Development Of A Rapid Detection Assay For Coxsackievirus A6 Based On Recombinase Polymerase Amplification Technique

Background and purpose:Hand foot mouth disease(HFMD)is an infectious viral disease,which has led to several outbreak all over the world and become more predominant in Asia-Pacific region.HFMD mainly infect infant and children under 5,its worldwide epidemic threats the infant and children's health and life.HFMD is caused by a variety of virus,mainly including enterovirus 71(EV71),coxsakievirus A4,A6,A10,B3,B5(CA4,CA6,CA10,CB3,CB5)and echovirus 30 et al.In recent years,the pathogenic spectrum of agents which could cause HFMD has changed.CA6 has become a more and more important pathogenic agent,which caused several outbreak worldwide.CA6 could lead to severe HFMD cases and even dead cases.CA6 always cause atypical HFMD,which could be misdiagnosed.At present,there is no effective drugs for HFMD,monovalent and bivalent vaccines for EV71 and CA16 is being evaluated in clinical trials.But progress for multivalent vaccine containing multiple viruses is poor.The measurements to prevent and control HFMD mainly depend on cutting transmission and symptomatic treatment to prevent complications.It is beneficial for prevent and control of HFMD to enhance worldwide epidemiological and laboratory etiological surveillance for anticipating new outbreak.Recombinase polymerase amplification(RPA)is a new technique for nucleic acid amplification,which was commercialized by Twist DX company.RPA do not depend on sophisticated equipment,it could work well at 37-42 degree centigrade.And there are a variety of probes to detect the amplicon,which could largely improve specificity.Meanwhile,the amplicon could be sequenced,which makes you can check the result.Therefore,RPA is suitable for diagnose of pathogenic agents in point of care.Pathogenic agents could be detected by RPA in 20min,which makes it the most quick assay for nucleic acid detection.Methods:The genetic sequences of VP 1 of CA6 was downloaded from GenBank database,which were subsequently blasted for searching conserved region.The specific primers were designed based on conserved region,and specific probe was designed after primer screening.Subsequently,real time RT-RPA for detection of CA6 was established.The cDNA containing target fragment was cloned into vector for transcript in vitro.The concentration of RNA was calculated and transformed to copies/?1.The RNA panel was done by ten times step dilution with each 8 duplicates for every concentration.The RNA panel was detected by commercial real time PCR kit for detection of CA6 and real time RT-RPA for detection CA6,respectively.Probit analysis was performed to calculate 95%detection limit.The CA6 reference strains and control strains related to HFMD were detected by real time RT-RPA to calculate it's specificity.To determine the clinical performance,EV71 and CA16 negative stool samples from 234 HFMD cases was detected using commercial real time PCR kit for detection of CA6 and real time RT-RPA for detection CA6,respectively,and the Kappa was calculated.Results:The time for RT-RPA assay was 20min.Only CA6 reference strains were amplified to generate amplification curves and there were no any were amplified,the specificity was 100%.The 95%detection limit of real time RT-RPA was 231 copies/reaction,with no difference to commercial real time PCR kit.For detecting clinical samples,there was no difference between real time RT-RPA and commercial real time PCR kit,Kappa was 0.93(p<0.001),suggesting high consistence between them.Conclusion:The real time RT-RPA for detection CA6 established in this study has high specificity and sensitivity,short time for detection,therefore would be helpful for surveillance and diagnosis of HFMD.