多重荧光定量RT-PCR检测高致病性H5N6禽流感病毒

Detection of a highly pathogenic avian influenza H5N6 virus by the multiplex real-time RT-PCR assay

目的建立一种快速检测流感病毒A型(FluA)及高致病性禽流感(HPAI)H5N6病毒感染的荧光定量RT-PCR法。方法以人细胞核糖核酸酶P(RP)基因作为临床样本的内参基因,用Primer Express 3.0软件设计PCR特异性引物和探针,建立一种检测FluA及HPAI-H5N6病毒的一步法多重荧光定量RT-PCR方法,评估其灵敏度、特异性,并与上海之江公司提供的试剂及测序法进行比较。结果建立的多重荧光定量RT-PCR法检测FluA、H5、N6及RP质粒标准品的灵敏度均达102copies/m L。各对引物和相应探针仅检测出相应的病毒,在这些病毒和常见病原分析中未发现存在交叉反应,特异性达100%。用该法检测135例临床标本,结果显示FluA为23例,未发现HPAI-H5N6病毒的感染者;与上海之江公司提供的试剂的检测结果及基因测序结果一致。结论建立的一步法多重荧光定量RT-PCR法可用于FluA及HPAI-H5N6病毒感染患者的早期诊断。

Objective To rapidly detect the infections of influenza A virus（ FluA） and highly pathogenic avian influenza（ HPAI）H5N6 virus. Methods The specific primers and probes for FluA and HPAI-H5N6 virus were designed by the Primer Express 3. 0software,and human RNase P（ RP） gene was selected as the internal control. Then,a one-step multiplex real-time RT-PCR assay was established for detecting the FluA and HPAI-H5N6 virus in clinical specimens,and its sensitivity and specificity were evaluated. The regents from Shanghai ZJ Bio-Tech Co.,Ltd,and the sequencing methods was compared with this method. Results The sensitivity of the established multiplex real-time RT-PCR assay for detecting FluA,H5,N6 and RP genes reached to 102 copies / m L. Each pair of primers and probes only detected the corresponding virus. No cross-reaction was observed between these viruses and common respiratory pathogens,and the specificity of the established assay reached to 100%. A total of 135 clinical specimens were detected by the established assay,and the results showed that 23 were detected FluA,and no HPAI-H5N6 virus was found. The results detected by the established assay were completely consistent with those by the regents from Shanghai ZJ Bio-Tech Co.,Ltd,and the sequencing results of5 FluA-positive speciemens also coincided with the target gene. Conclusion The established multiplex real-time RT-PCR assay may be applied to the early diagnosis of the patients with FluA and / or HPAI-H5N6 virus infection.