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.

Shiyu He,Juan Wang,Xiuling Song,Bo Pang,Hui Li,Yanling Zhao,Juan Li,Liwei Sun,Lixue Wang,Yanzhi Huang,Haoyan Yu

doi:10.3389/fmicb.2020.00734

Shiyu He, Juan Wang + Show 9 more

Open Access

https://doi.org/10.3389/fmicb.2020.00734

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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 transcription-polymerase 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 × 102 and 2.4 × 101 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.

Highlights

Hand, foot, and mouth disease (HFMD) has long been a focus of global public health, especially for children aged < 5 years (Huang et al, 2018; Qi et al, 2018)
In reverse transcriptionpolymerase spiral reaction (RT-Polymerase spiral reaction (PSR)), under the action of reverse transcriptase, viral RNA was reverse transcribed into cDNA
Primer 2 was selected as the best Reverse transcription (RT)-PSR primer for CVA-16 detection

Summary

Introduction

Foot, and mouth disease (HFMD) has long been a focus of global public health, especially for children aged < 5 years (Huang et al, 2018; Qi et al, 2018). Enteroviruses (EVs), especially EV-A71 and Coxsackievirus A16 (CVA-16), are the main etiological agents involved in HFMD (Solomon et al, 2010; Chen et al, 2013). Since around 2014, CVA-10 and a new CVA-6 genotype have become widespread (Chen et al, 2017; Broccolo et al, 2019). Rapid Detection of Coxsackievirus A16 by PSR rate of HFMD caused by this strain has decreased significantly (Chong et al, 2012; Yi et al, 2017; Li Y. et al, 2019).

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