H3亚型禽流感病毒巢式PCR检测方法的建立

为建立一种H3亚型禽流感病毒(AIV)的检测方法,本研究针对H3亚型AIV HA基因保守序列,设计并筛选出2对特异性引物,通过优化反应条件,建立了H3亚型AIV巢式PCR检测方法。对该法进行特异性和敏感性检验,并用该法对96份临床样品进行检测。特异性试验结果表明该法只能检测到H3亚型AIV,对其他常见禽病病原体不扩增;敏感性试验结果表明该巢式PCR对H3亚型AIV检测下限为1×10³拷贝/μL,灵敏度比常规PCR高100倍;96份临床样品检测结果与病毒分离结果一致。本研究所建立的巢式PCR为H3亚型AIV的诊断提供了一种准确、有效的检测方法。

Establishment of Nested PCR for Diagnosis of H3 Subtype Avian Influenza Virus

In order to establish a method to detect H3 subtype avian influenza virus (AIV), two pairs of specific primers were designed and selected according to the conserved sequences of the hemagglutinin (HA) genes of H3 subtype AIV, the reaction conditions were optimized to establish the nested PCR.The specificity and sensitivity of assays were evaluated, and 96 clinical samples were detected by nested PCR. The results showed that the nested PCR was only specific to H3 subtype AIV, without amplification of other viruses; the detection limit of nested PCR was 1×10³ copies/μL which was 100 fold higher than the routine PCR; the detection of clinical samples were consistent with the viral isolation completely. This study provided a accurate and effective method for the detection of H3 subtype AIV.