H5N1虎源流感病毒荧光定量RT-PCR检测方法的建立及临床应用

根据本室分离获得的虎源H5N1流感病毒的HA基因序列测定结果,结合GenBank中报道的H5亚型禽流感病毒HA基因序列进行同源性比较分析,选择保守序列区作为扩增区域,利用Primer5·0引物设计软件和BLAST软件程序设计出特异性扩增引物。采用成本较低、不需要特异探针引物、优化周期短,能区分病毒变异株的SYBRGreenI随机掺入法建立定量PCR反应体系,并对反应条件进行优化。试验结果表明应用该方法对虎源H5流感病毒的检测具有高度的特异性,检测的灵敏度为101~102拷贝数。对20份病死老虎临床病料和24份人工感染的小鼠脏器病料用荧光定量RT-PCR方法、常规RT-PCR方法和病毒分离方法进行检测,荧光定量RT-PCR方法检测结果略高于常规RT-PCR方法,但与病毒分离方法结果相一致。这说明荧光定量RT-PCR方法可以对临床上H5N1虎源流感病毒检测提供参考。

Development and clinical application of fluorogenetic quantitative RT-PCR for detection of H5N1 tiger influenza A virus

To establish a fluorogenetic quantitative RT-PCR assay for detection of H5N1 tiger influenza A virus (TIV), the HA gene of TIV was aligned using the biologic software and the specific primers were designed in the conserved region of the HA gene by Primer 5.0. For SYBR Green I was cheaper and can be used to distinguish variant of virus, the SYBR Premix Ex TaqTM system was selected. The primers and the reactive condition were optimized to improve the sensitivity and specificity of the assay. The sensitivity of the assay was 10~1~10~2 copies. Twenty clinical samples from died tigers and 24 samples of mice infected with A/Tiger/Harbin/01/2003 (H5N1) were detected by the fluorogenetic quantitation RT-PCR, routine RT-PCR and virus isolation,recpectively. The sensitivity of the method of fluorogenetic quantitation RT-PCR was higher than routine RT-PCR, but similar with virus isolation. The result indicated that fluorogenetic quantitation RT-PCR may be used as a good reference in detection of H5N1 TIV.