Abstract: Objective To designe and use new primers in the detection of pan-enterovirus(PE),coxsackievirus A16(CVA16) and enterovirus 71(EV71) for the diagnosis of hand,food and mouth disease(HFMD) using both reverse transcription-PCR(RT-PCR) and real-time RT-PCR.Methods Both RT-PCR and real-time RT-PCR were performed for the detection of PE,CVA16 and EV71 in 100 stool specimens from cases of HFMD and 50 normal control samples.The primers were refined to reexamine CVA16 in RT-PCR.The specificity of primers was checked with Basic Local Aligment Search Tool(BLAST) of National Center and analyzed with Primer Premier 6.0 and Oligo 6.62.Results The positive rates of the samples were 36%,33%,71% and 20%,27%,64% for CVA16,EV71 and PE with real-time RT-PCR and RT-PCR assays,respectively.Except CVA16,no significant difference was found in the detection rates of PE or EV71 between the two methods.The utilization of refined primers in RT-PCR could significantly increase the positive rate for CVA16 up to 35%(P<0.05),and the coincidence rate with clinic diagnosis increased by 10%,which was close to that of real-time RT-PCR assay.There were no significant differences between the two methods in specificity,misdiagnosis rate and missed diagnosis rate.Conclusion Routine RT-PCR method has a low sensitivity in detectoin of CVA16 and the detection rate could be increased to the level of real-time RT-PCR with newly designed primers.