H4、N2和所有亚型禽流感三重RT-PCR检测方法的建立

Development of a Triplex RT-PCR Assay for Detection of H4, N2 and all Subtypes Avian Influenza

关键词:  禽流感  H4亚型  N2亚型  三重RT-PCR

Key words:  H4 subtype AIV  N2 subtype AIV  Triplex RT-PCR  Method

摘要:

为建立一种同时检测H4、N2和所有亚型禽流感的方法，分别针对H4亚型禽流感病毒(AIV) HA基因、N2亚型AIV NA基因和所有亚型AIV M基因保守序列，设计筛选出3对特异性引物，优化引物之间的浓度，对三重反应体系进行特异性和敏感性验证，建立了H4、N2和所有亚型AIV三重RT-PCR检测方法，用该法对临床样品进行检测。建立的方法能特异性扩增H4、N2和所有亚型AIV，与其它禽病病原体不发生交叉反应；对H4、N2和所有亚型AIV至少能检测到6 pg/μL。在185份临床样品的检测中，4份H4，10份N2，19份AIV阳性。所建立的三重RT-PCR方法特异性强、灵敏度高，为快速检测H4、N2和所有亚型AIV提供了快速有效的方法。

Abstract:

To develop a simultaneous detection method for H4, N2 and all subtypes avian influenza, the conserved sequences of HA gene of H4 subtype avian influenza virus(AIV), NA gene of N2 subtype AIV, and the M gene of all subtypes AIV were targeted, respectively, and three pairs of specific primers were designed and screened out. The concentration between primers was optimized. The specificity and sensitivity of the triplex assay were validated. A triplex RT-PCR assay for detection of H4, N2 and all subtypes AIV was developed. Clinical samples were detected with developed method. The method amplified specifically H4, N2 and all subtypes AIV, and had no cross reaction with common avian pathogens, and at least can detect 6 pg/μL for H4, N2 and all subtypes AIV. Among 185 clinical samples, positive samples included H4 (4), N2 (10) and AIV (19). This developed triplex RT-PCR assay is specific and sensitive, and provides a rapid and effective method for detection of H4, N2 and all subtypes AIV.