Establishment and Application Prospect of Reverse Transcriptase Recombinase-Aided Amplification Assay for Subgroup C Avian Metapneumovirus

Simple Summary The emergence of aMPV-C will have a detrimental impact on egg quality, elevate chicken mortality rates, and severely impede the progress of the chicken industry. Hence, there is an urgent need to establish a rapid and convenient method for aMPV-C detection. Traditional approaches such as PCR, real-time PCR, ELISA, and virus neutralization are associated with expensive equipment requirements and intricate operational procedures that impose certain limitations. In this study, we have developed an RT-RAA method utilizing fluorescent probe technology for isothermal nucleic acid amplification. This novel approach enables the detection of aMPV-C within just 30 min at 41 degrees C while exhibiting exceptional sensitivity and specificity.Abstract Among broilers, the main pathogen that leads to swollen head syndrome (SHS) is the subgroup C avian metapneumovirus (aMPV-C). The aMPV-C infection can lead to an upsurge in the rate of soft-shell eggs, resulting in reduced egg production and seriously affecting the economy of the livestock industry. Therefore, a rapid method for aMPV-C detection needs to be invented. According to the N gene of aMPV-C, we designed the specific probe and primer and created a reverse transcription recombinase-aided amplification assay (RT-RAA) for the detection of aMPV-C. aMPV-C could be detected quickly and specifically by this method at 41 degrees C for 30 min. The sensitivity assay inferred that the minimum detection threshold of RT-RAA was 3.38 x 101 copies/mu L. A specificity assay showed that the RT-RAA method did not cross-react with other subgroups (aMPV-A, aMPV-B, aMPV-D) or other viruses (H9N2, NDV, IBV, IBDV). Forty samples of known clinical background were tested by RT-RAA and RT-qPCR. The two approaches had a 100% correlation rate. In conclusion, this research successfully created an RT-RAA assay for aMPV-C.