Rapid detection of enterovirus with TaqMan real-time RT-PCR

Objective To establish a specific,sensitive and rapid method for detecting enterovirus nucleic acid with quantitative real-time RT-PCR and laboratory diagnosis for rapid response of enterovirus outbreaks.Methods Based on the alignment result of enterovirus sequences downloaded from GenBank,the conserved region was used for the design of primers and TaqMan probe.The reaction conditions were optimized using different concentrations of primers and probe.Specificity and sensitivity evaluations of the TaqMan real-time RT-PCR were also included.Laboratory diagnoses of some clinical specimens from suspected cases of enterovirus infection were conducted using the real-time RT-PCR method.Results The real-time RT-PCR was proved to be a high specificity method which could detect enteroviruses such as poliovirus,coxsackie virus,EHCO virus and so on.Cross-reactivity with mumps,measles,rubella or Japanese encephalitis virus was not observed.The sensitivity for detection was 0.1 TCID50.The detection of enteroviruses could be performed using nucleic acid directly extracted from cerebrospinal fluid,herpes fluid or stool samples of suspected cases.The time duration was about 3h from the viral RNA extraction to the completion of detection.Conclusion The established TaqMan quantitative real-time RT-PCR was a specific and sensitive method for rapid detection of enteroviruses.It could be used for early laboratory diagnosis in response to enterovirus epidemics.