Detection of Coxsackievirus A16 by Reverse Transcription Loop-Mediated Isothermal Amplification

Abstract

Aims: A one-step, single-tube reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed and validated for the detection of Coxsackievirus A16 (CA16). Methods & materials: An optimized RT-LAMP assay was tested for in its sensitivity, primers specificity, products specificity and reproducibility. Results: The detection limit of the RT-LAMP assay was 10⁶-fold dilution of stock virus or 81 copies in samples after RNA extraction, which was tenfold higher in sensitivity than the traditional reverse transcription PCR (RT-PCR) and equal to real-time RT-PCR. Digestion with a specific restriction enzyme EcoRI demonstrated that the amplified product was unique. The specificity of the assay was confirmed as it was demonstrated that the positive amplification only appeared among all CA16 stains, while no amplification was achieved in other viruses genetically related to hand, foot and mouth disease or similar clinical features. A good correlation between RT-LAMP and real-time RT-PCR was observed on the basis of the analysis of 33 clinical samples. Conclusion: RT-LAMP is a novel, alternative microbiological approach for rapid, sensitive and specific detection of CA16 in hand, foot and mouth disease.

Keywords:

• CA16
• Coxsackievirus A16
• hand, foot and mouth disease
• reverse transcription loop-mediated isothermal amplification
• RT-LAMP

Financial & competing interests disclosure

The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.

No writing assistance was utilized in the production of this manuscript.

Ethical conduct of research

The authors state that they have obtained appropriate institutional review board approval or have followed the principles outlined in the Declaration of Helsinki for all human or animal experimental investigations. In addition, for investigations involving human subjects, informed consent has been obtained from the participants involved – in this case the parents or guardians were given written informed consents that are approved by the Ethics Committee at Southern Medical University.