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Comparative Study
. 2019 Dec 30;16(1):166.
doi: 10.1186/s12985-019-1264-z.

Applicability of duplex real time and lateral flow strip reverse-transcription recombinase aided amplification assays for the detection of Enterovirus 71 and Coxsackievirus A16

Xin-Na Li  1 , Xin-Xin Shen  1 , Ming-Hui Li  2 , Ju-Ju Qi  1 , Rui-Huan Wang  1 , Qing-Xia Duan  1 , Rui-Qing Zhang  1 , Tao Fan  1 , Xue-Ding Bai  1 , Guo-Hao Fan  1 , Yao Xie  3 , Xue-Jun Ma  4
Affiliations
Comparative Study

Applicability of duplex real time and lateral flow strip reverse-transcription recombinase aided amplification assays for the detection of Enterovirus 71 and Coxsackievirus A16

Xin-Na Li et al. Virol J. .

Abstract

Background: Enterovirus 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.

Methods: Duplex 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.

Results: The 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.

Conclusions: The developed duplex real time RT-RAA and LFS RT-RAA assays for detection of EV71 and CA16 are potentially suitable in primary clinical settings.

Keywords: Coxsackievirus A16; Duplex; Enterovirus 71; Hand foot and mouth disease; Internal amplification controls (IAC); Lateral flow strip; Reverse-transcription recombinase aided amplification assays.

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Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Fig. 1
Fig. 1
Detection of the RAA amplicons by lateral flow strip. The sample pad contains gold-labeled anti-FAM antibodies, the test line was coated with biotin-ligands, and control line was coated with anti-rabbit antibodies. The double-labeled amplicons (FAM and biotin) were diffused through the chromatographic membrane, and when they diffused to the test line, the products were captured by the biotin -ligands, resulting in an appearance of red-pink color. Non-captured particles will be fixed at the control line by anti-rabbit antibodies. In the absence of target amplicons, color will appear at control line only [26]
Fig. 2
Fig. 2
Schematic diagram of duplex real time RAA assays for detection of EV71 and CA16
Fig. 3
Fig. 3
The amplification curves of the duplex real time RT-RAA assays using 10-fold dilution series of plasmid DNA containing the VP1gene of EV 71 (a) and CA16 (b). Fluorescence signals from target amplification were recorded in the FAM detection channels of the QT-RAA1620 device, while IAC amplification was recorded in the HEX detection channel. The development of fluorescence using a dilution range of 106 copies/μL − 1 copy/μL of the target recombinant plasmid standards. a represents the duplex RT- RAA of EV71 with 50copies IAC plasmid per reaction. The sensitivity of the assay is 10 copies. High concentrations of target plasmids (106copies/μL) completely inhibit the amplification of IAC plasmid. EV71 amplification curves of high concentration plasmid (106 copies per reaction) occurred early and even overlapped, while for the IAC, no amplification curves were observed in the HEX channel. EV71 amplification curves of medium and low concentration plasmids appeared slightly later than the high concentration, while for the IAC, amplification curves were observed in the HEX channel. b represents the duplex RT-RAA of CA16 with 100 copies IAC plasmid per reaction. The sensitivity of the assay is 10 copies. High concentrations of target plasmids (106-105copies/μL) completely inhibit the amplification of IAC plasmid. The amplification curves of CA16 plasmids with high concentration (106 -105copies per reaction) appeared early and even overlapped, while for the IAC, no amplification curves were observed in the HEX channel. CA16 plasmids with medium and low concentrations revealed no significant interference with IAC amplification curves in the HEX channel
Fig. 4
Fig. 4
Performance of EV71 LFS RT-RAA assay. a Optimization experiment of LFS RT-RAA reaction time. When the reaction time was longer than 20 min, the test line was visible. b Analytical sensitivity of the LFS RT- RAA assay. Lane1, 1 × 106 copies, Lane2, 1 × 105copies, Lane3, 1 × 104copies, Lane4, 1 × 103copies, Lane5, 1 × 102 copies, Lane6, 1 × 101 copies, Lane7, 1 copy, Lane8, no template control. c Analytical specificity of the LFS RT-RAA assay. Only EV71 specimen was amplified. The other samples were not amplified. Lane1, EV71, Lane2, CA 16, Lane3, CA6, Lane4, CA10, Lane5, CA5, Lane6, CA9, Lane7, CA24, Lane8, CB2, Lane9, CB4, Lane10, PV2, Lane11, PV3, Lane12, Eco30, Lane13, HEV14, Lane14, no template control
Fig. 5
Fig. 5
Performance of CA16 LFS RT-RAA assay. a Optimization experiment of LFS RT-RAA reaction time. When the reaction time was longer than 20 min, the test line was visible. b Analytical sensitivity of the LFS RT-RAA assay. Lane1, 1 × 106 copies, Lane2,1 × 105copies, Lane3, 1 × 104copies, Lane4, 1 × 103copies, Lane5, 1 × 102 copies, Lane6, 1 × 101 copies, Lane7, 1copy, Lane8, no template control. c Analytical specificity of the LFS RT-RAA assay. Only CA16 specimen was amplified. The other samples were not amplified. Lane1, CA16, Lane2, EV71, Lane3, CA6, Lane4, CA10, Lane5, CA5, Lane6, CA9, Lane7, CA24, Lane8, CB2, Lane9, CB4, Lane10, PV2, Lane11,PV3, Lane12, Eco30, Lane13, HEV14, Lane14, no template control
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