鸡新城疫病毒、H9亚型禽流感病毒和禽肺炎病毒三重RT-PCR检测方法的建立

Development of a Rapid and Sensitive Test for Identification of New castle disease,H9 subtype Avian Influenza virus and Avian pneumovirus by Triplex RT-PCR

本研究建立三重RT-PCR快速检测鉴别鸡新城疫病毒(NDV)、H9亚型禽流感病毒(AIV)和禽肺炎病毒(APV)的方法。根据Gen Bank中H9亚型AIV HA基因和鸡NDV以及APV F基因的保守序列分别设计合成了三对特异性扩增引物,通过优化反应条件建立了三重RT-PCR检测鉴别鸡NDV、H9亚型AIV和APV的方法。对该方法进行特异性、敏感性和临床样品检测。特异性试验结果显示,所有参试的NDV、H9亚型AIV、APV分别扩增出大小为247 bp、569 bp和424 bp的片段,而对照的其他毒株不扩增;敏感性试验结果表明,本试验建立的方法能检测到10 pg的各自RNA模板。检测临床样品132份,NDV阳性26份,H9亚型AIV阳性6份,APV阳性2份,结果与病毒分离结果一致。PCR阳性产物测序结果与各自毒株序列100%同源。表明本试验建立的三重RT-PCR检测鉴别鸡NDV、H9亚型AIV和APV方法具有特异、敏感、快速的特点,可用于临床快速、准确的鉴别检测。

A rapid and specified method for detection of New castle disease virus（NDV）,H9 subtype Avian Influenza virus（AIV H9） and Avian pneumovirus（APV） by triplex reverse transcriptase polymerase chain reaction（RT-PCR） was developed.Three pair primers were designed and synthesized based on HA gene of NDV and AIV H9,and the Fusion gene of APV.Triplex RT-PCR was set to detect NDV,AIV H9 and APV with three pair primers.247 bp in length of NDV,569 bp in length of AIV H9,and 424 bp in length of APV were amplified.Specified test results showed that all test strains from NDV,AIV H9,and APV were positive.Other pathogen strains as controls were negative.This Triplex RT-PCR using three pair primers was able to detect at least 10 pg template of NDV,AIV H9,and APV.For detection clinical samples,26 of 132 samples were positive for NDV,6 of 132 samples were positive for AIV H9,and 2 of 132 samples were positive for APV.Homology analysis showed that it was 100% homology with NDV,AIV H9,and APV.A rapid and specified method was developed to detect NDV,AIV H9,and APV.