A Reverse Transcription-Polymerase Spiral Reaction (RT-PSR)-Based Rapid Coxsackievirus A16 Detection Method and Its Application in the Clinical Diagnosis of Hand, Foot, and Mouth Disease

Abstract

Hand, foot, and mouth disease (HFMD) is a common viral illness affecting infants and children that is usually caused by Coxsackievirus A16 (CVA-16). To diagnose HFMD, we developed a method for rapid detection of CVA-16 based on reverse transcriptionpolymerase spiral reaction (RT-PSR). We used two pairs of primers that specifically recognize the conserved sequences of VP1 coding region of CVA-16, and template RNA was reverse transcribed and amplified in a single tube under isothermal conditions, total reaction time could be reduced to less than 40 min. The detection limit of this method was between 2.4 × 10 2 and 2.4 × 10 1 copies/µl with excellent specificity. To test the clinical applicability of the method, 40 clinical stool samples were analyzed using RT-PSR and quantitative reverse transcription-polymerase chain reaction, and comparison showed that the coincidence rate was 100%. Compared with other similar detection methods, RT-PSR requires less time, simpler operation, and lower cost. These results prove that our novel, simple, and reliable isothermal nucleic acid testing assay has potential application for clinical detection of CVA-16.

Evaluation of reverse transcriptase-polymerase spiral reaction assay for rapid and sensitive detection of severe acute respiratory syndrome coronavirus 2

“…Previous studies on porcine epidemic diarrhoea virus, canine parvovirus 2, and coxsackie virus have shown RT-PSR assay to be highly sensitive with an ability to amplify even a single copy of RNA [12] , [23] , [24] . This study also reflected the high sensitivity of the test with the ability to amplify RNA as low as 5 ng/μL.…”

Isothermal amplification‐based assays for rapid and sensitive detection of severe acute respiratory syndrome coronavirus 2: Opportunities and recent developments

“…RT-PSR assay was developed for the detection of RNA viruses like porcine epidemic diarrhoea virus (PEDV). 39 In another study by He et al, 40 RT-PSR was found to be 10 times more sensitive than the RT-qPCR for the detection of coxsackievirus. Recently Tran et al 41 have developed PSR along with another isothermal assay that is, colorimetric isothermal NAATs, for the detection of SARS-CoV-2 targeting N gene and OFR1ab region.…”

“…In general, isothermal assays are reported to be an easy and less time‐consuming method for the detection of viral pathogen. 59 The isothermal amplification‐based assays can be further simplified by modifying the end‐point detection process of nucleic acid amplicons. Various end‐point detection methods have been optimized, such as using various dye indicators, labelled probe lateral flow, CRISPR‐Cas etc, can be used as POC diagnostics to detect SARS‐CoV‐2.…”

“…Various end-point detection methods have been optimized, such as using various dye indicators, labelled probe lateral flow, CRISPR-Cas etc, can be used as POC diagnostics to detect SARS-CoV-2. 39,40,43,58,60,61…”