Development Of A Reverse Transcription Recombinase-aid Amplification Assay For The Detection Of Coxsackievirus A10 And Coxsackievirus A6 Virus

Objectve: To develop RT-RAA assays for the detection of CVA10 and CVA6 respectively and the Real-time RT-RAA and RT-RAA-LFD were adopted to detect the amplification products of CVA10 and CVA6.Methods: All the target sequences available were obtained from the National Center for Biotechnology Information database(NCBI).Conserved regions of the sequences validated by BioEdit Sequence Alignment Editor were used to design primers and probes.Primers candidates were further validated using Primer-Blast.According to the different principles of Real-time RT-RAA and RT-RAA-LFD,the primers and probes were subjected to different modifications.The analytical specificity and sensitivity of the RT-RAA assays were evaluated by recombinant plasmids or control virus strains.And 455 clinical samples collected were detected by the RT-RAA assay of CVA10 and CVA 6.The performance of the RT-RAA assays were compared to those obtained from the commercial RT-qPCR assays of CVA10 and CVA6 respectively.A probit analysis for the detection limit of RT-RAA was done at a 95% probability level.The Kappa and p values of RT-qPCR and RT-RAA were calculated.The statistical analyses were all operated by SPSS 21.0(IBM,USA).Results:1.Real-time RT-RAA assay for CVA10The analytical sensitivities of Real-time RT-RAA assay for CVA10 at 95% probability by probit regression analysis were 35 copy /?l and with 100% specificity.Compared with commercial RT-qPCR assay on the testing of 455 fecal specimens,the kappa value of the Real-time RT-RAA assay for CVA10 was 0.920(P<0.001).2.Real-time RT-RAA assay for CVA6The analytical sensitivities of Real-time RT-RAA assay for CVA6 at 95% probability by probit regression analysis were 38 copy /?l and with 100% specificity.Compared with commercial RT-qPCR assay on the testing of 455 fecal specimens,the kappa value of the Real-time RT-RAA assay for CVA6 was 0.952(P<0.001).3.RT-RAA-LFD assay for CVA10The analytical sensitivities of RT-RAA-LFD assay for CVA10 at 95% probability by probit regression analysis were 38 copy /?l and with 100% specificity.Compared with commercial RT-qPCR assay on the testing of 455 fecal specimens,the kappa value of the RT-RAA-LFD assay for CVA10 was 0.948(P<0.001).4.RT-RAA-LFD assay for CVA6The analytical sensitivities of RT-RAA-LFD assay for CVA6 at 95% probability by probit regression analysis were 38 copy /?l and with 100% specificity.Compared with commercial RT-qPCR assay on the testing of 455 fecal specimens,the kappa value of the RT-RAA-LFD assay for CVA6 was 0.952(P<0.001).Conclision: The proposed RT-RAA assays are very valuable tools for the detection of CVA10 and CVA6 and have the potential of route use in the resource-limited settings.The Real-time RT-RAA and RT-RAA-LFD assays are two different methods for the detections of the amplification products and can be applied according different situations.