Using Semi-nest Reverse Transcription-PCR And A Diagnostic Microarray To Detect And Differentiate Enterovirus71and CoxsackievirusA16

Hand, foot and mouth disease (HFMD) is generally a benign febrile exanthematous childhood disease caused by many human enterovirus. The two main etiologic agents are enterovirus71and coxsackievirus A16. EV71and CAV16are closely related genetically, the clinical symptoms of EV71-and CAV16-associated HEMD are similar, but EV71infection may develop severe neurologic diseases or die.While the detecting and 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. DNA microarray is able to detect several serotypes with high accuracy. In this study, we combined an enterovirus microarray with seminestd reverse transcription-PCR to detect and serotype EV71and CAV16. Primers and oliginucleotide probes were designed and synthesized based on the region of VP1gene.399stool specimens from patients infected with HFMD or suspected to have HFMD were examined with seminested reverse transcription-PCR and139specimens were positive for enterovirus.399clinical samples were analyzed by this DNA microarray, and the results were compared with those of DNA sequencing. The sensitivity and specificity of the DNA microarray technology was100%and92.5%to EV71. The sensitivity and specificity of the DNA microarray technology was100%and95.9%to CAV16. The detection limits were101copies/μl and the test showed no cross-reaction to other serotypes. This highly sensitive DNA microarray may become a useful alternative in clinical diagnostics of EV71and CAV16.