RESUMO
Large outbreaks of hand, foot, and mouth disease have been reported in the Asia Pacific region over the last few years and resulted in significant fatalities. The 2 main etiologic agents are Enterovirus 71 (EV71) and Coxsackievirus A16 (CA16). Both viruses are closely related genetically and show similar clinical symptoms. However, EV71 are associated with neurologic complications and can lead to fatalities. In this study, we developed a multiplex real-time hybridization probe reverse transcriptase polymerase chain reaction to detect and differentiate EV71 from CA16 using the LightCycler (Roche Molecular Biochemicals). Specific primers and hybridization probes were designed based on highly conserved VP1 region of EV71 or CA16. Our results showed high specificity and sensitivities in detecting EV71 or CA16 from 67 clinical specimens, and no other enterovirus serotype was detected. Rapid diagnosis to differentiate EV71 from CA16 in outbreak situations will enable pediatricians to identify and manage the patients more effectively.
Assuntos
Infecções por Coxsackievirus/diagnóstico , Infecções por Enterovirus/diagnóstico , Enterovirus/classificação , Enterovirus/isolamento & purificação , Hibridização de Ácido Nucleico/métodos , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Infecções por Coxsackievirus/virologia , Enterovirus/genética , Infecções por Enterovirus/virologia , Genes Virais , Humanos , Sondas de Oligonucleotídeos/genética , Sensibilidade e EspecificidadeRESUMO
BACKGROUND: Enterovirus 71 (EV71) is the main etiological agent of Hand, Foot and Mouth Disease (HFMD) and has been associated with neurological complications which resulted in fatalities during recent outbreaks in Asia Pacific region. OBJECTIVE: Develop a real-time TaqMan RT-PCR for rapid detection of EV71. STUDY DESIGN: Specific primers and probe were designed based on highly conserved VP1 region of EV71. The sensitivity of the real-time RT-PCR was evaluated with 67 clinical specimens collected from pediatric patients with suspected HFMD. RESULTS: Our real-time TaqMan RT-PCR showed 100% specificity in detecting EV71 and showed an analytical sensitivity of 5 viral copies. High sensitivity was also achieved in detecting EV71 directly from clinical specimens. CONCLUSIONS: Real-time TaqMan RT-PCR offers a rapid and sensitive method to detect EV71 from clinical specimens, and will allow quarantine measures to be taken more effectively during outbreaks.
