背景:禽流感病毒(AIV)不仅给养禽业造成巨大的经济损失,但也威胁着人类健康。逆转录重组酶辅助扩增(RT-RAA)是一种新型的等温核酸扩增技术。本研究旨在提高H5、H7、H9亚型AIV的检测效率,及时发现疾病。本研究建立了RT-RAA-LFD和实时荧光RT-RAA(RF-RT-RAA)检测方法,分别将RT-RAA与侧向流动试纸(LFD)和外部探针相结合,同时根据RT-RAA的反应原理设计引物和探针。
结果:结果表明,RT-RAA-LFD可以在37°C时特异性扩增AIV的H5,H7和H9亚型,18分钟,39°C,20分钟,和38°C,18分钟,分别。所有三种亚型对RT-RAA-LFD的灵敏度为102拷贝/微升,比逆转录聚合酶链反应(RT-PCR)琼脂糖电泳方法高10~100倍。RF-RT-RAA可以在40°C特异性扩增AIV的H5、H7和H9亚型,20分钟,38°C,16分钟,39°C,17分钟,分别。所有三种亚型对RF-RT-RAA的灵敏度为101拷贝/微升,与实时荧光定量RT-PCR结果一致,比RT-PCR-琼脂糖电泳方法高100~1000倍。两种方法与RT-PCR-琼脂糖电泳检测临床样本的总符合率均高于95%。
结论:本实验成功建立了RT-RAA-LFD和RF-RT-RAA,快速反应,操作简单,特异性强,高灵敏度,良好的重复性,和稳定性。它们适用于禽流感的早期和快速诊断,对预防具有积极意义,控制疾病,和公共卫生安全。
BACKGROUND: Avian influenza virus (AIV) not only causes huge economic losses to the poultry industry, but also threatens human health. Reverse transcription recombinase-aided amplification (RT-RAA) is a novel isothermal nucleic acid amplification technology. This study aimed to improve the detection efficiency of H5, H7, and H9 subtypes of AIV and detect the disease in time. This study established RT-RAA-LFD and real-time fluorescence RT-RAA (RF-RT-RAA) detection methods, which combined RT-RAA with lateral flow dipstick (LFD) and exo probe respectively, while primers and probes were designed based on the reaction principle of RT-RAA.
RESULTS: The results showed that RT-RAA-LFD could specifically amplify H5, H7, and H9 subtypes of AIV at 37 °C, 18 min, 39 °C, 20 min, and 38 °C, 18 min, respectively. The sensitivity of all three subtypes for RT-RAA-LFD was 102 copies/µL, which was 10 ∼100 times higher than that of reverse transcription polymerase chain reaction (RT-PCR) agarose electrophoresis method. RF-RT-RAA could specifically amplify H5, H7, and H9 subtypes of AIV at 40 °C, 20 min, 38 °C, 16 min, and 39 °C, 17 min, respectively. The sensitivity of all three subtypes for RF-RT-RAA was 101 copies/µL, which was consistent with the results of real-time fluorescence quantification RT-PCR, and 100 ∼1000 times higher than that of RT-PCR-agarose electrophoresis method. The total coincidence rate of the two methods and RT-PCR-agarose electrophoresis in the detection of clinical samples was higher than 95%.
CONCLUSIONS: RT-RAA-LFD and RF-RT-RAA were successfully established in this experiment, with quick response, simple operation, strong specificity, high sensitivity, good repeatability, and stability. They are suitable for the early and rapid diagnosis of Avian influenza and they have positive significance for the prevention, control of the disease, and public health safety.