. Homology analysis showed 100% homology with AIV H9 （No. JF715045.1）and APV（No. AF187154）. A rapid and specific method was set to detect AIV H9 and APV.
出处
《动物医学进展》
北大核心
2016年第12期7-12,共6页
Progress In Veterinary Medicine
基金
广西科技厅专项(桂科AD16380009,桂科合14123001-8,桂科重14121003-4-2,桂科专项16-1)
广西自然科学基金项目(2013GXNSFDA019015)
国家万人计划领军人才专项(2016-37)
