Establishment of a Real-Time Recombinase Polymerase Amplification Assay for the Detection of Avian Reovirus

Abstract

Avian reovirus (ARV) 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 ARV. The limit of detection of the real-time RT-RPA was 10 2 copies/µL of ARV 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 to the RT-RPA test, only ARV 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 ARV in resource-limited settings.

Real-time detection of Seneca Valley virus by one-tube RPA-CRISPR/Cas12a assay

“…In the last 10 years, the short reaction time and high sensitivity of the RPA method have promoted its rapid development ( Li et al., 2018 ; Tan et al., 2022 ). To date, more than one hundred detection methods based on RPA technology have been established for the diagnosis of various kinds of infectious pathogens ( Ma et al., 2019 ; Ma et al., 2020 ; Ma L. et al., 2022 ; Zhu et al., 2022 ). The RPA assay is currently the fastest among molecular detection methods.…”

The Establishment of a Recombinant Polymerase Amplification Technique for the Detection of Mouse Poxvirus

“…These allow for easy transportation and long-term storage at room temperature [29] .Another advantage of isothermal assays is that RPA assays can withstand a wide range of reaction temperatures and do not require precise control of reaction temperatures [30]. Previous studies have shown that RPA retains reliable functionality between 31°C and 43°C [31] and even between 30°C and 45°C [32,33]. The technique can accomplish exponentially ampli ed target sequences in 30 minutes at relatively low temperatures, making it suitable for use in resource-poor environments with minimal training and equipment.…”

Visual detection of porcine epidemic diarrhea virus by recombinase polymerase amplification combined with lateral flow dipstrip

“…Therefore, many diagnostic methods based on RPA technology have been developed to detect various human and animal pathogens. Generally, a RPA product is detected by a fluorescence detector or a LFD [ 18 , 27 – 30 ]. Real-time detection of PEDV by the RT-RPA assay has been reported [ 31 ].…”