College of Animal Science and Technology, Guangxi University, Nanning 530004, China.
College of Animal Science and Technology, Guangxi University, Nanning 530004, China; Guangxi Zhuang Autonomous Region Engineering Research Center of Veterinary Biologics, Nanning 530004, China; Guangxi Key Laboratory of Animal Breeding, Disease Control and Prevention, Nanning 530004, China; Guangxi Colleges and Universities Key Laboratory of Prevention and Control for Animal Disease, Nanning 530004, China.
J Virol Methods. 2024 Jul;328:114955. doi: 10.1016/j.jviromet.2024.114955. Epub 2024 May 18.
Infectious bronchitis (IB) is an acute, highly contagious contact respiratory disease of chickens caused by infectious bronchitis virus (IBV). IBV is very prone to mutation, which brings great difficulties to the prevention and control of the disease. Therefore, there is a pressing need for a method that is fast, sensitive, specific, and convenient for detecting IBV. In this study, a real-time fluorescence-based recombinase-aided amplification (RF-RAA) method was established. Primers and probe were designed based on the conserved regions of the IBV M gene and the reaction concentrations were optimized, then the specificity, sensitivity, and reproducibility of this assay were tested. The results showed that the RF-RAA method could be completed at 39℃ within 20 min, during which the results could be interpreted visually in real-time. The RF-RAA method had good specificity, no cross-reaction with common poultry pathogens, and it detected a minimum concentration of template of 2 copies/μL for IBV. Besides, its reproducibility was stable. A total of 144 clinical samples were tested by RF-RAA and real-time quantitative PCR (qPCR), 132 samples of which were positive and 12 samples were negative, and the coincidence rate of the two methods was 100 %. In conclusion, the developed RF-RAA detection method is rapid, specific, sensitive, reproducible, and convenient, which can be utilized for laboratory detection and clinical diagnosis of IBV.
传染性支气管炎（IB）是一种由传染性支气管炎病毒（IBV）引起的鸡急性、高度接触传染性呼吸道疾病。IBV 非常容易发生突变，这给疾病的防控带来了很大的困难。因此，迫切需要一种快速、灵敏、特异、方便的 IBV 检测方法。本研究建立了一种基于实时荧光重组酶辅助扩增（RF-RAA）的检测方法。根据 IBV M 基因的保守区设计引物和探针，并优化反应浓度，然后测试该方法的特异性、敏感性和重现性。结果表明，RF-RAA 法可在 39℃下 20 min 内完成，实时可进行肉眼判读。该方法具有良好的特异性，与常见家禽病原体无交叉反应，对 IBV 的最小模板检测浓度为 2 拷贝/μL。此外，其重现性稳定。用 RF-RAA 和实时荧光定量 PCR（qPCR）法检测了 144 份临床样本，其中 132 份为阳性，12 份为阴性，两种方法的符合率为 100%。综上所述，所建立的 RF-RAA 检测方法具有快速、特异、灵敏、重现性好、方便等特点，可用于 IBV 的实验室检测和临床诊断。
