Development of a GeXP based multiplex RT-PCR assay for simultaneous differentiation of nine human hand food mouth disease pathogens.
- Author:
Xiu-Mei HU
1
;
Yong ZHANG
;
Bang-Lao XU
;
Meng-Jie YANG
;
Miao WANG
;
Chen ZHANG
;
Jin LI
;
Ru-Yin BAI
;
Xiao-Mian ZHOU
;
Wen-Bo XU
;
Xue-Jun MA
Author Information
1. 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.
- Publication Type:Journal Article
- MeSH:
DNA Primers;
genetics;
Enterovirus;
classification;
genetics;
isolation & purification;
Hand, Foot and Mouth Disease;
diagnosis;
virology;
Humans;
Reverse Transcriptase Polymerase Chain Reaction;
methods
- From:
Chinese Journal of Virology
2011;27(4):331-336
- CountryChina
- Language:Chinese
-
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.
