Establishment of a Real-Time Recombinase Polymerase Amplification Assay for the Detection of Avian Reovirus
Lei Ma, Hongfei Shi, Mingliang Zhang, Yuwei Song, Kunpeng Zhang, Feng Cong
Abstract
Chicken reovirus (CRV) infection results in multiple disease manifestations in chicken. A rapid detection method will contribute to early diagnosis and control of the virus infection. The recombinase polymerase amplification (RPA) technology is a nucleic acid amplification method which is experiencing rapid development. In present study, a real-time reverse transcription (RT)-RPA assay was developed for the detection of CRV. The limit of detection of the real-time RT-RPA was 102 copies/μL of CRV genomic RNA standard in 95% of cases. The RT-RPA assay also exhibited remarkable specificity. When the nucleic acids of CRV and other common avian pathogens were subjected the RT-RPA test, only CRV tested positive, all the other pathogens tested negative. Furthermore, the practicality of the RT-RPA assay in field was confirmed by testing 86 clinical samples. The clinical samples were also detected by qRT-PCR. The detection result by RT-RPA was 96.5% agreement with that of qRT-PCR. As a result of the simplicity and convenience of the assay with high sensitivity and specificity, the probe-based RT-RPA will be an alternative diagnostic assay for the detection of CRV in resource-limited settings.