Development And Evaluation Of A Rapid Recombinase Polymerase Amplification Assay For The Detection Of Human Enterovirus 71

Background and objectiveHand,foot and mouth disease(HFMD)has been a major public health concern in the Asia-Pacific region.Young children and infants(? 5 years old)are among the most susceptible population.The main pathogens include coxsackievirus A16 and enterovirus 71(EV71).EV71 is a highly contagious virus and neurotrophic enterovirus.EV71 has caused a series of HFMD outbreaks and epidemics in the Asia-Pacific region and is the predominant pathogen accounting for severe cases and deaths in HFMD patients.Since EV71 was first reported,EV71-related HFMD epidemic has been on the rise in China.Indeed,EV71 has caused HFMD epidemics in China every year.HFMD caused by EV71-C4 has placed an extremely heavy burden on the economy and health and has been a major public health concern in China.According to previous reports and data,EV71 subgenotype C4(EV71-C4)has been the only reported EV71 subgenotype in China since 1998.Currently,no effective antiviral drugs against EV71 have been developed.Early diagnosis and treatment can control spreading of the disease.Thus,identifying a simple,rapid and accurate detection assay of EV71-C4 will have great significance to prevent severe HFMD cases,reduce mortality,and control spreading of the disease in China.Recombinase polymerase amplification(RPA),a novel isothermal amplification technology,is characterized by a fast reaction speed,simple operation process,and a high sensitivity and specificity.For this technology,complex equipment and a skilled operator are not required.Since the introduction of RPA technology,it has been widely applied in the detection of different types of pathogens,such as HIV,dengue virus,and noroviruses.So far,no RT-RPA assay has been applied to detect EV71.In this study,a RT-RPA assay was established and evaluated to determine EV71-C4.The purpose of this study was to establish a rapid and low-cost method for the detection of EV71-C4 in the field during HFMD outbreaks.MethodsThe strain SZ/HK08-6(GenBank accession No.GQ279370.1)was considered as a reference sequence.Primers and probes were designed using the conserved area of the VP1 gene.A RT-RPA assay was established to determine EV71-C4 and its sensitivity,specificity and clinical performance were evaluated.Serial dilutions of sample RNA were simultaneously detected by RT-RPA,RT-qPCR,and RT-PCR.The 95%detection limit of the three detection methods were calculated and compared.Control virus,including coxsackievirus group A serotypes(CV-A2,-4,-5,-6,-9,-10,and-16),coxsackievirus group B serotypes(CV-B2,and-4),enteric cytopathic human orphan(ECHO)virus serotype 9,and norovirus GIL 17,were applied for the specificity evaluation in the RT-RPA assay.The clinical significance of the EV71-C4 RT-RPA assay was evaluated from 178 suspicious HFMD stool samples and compared by RT-PCR and a commercial RT-qPCR diagnostic kit.In this study,RT-qPCR was used as the gold standard for diagnosing EV71.ResultsThe experimental results showed that the reaction time of the EV71-C4 RT-RPA assay at 40? was less than 10 min.The EV71-C4 RT-RPA assay was highly specific,and no reaction with the control virus and clinically negative samples were observed.Using the probit analysis,the 95%detection limit of RT-RPA,RT-qPCR,and RT-PCR were as follows:3.767(95%CI,3.489 to 4.283),2.026(95%CI,1.645 to 2.710),and 4.921(95%CI,4.628 to 5.382)log10 genomic copies(LGC)per reaction,respectively.In the evaluation of clinical performance,RT-qPCR was used as the gold standard for diagnosing EV71.The test results of 44 stool samples indicated that both the sensitivity and specificity of the RT-RPA assay were 100.0%,whereas the sensitivity and specificity of the RT-PCR were 90.0%(18/20)and 91.7%(22/24),respectively.Based on the assay results of another 134 stool samples,the sensitivity and specificity of the RT-RPA assay were 89.5%(68/76)and 100.0%(58/58),while the sensitivity and specificity of RT-PCR were 63.2%(48/76)and 89.7%(52/58),respectively.The clinical performance of the RT-RPA assay was not significantly different with the RT-qPCR and showed a better clinical performance compared to the RT-PCR assay.ConclusionIn this study,a simple,rapid,and accurate EV71-C4 RT-RPA approach was established.In the evaluation of clinical performance,the EV71-C4 RT-RPA assay showed highly specificity and sensitivity.Thus,EV71-C4 RT-RPA approach may become a promising alternative for the detection of EV71-C4 especially in underdeveloped areas and in the field of HFMD outbreaks.