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One-Step Reverse-Transcription Recombinase Polymerase Amplification Using Lateral Flow Strips for the Detection of
Jia Xie1, Xiaohan Yang1,2, Lei Duan1
1Medical Genetic Centre, Guangdong Women and Children's Hospital, Guangzhou Medical University, Guangzhou, China.
Abstract:
Hand, foot, and mouth disease (HFMD) is a common infectious disease affecting mainly children under 5 years of age. Coxsackievirus A6 (CVA-6), a major causative pathogen of HFMD, has caused outbreaks in recent years. Currently, no effective vaccine or antiviral treatments are available. In this study, one-step reverse-transcription recombinase polymerase amplification (RT-RPA), combined with a disposable lateral flow strip (LFS) assay, was developed to detect CVA-6. This assay can be performed in less than 35 min at 37°C without expensive instruments, and the result can be observed directly with the naked eye. The sensitivity of the RT-RPA-LFS was 10 copies per reaction, which was comparable to that of the conventional real-time quantitative polymerase chain reaction (qPCR) assays. Moreover, the assay specificity was 100%. The clinical performance of the RT-RPA-LFS assay was evaluated using 142 clinical samples, and the coincidence rate between RT-RPA-LFS and qPCR was 100%. Therefore, our RT-RPA-LFS assay provides a simple and rapid approach for point-of-care CVA-6 diagnosis.
Insights
A new rapid diagnostic test for Hand, Foot, and Mouth Disease (HFMD) caused by Coxsackievirus A6 (CVA-6) has been developed. This simple, instrument-free assay offers accurate detection at the point-of-care.
Area of Science:
- Virology
- Molecular Diagnostics
- Public Health
Background:
- Hand, Foot, and Mouth Disease (HFMD) is a prevalent childhood illness, frequently caused by Coxsackievirus A6 (CVA-6).
- Recent CVA-6 outbreaks highlight the urgent need for effective diagnostic tools, as no specific vaccines or antiviral treatments are currently available.
- Existing diagnostic methods may be time-consuming or require specialized laboratory equipment.
Purpose of the Study:
- To develop a rapid, sensitive, and specific diagnostic assay for Coxsackievirus A6 (CVA-6).
- To create a point-of-care testing method that is accessible and easy to use, even without advanced instrumentation.
Main Methods:
- Development of a one-step reverse-transcription recombinase polymerase amplification (RT-RPA) assay.
- Integration of the RT-RPA assay with a disposable lateral flow strip (LFS) for visual detection.
- Validation of the assay's performance using synthetic CVA-6 RNA and 142 clinical samples.
Main Results:
- The developed RT-RPA-LFS assay demonstrated high sensitivity (10 copies/reaction), comparable to real-time quantitative polymerase chain reaction (qPCR).
- The assay achieved 100% specificity, ensuring accurate identification of CVA-6.
- Clinical evaluation showed a 100% coincidence rate with qPCR, confirming its reliability in real-world settings.
- The entire testing process, from sample to result, takes less than 35 minutes at 37°C.
Conclusions:
- The RT-RPA-LFS assay provides a simple, rapid, and accurate method for CVA-6 detection.
- This assay is suitable for point-of-care diagnosis of HFMD, particularly in resource-limited settings.
- The developed diagnostic tool can aid in timely disease management and outbreak surveillance of CVA-6.
