Establishment of a rapid real-time fluorescence-based recombinase-aided amplification method for detection of avian infectious bronchitis virus

Taoni Zhang; Jinwen Tang; Yu Zhang; Yinghao Jin; Zixue Lin; Jiming Chen; Jianni Huang; Meilan Mo

doi:10.1016/j.jviromet.2024.114955

Establishment of a rapid real-time fluorescence-based recombinase-aided amplification method for detection of avian infectious bronchitis virus

J Virol Methods. 2024 May 18:328:114955. doi: 10.1016/j.jviromet.2024.114955. Online ahead of print.

Authors

Taoni Zhang¹, Jinwen Tang¹, Yu Zhang¹, Yinghao Jin¹, Zixue Lin¹, Jiming Chen¹, Jianni Huang¹, Meilan Mo²

Affiliations

¹ College of Animal Science and Technology, Guangxi University, Nanning 530004, China.
² 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. Electronic address: momeilan@gxu.edu.cn.

PMID: 38768869
DOI: 10.1016/j.jviromet.2024.114955

Abstract

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.

Keywords: Infectious bronchitis virus; Rapid detection; Real-time recombinase-aided amplification.