双重荧光实时RT-PCR技术鉴定H9N2亚型禽流感病毒

Detection of avian influenza A subtype H9N2 by duplex real-time reverse transcription polymerase chain reaction

采用双重荧光实时RT-PCR技术,建立一种简便易行的H9N2亚型禽流感病毒的快速鉴定方法。通过比对GenBank甲型禽流感病毒H9N2亚型的HA和NA基因序列,设计特异性针对HA和NA的引物,分别选用FAM和JOE两种不同荧光标记探针,构建双重实时荧光PCR一步法反应体系,同时检测样本中的HA和NA基因。结果显示:扩增曲线和特异性实验结果显示,该体系具有很好的扩增效率,且仅特异性识别H9N2亚型AIV的HA、NA基因,与相关病毒或其他亚型无交叉反应。采用重组质粒pMD19-T构建HA、NA阳性质粒,10倍梯度稀释液为模板,进行荧光定量PCR。结果证实,本研究所建立的双重荧光定量RT-PCR体系敏感性能达到10个RNA拷贝数。采用本方法检测60份感染动物样品及60份环境样品,与传统PCR方法相比,检测敏感性提高了100倍;与病毒分离鉴定方法比较,二者的鉴定结果完全吻合。该方法具有特异性强、灵敏度高、快速易操作等优点,是H9N2亚型禽流感病毒鉴定的有效方法。

The objective of the present study was to develop a duplex real-time chain reaction （rRT-PCR） assay for the simultaneous detection of both hemaggh reverse transcription polymerase ntinin （HA） and neuraminidase （NA） genes of H9N2 avian influenza viruses. In order to design primers and probes for the detection of HA and NA genes of avian influenza virus subtype H9N2, current available HA and NA gene sequence of avian influenza virus subtype H9N2 from GenBank were aligned, and two different fluorescein （ FAM and JOE） were used to label HA and NA probe, respectively. A one-step duplex Taq Man rRT-PCR assay, which could detect both HA and NA genes of the avian influenza virus subtype H9N2 simultaneously was then established. The amplification curve showed that the duplex rRT-PCR assay had a very good amplification efficiency. The that the duplex rRT-PCR assay could identify HA and NA gene of the avian influenza specificity detection showed virus subtype H9N2 specifically, with no cross reactivity being observed with other influenza virus subtypes or respiratory sensitivity of the duplex rRT-PCR assay was determined to be 10 RNA copies per reaction for genes. Sixty H9N2-infected mice and sixty environmental specimens were tested and compared tract viruses. The both HA and NA with conventional PCR. the sensitivity of the duplex rRT-PCR was 100-fold higher than conventional PCR. The results of the duplex rRT-PCR were completely consistent with those of virus isolation. It was concluded that the duplex rRT-PCR assay established in this study was a highly specific and sensitive method that could be used for the avian influenza virus subtype H9N2.