Microchip capillary electrophoresis with laser‐induced fluorescence combined with one‐step duplex reverse‐transcription polymerase chain reaction for the rapid detection of Enterovirus 71 and Coxsackievirus A16 in throat swab specimens

Abstract

Enterovirus 71 and Coxsackievirus A16 are the main pathogens causing hand-foot-mouth disease. In this paper, microchip capillary electrophoresis with laser-induced fluorescence combined with one-step duplex reverse transcript-polymerase chain reaction has been developed for the detection of Enterovirus 71 and Coxsackievirus A16 in throat swab specimens. The specific reverse transcription-polymerase chain reaction amplicons labeled with SYBR Orange were separated by microchip capillary electrophoresis and detected by laser induced fluorescence detector within 7 min. The intraday and interday relative standard deviation of migration time for DNA Marker was in the range of 1.36-2.94 and 2.78-3.96%, respectively. The detection limits were as low as 2.06 × 10(3) copies/mL for Enterovirus 71 and 5 × 10(3) copies/mL for Coxsackievirus A16. No cross-reactivity was observed with rotavirus, astrovirus, norovirus, and adenovirus, which showed good specificity of the method. This assay was validated using 100 throat swab specimens that were detected by real-time reverse-transcript polymerase chain reaction in parallel and the two methods produced the same results. This study provided a rapid, sensitive and specific method for the detection of Enterovirus 71 and Coxsackievirus A16, which make a contribution to significant time and cost saving for the identification and treatment of patients.

Microchip Electrophoresis for Fluorescence-Based Measurement of Polynucleic Acids: Recent Developments

“…Jia, R.; Chengjun, S.; Heng, C.; Chen, Z.; Yuanqian, L.; Yongxin, L. Journal of Separation Science Vol. 38 , issue 14 (ref ). Copyright 2015 Wiley.…”

“…Integration of this system with an upstream immunoaffinity extraction process will create a sample-to-answer microfluidic device for preterm birth biomarkers. Jai et al detected two major pathogens, Enterovirus 71 and Coxsackievirus A16, that cause hand-foot-and-mouth disease by combining one-step duplex reverse transcript-PCR and ME-LIF detection (Figure F) . With an electrophoresis channel having a L eff of 45 mm, the separation was complete in under 7 min.…”

“…Jai et al detected two major pathogens, Enterovirus 71 and Coxsackievirus A16, that cause hand-foot-and-mouth disease by combining one-step duplex reverse transcript-PCR and ME-LIF detection (Figure 8F). 106 With an electrophoresis channel having a L eff of 45 mm, the separation was complete in under 7 min. The assay yielded high specificity with reported limits of detection of 2.06 × 10 3 copies/mL for Enterovirus 71 and 5 × 10 3 copies/mL for Coxsackievirus A16.…”

Recent advances in CE and microchip‐CE in clinical applications: 2014 to mid‐2017

“…Ruan et al. used both CE‐LIF and ME‐LIF to study PCR products derived from adenovirus DNA in fecal samples. PCR products were detected within 9 min by CE‐LIF and 6 min by ME‐LIF, with intra‐ and interday RSD's of 1.14–1.34 and 1.27–2.76%, respectively, for CE‐LIF, and 1.18–1.48 and 2.85–4.06%, respectively, for the ME‐LIF.…”

Capillary Electrophoresis Based on Nucleic Acid Detection as Used in Food Analysis

“…Small DNA fragments were analyzed using MCE‐AD without requiring any preconditioning or use of polyacrylamide gel in the microchannel (Jang and others ). MCE‐LIF was combined with 1‐step duplex RT‐PCR to rapidly detect Enterovirus 71 and Coxsackievirus A16 (Jia and others ). Finally, PCR‐RFLP‐MCE was used to identify 9 fish species (Li and others ).…”