College of Veterinary Medicine, Hebei Agricultural University, Baoding, China.
Institute of Special Animal and Plant Sciences, Chinese Academy of Agricultural Sciences, Changchun, China.
BMC Vet Res. 2024 May 16;20(1):203. doi: 10.1186/s12917-024-04040-9.
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.
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 10 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 10 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%.
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.
禽流感病毒（AIV）不仅给家禽业造成巨大的经济损失，还威胁人类健康。逆转录重组酶辅助扩增（RT-RAA）是一种新型等温核酸扩增技术。本研究旨在提高对 H5、H7 和 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 与侧流试纸条（LFD）和外切探针结合，同时根据 RT-RAA 的反应原理设计引物和探针。
结果表明，RT-RAA-LFD 可在 37°C、18 分钟、39°C、20 分钟和 38°C、18 分钟特异性扩增 H5、H7 和 H9 亚型 AIV。三种亚型的 RT-RAA-LFD 灵敏度均为 10 拷贝/μL，比逆转录聚合酶链反应（RT-PCR）琼脂糖电泳法高 10100 倍。RF-RT-RAA 可在 40°C、20 分钟、38°C、16 分钟和 39°C、17 分钟特异性扩增 H5、H7 和 H9 亚型 AIV。三种亚型的 RF-RT-RAA 灵敏度均为 10 拷贝/μL，与实时荧光定量 RT-PCR 结果一致，比 RT-PCR-琼脂糖电泳法高 1001000 倍。两种方法与 RT-PCR-琼脂糖电泳法在临床样本检测中的总符合率均高于 95%。
本实验成功建立了 RT-RAA-LFD 和 RF-RT-RAA，具有反应迅速、操作简单、特异性强、灵敏度高、重复性好、稳定性强等特点。适用于禽流感的早期快速诊断，对疾病的防控和公共卫生安全具有积极意义。
