1.Identification And Typing Of Human Enteroviruses Using A RT-PCR Assay 2.The Contruct Of Expression Vector Of Multi-epitope Antigen Of HBV In P.pastoris

Human enteroviruses(HEV) are a large genus belonging to Picornaviridae family, and 91 immunologically distinct serotypes are known to cause infections in human. Most infections are mild or asymptomatic. In some cases, however,HEV may cause serious and even fatal diseases;they are the major cause of paralysis, aseptic meningitis, acute myocarditis and hand-foot-and-mouth disease. The traditional method for the diagnosis of HEV infections is virus isolation from clinical specimens in cell culture, followed by analysis with neutralizing antisera. Since there are a lot of serotypes,HEV typing by neutralization assay is expensive, time-consuming and labor-intensive. The aim of this study is to establish a molecular detection and typing assay for identification and typing of human enteroviruses(HEV) which is suitable for clinical detection and epidemic research.According to HEV sequences and literature, we synthesize a pair of primers"EVl/EV2"specific for HEV and 5 pairs of typing primers: "P1/P2', "P1/P3', "P4/P7',"P5/P7" and "P6/P7". Nucleic acid was extracted from 18 suspicious clinical HEV samplesand reverse transcription polymerase chain reaction(RT-PCR) was used to amply the cDNAusing both primers specific for HEV genus and HEV typing primers.17 clinical samples were judged as HEV by agarose gel electrophoresis of RT-PCR amplicons. The serotypes of all 18 clinical samples were identified through nucleotide sequence analysis of RT-PCR amplicons: 4 Coxackievirus type A24(CVA24), 3 CVB3, 1 CVB2, 1 CVA9, 1 CVA15, 1 Echovirus type 3(E3), 1 E6, 1 E9, 1 Ell, 1 E14, 1 E33 and 1 Rhinovirus type 9. The result is validated by monospecific antisera neutralization.This RT-PCR assay for identification and typing of human enteroviruses is more sensitive and rapid than the previously used neutralization methods. Moreover, this assaycan be used to study molecular epidemiological analysis of the viruses. So this RT-PCR assay for HEV detection and typing is fit for clinical detection and epidemic research in routine practice.