Study On Detection Of Coxsackievirus A6by Reverse Transcription-loop-mediated Isothermal Amplification Assay

Aims: Coxsackievirus A6(CVA6) is a major pathogen of Hand-Foot-Mouth disease(HFMD). HFMD caused by CVA6is significantly associated with developing nailabnormalities. In this study, a one-step, single-tube reverse transcriptionloop-mediated isothermal amplification (RT-LAMP) assay with high specificity,sensitivity, efficiency and rapidity was developed and validated for the detection ofcoxsackievirus A6(CVA6).Methods: Stool specimens were collected from HFMD patients and subjected toone-step reverse transcription polymerase chain reaction (RT-PCR) and sequenceanalysis. These stool specimens served as templates in our study. The CVA6capsidprotein VP1sequences were available in the GenBank database and had been alignedto identify conserved regions. A set of primers were design to target the conservedregions of CVA6VP1gene. RT-LAMP assay was developed to amplify VP1gene inthe presence of the primer set and Bst DNA polymerase, which had strongstranddisplacement activity. The RT-LAMP reaction conditions were optimized byvarying the amplification temperature, concentration of primers, Mg2+and dNTP. The amplified products could be visualized directly with naked eyes or electrophoresed inagarose gel. The sensitivity and specificity of CVA6-RT-LAMP were tested. Meltingcurve analysis was done on an Applied Biosystems7500Real-Time PCR System.Results: The specific positive amplification was only observed in CVA6samples,while no amplification was detected in other tested enteroviruses. The detection limitof CVA6-RT-LAMP assay exceeded that of conventional CVA6-RT-PCR, by at least100times.40min were required for RT-LAMP to reach a plateau phase. Only onemelting peak was observed, indicating the specificity in other way.Conclusions: With the advantage of its rapidity, high efficiency, sensitivity,specificity and ease of use and result judging, CVA6-RT-LAMP could be a routinediagnostic tool for CVA6infection, especially when sophisticated and expensivelaboratory equipments are hardly affordable.