Development Of A Reverse Transcription Recombinase Aid Amplification Assay For The Detection Of Respiratory Syncytial Virus

Objective: Respiratory syncytial virus(RSV)is a common viral pathogen that causes lower respiratory tract infections in infants and children globally.Recombinase aided Amplification(RAA)is a rapid isothermal nucleic acid detection technique with China own independent intellectual property rights.The detection of RAA amplification products mainly includes real-time fluorescent detection and end-point lateral flow dipstick(LFD).In this study,we attempt to develop two methods for rapid detection of respiratory syncytial virus.Of which one is a duplex reverse-transcription recombinase aided amplification assay containing an internal control(IC-rtRAA)in one single closed tube and the other is the LFD-rtRAA assay,which combines RAA assay with LFD.Then,we evaluated the analytical sensitivity,specificity and the clinical performance of the two methods.Methods: We download all of the RSV complete sequences from the NCBI.After that,we conducted multiple sequence alignment to locate highly conserved regions covering universal genotypes of RSV sequences for the design of universal primers and probes.1.Development of IC-rtRAA for RSV detection: According to the design principles of RAA primer and probe,we evaluated and designed the primers and probes in all the conserved regions,then screened the optimal primer combinations.Here,we firstly developed and evaluated universal singleplex-rtRAA assay for RSV.An IC-rtRAA assay with internal control was then established.2.Development of LFD-rtRAA for RSV detection: According to the design principles of RAA-LFD primer and probe,we designed and evaluated the primers and probes suitable for LFD-rtRAA assay,and the LFD-rtRAA assay was then established.The analytical sensitivity of the two methods was evaluated by the recombinant plasmid standard containing the target gene,and the analytical specificity was evaluated with other respiratory viruses.The clinical performance of the two assays was evaluated using 278 clinical specimens and compared to RT-PCR.Results:1.IC-rtRAA assayThe analytical sensitivity of IC-rtRAA assay for RSV at 95% probability by probit regression analysis was 5.0 copies per reaction,and no cross-reaction with other common respiratory viruses was observed.The IC-rtRAA assay was used to detect 278 clinical specimens and the results showed absolute consistency with RSV RT-qPCR analysis,demonstrating 100% diagnostic sensitivity and specificity.2.LFD-rtRAA assayThe analytical sensitivity of LFD-rtRAA assay for RSV was 10 copies per reaction,and no cross-reaction with other common respiratory viruses was observed.The LFD-rtRAA assay was used to detect 278 clinical specimens and the results showed absolute consistency with RSV IC-rtRAA assay and RT-qPCR assay,demonstrating 100% diagnostic sensitivity and specificity.Conclusion: The two RAA detection methods established in this study have the advantages of high sensitivity and simplicity and have great potential application in the rapid detection of respiratory syncytial virus and they provided new tools for rapid detection of human respiratory syncytial virus.The strategy we adopted in the designing of an internal control is of a valuable reference for the practical applications of RAA-based detection.The incorporation of internal control facilitates the clinical use of IC-rtRAA assay.LFD-rtRAA assay is more suitable for use in underdeveloped areas or rapid on-site testing.