Abstract
BackgroundEnterovirus 71 (EV71) and coxsackievirus A16 (CA16) are the two main etiological agents of Hand, Foot and Mouth Disease (HFMD). Simple and rapid detection of EV71 and CA16 is critical in resource-limited settings.MethodsDuplex real time reverse-transcription recombinase aided amplification (RT-RAA) assays incorporating competitive internal amplification controls (IAC) and visible RT-RAA assays combined with lateral flow strip (LFS) for detection of EV71 and CA16 were developed respectively. Duplex real time RT-RAA assays were performed at 42 °C within 30 min using a portable real-time fluorescence detector, while LFS RT-RAA assays were performed at 42 °C within 30 min in an incubator. Recombinant plasmids containing conserved VP1 genes were used to analyze the sensitivities of these two methods. A total of 445 clinical specimens from patients who were suspected of being infected with HFMD were used to evaluate the performance of the assays.ResultsThe limit of detection (LoD) of the duplex real time RT-RAA for EV71 and CA16 was 47 copies and 38 copies per reaction, respectively. The LoD of the LFS RT-RAA for EV71 and CA16 were both 91 copies per reaction. There was no cross reactivity with other enteroviruses. Compared to reverse transcription-quantitative PCR (RT-qPCR), the clinical diagnostic sensitivities of the duplex real time RT-RAA assay were 92.3% for EV71 and 99.0% for CA16, and the clinical diagnostic specificities were 99.7 and 100%, respectively. The clinical diagnostic sensitivities of the LFS RT-RAA assay were 90.1% for EV71 and 94.9% for CA16, and the clinical diagnostic specificities were 99.7 and 100%, respectively.ConclusionsThe developed duplex real time RT-RAA and LFS RT-RAA assays for detection of EV71 and CA16 are potentially suitable in primary clinical settings.
Highlights
Enterovirus 71 (EV71) and coxsackievirus A16 (CA16) are the two main etiological agents of Hand, Foot and Mouth Disease (HFMD)
EV71-related HFMD can be accompanied by serious complications, such as myocarditis, pulmonary edema, aseptic meningitis, a proportion of which are fatal [7, 8], while CA16-related HFMD is usually mild and self-limiting
The objective of the research is to establish sensitive and rapid Recombinase aided amplification (RAA) assays for the detection of EV71 and CA16, including duplex real time reverse-transcription recombinase aided amplification (RT-RAA) assay containing internal amplification controls (IAC) to reduce the false negative rates and lateral flow strip (LFS) RT-RAA assay suitable for field detection in resource limited areas
Summary
Enterovirus 71 (EV71) and coxsackievirus A16 (CA16) are the two main etiological agents of Hand, Foot and Mouth Disease (HFMD). Foot and mouth disease (HFMD) is a common acute infectious disease with typical rash distribution characteristics in the mouth, hands and feet, and occurs mainly in the children under 5 years of age Enteroviruses such as enterovirus 71 (EV71), coxsackievirus A16 (CA16), coxsackievirus A6 (CA6) and coxsackievirus A10 (CA10) are the main pathogens causing the disease. Many isothermal methods have emerged, such as nucleic acid sequence based amplification [18, 19], loop mediated isothermal amplification [20, 21], and recombinase polymerase amplification [22] These assays are performed at a constant temperature for less than 1 h with high sensitivities, specificities and do not require use of thermal cycler, which represents valuable alternatives to carry out simple and rapid pathogen detection
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