title: [Development of a GeXP based multiplex RT-PCR assay for simultaneous differentiation of nine human hand food mouth disease pathogens].
abstract: A multiplex RT-PCR assay based on GeXP system was developed in order to detect simultaneously human enterovirus 71 (EV71) and coxsackievirus A16 (CVA16) and other coxsackieviruses (CVA4, 5, 9 and 10, CVB1, 3 and 5). Enterovirus detection was performed with a mixture of 12 pairs of oligonucleotide primers including one pair of published primers for amplifying all known pan-enterovirus genomes and eleven primer pairs specific for detection of the VP1 genes of EV71, C A16, CVA4, CVA5, CVA9, CVA10, CVB1, CVB3 and CVB5, respectively. The specificity of multiplex RT-PCR system was examined using enterovirus cell cultures and positive strains identified previously from hand-foot-and-mouth disease (HFMD) patients. Serial dilution of titrated EV71 and C A16 cell cultures and in vitro transcripted RNA of enterovirus VP1 regions were used to detect the sensitivity of the multiplex RT-PCR system. The limit of detection for this multiplex RT-PCR system was 10(0.5) TCID50/microL for EV71 and C A16 cell cultures and 1000 copies for in vitro transcripted RNA of nine viruses per assay. This multiplex RT-PCR assay is a rapid, sensitive and specific assay for the diagnosis of common enterovirus infection in cases of HFMD outbreak and is also potentially useful for molecular epidemiological investigation.
authors.affiliation: State Key Laboratory for Genetic Engineering and Molecular Virology, National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing 102206, China.
authors.forename: Xiu-Mei
authors.initials: XM
authors.lastname: Hu
authors.forename: Yong
authors.initials: Y
authors.lastname: Zhang
authors.forename: Bang-Lao
authors.initials: BL
authors.lastname: Xu
authors.forename: Meng-Jie
authors.initials: MJ
authors.lastname: Yang
authors.forename: Miao
authors.initials: M
authors.lastname: Wang
authors.forename: Chen
authors.initials: C
authors.lastname: Zhang
authors.forename: Jin
authors.initials: J
authors.lastname: Li
authors.forename: Ru-Yin
authors.initials: RY
authors.lastname: Bai
authors.forename: Xiao-Mian
authors.initials: XM
authors.lastname: Zhou
authors.forename: Wen-Bo
authors.initials: WB
authors.lastname: Xu
authors.forename: Xue-Jun
authors.initials: XJ
authors.lastname: Ma
mesh_terms: D017931:DNA Primers / Q000235:genetics; D004770:Enterovirus / Q000145:classification / Q000235:genetics / Q000302:isolation & purification*; D006232:Hand, Foot and Mouth Disease / Q000175:diagnosis / Q000821:virology*; D006801:Humans; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*
chemical_list: D017931:DNA Primers