Guangxi Key Laboratory of Veterinary Biotechnology, Guangxi Veterinary Research Institute, 51 You Ai North Road, Nanning, Guangxi, 530001, China.
Sci Rep. 2018 Apr 18;8(1):6183. doi: 10.1038/s41598-018-24620-8.
Recent studies have demonstrated that at least eight subtypes of avian influenza virus (AIV) can infect humans, including H1, H2, H3, H5, H6, H7, H9 and H10. A GeXP analyser-based multiplex reverse transcription (RT)-PCR (GeXP-multiplex RT-PCR) assay was developed in our recent studies to simultaneously detect these eight AIV subtypes using the haemagglutinin (HA) gene. The assay consists of chimeric primer-based PCR amplification with fluorescent labelling and capillary electrophoresis separation. RNA was extracted from chick embryo allantoic fluid or liquid cultures of viral isolates. In addition, RNA synthesised via in vitro transcription was used to determine the specificity and sensitivity of the assay. After selecting the primer pairs, their concentrations and GeXP-multiplex RT-PCR conditions were optimised. The established GeXP-multiplex RT-PCR assay can detect as few as 100 copies of premixed RNA templates. In the present study, 120 clinical specimens collected from domestic poultry at live bird markets and from wild birds were used to evaluate the performance of the assay. The GeXP-multiplex RT-PCR assay specificity was the same as that of conventional RT-PCR. Thus, the GeXP-multiplex RT-PCR assay is a rapid and relatively high-throughput method for detecting and identifying eight AIV subtypes that may infect humans.
最近的研究表明，至少有 8 种禽流感病毒（AIV）亚型可以感染人类，包括 H1、H2、H3、H5、H6、H7、H9 和 H10。我们最近的研究开发了一种基于 GeXP 分析器的多重逆转录（RT）-PCR（GeXP-多重 RT-PCR）检测方法，该方法使用血凝素（HA）基因同时检测这 8 种 AIV 亚型。该检测方法由基于嵌合引物的 PCR 扩增、荧光标记和毛细管电泳分离组成。从鸡胚尿囊液或病毒分离物的液体培养物中提取 RNA。此外，还使用体外转录合成的 RNA 来确定该检测方法的特异性和灵敏度。在选择引物对后，优化了其浓度和 GeXP-多重 RT-PCR 条件。建立的 GeXP-多重 RT-PCR 检测方法可检测到低至 100 个混合 RNA 模板的拷贝数。在本研究中，使用从活禽市场和野生鸟类收集的 120 份临床标本评估了该检测方法的性能。GeXP-多重 RT-PCR 检测方法的特异性与常规 RT-PCR 相同。因此，GeXP-多重 RT-PCR 检测方法是一种快速且高通量的方法，可用于检测和鉴定可能感染人类的 8 种 AIV 亚型。
