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Journal of Clinical Microbiology, June 2006, p. 2212-2219, Vol. 44, No. 6
0095-1137/06/$08.00+0     doi:10.1128/JCM.02393-05
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Combining Multiplex Reverse Transcription-PCR and a Diagnostic Microarray To Detect and Differentiate Enterovirus 71 and Coxsackievirus A16

Division of Molecular and Genomic Medicine, National Health Research Institutes, Miaoli, Taiwan,¹ Department of Life Science and Institute of Biotechnology, National Tsing Hua University, Hsinchu, Taiwan,² Department of Computer Science and Information Engineering, Chang Gung University, Taoyuan, Taiwan,³ Biostatistics and Bioinformatics, National Health Research Institutes, Miaoli, Taiwan,⁴ Center for Disease Control, Department of Health, Taipei, Taiwan,⁵ Department of Medical Biotechnology and Laboratory Science, Chang Gung University, Taoyuan, Taiwan⁶

Received 18 November 2005/ Returned for modification 16 January 2006/ Accepted 7 April 2006

Cluster A enteroviruses, including enterovirus 71 (EV71) and coxsackievirus A16 (CA16), are known to cause hand-foot-and-mouth disease (HFMD). Despite the close genetic relationship between these two viruses, EV71 is generally known to be a more perpetuating pathogen involved in severe clinical manifestations and deaths. While the serotyping of enteroviruses is mostly done by conventional immunological methods, many clinical isolates remain unclassifiable due to the limited number of antibodies against enterovirus surface proteins. Array-based assays are able to detect several serotypes with high accuracy. We combined an enterovirus microarray with multiplex reverse transcription-PCR to try to develop a method of sensitively and accurately detecting and differentiating EV71 and CA16. In an effort to design serotype-specific probes for detection of the virus, we first did an elaborate bioinformatic analysis of the sequence database derived from different enterovirus serotypes. We then constructed a microarray using 60-mer degenerate oligonucleotide probes covalently bound to array slides. Using this enterovirus microarray to study 144 clinical specimens from patients infected with HFMD or suspected to have HFMD, we found that it had a diagnostic accuracy of 92.0% for EV71 and 95.8% for CA16. Diagnostic accuracy for other enteroviruses (non-EV71 or -CA16) was 92.0%. All specimens were analyzed in parallel by real-time PCR and subsequently confirmed by neutralization tests. This highly sensitive array-based assay may become a useful alternative in clinical diagnostics of EV71 and CA16.