title: A one-step, triplex, real-time RT-PCR assay for the simultaneous detection of enterovirus 71, coxsackie A16 and pan-enterovirus in a single tube.
abstract: The recent, ongoing epidemic of hand, foot, and mouth disease (HFMD), which is caused by enterovirus infection, has affected millions of children and resulted in thousands of deaths in China. Enterovirus 71 (EV71) and coxsackie A16 (CA16) are the two major distinct pathogens for HFMD. However, EV71 is more commonly associated with neurologic complications and even fatalities. Therefore, simultaneously detecting and differentiating EV71 and CA16 specifically from other enteroviruses for diagnosing HFMD is important. Here, we developed a one-step, triplex, real-time RT-PCR assay for the simultaneous detection of EV71, CA16, and pan-enterovirus (EVs) in a single tube with an internal amplification control. The detection results for the serially diluted viruses indicate that the lower limit of detection for this assay is 0.001-0.04 TCID50/ml, 0.02 TCID50/ml, and 0.001 TCID50/ml for EVs, EV71, and CA16, respectively. After evaluating known HFMD virus stocks of 17 strains of 16 different serotypes, this assay showed a favorable detection spectrum and no obvious cross-reactivity. The results for 141 clinical throat swabs from HFMD-suspected patients demonstrated sensitivities of 98.4%, 98.7%, and 100% for EVs, EV71, and CA16, respectively, and 100% specificity for each virus. The application of this one-step, triplex, real-time RT-PCR assay in clinical units will contribute to HFMD surveillance and help to identify causative pathogen in patients with suspected HFMD.
authors.affiliation: National Institute of Diagnostics and Vaccine Development in Infectious Disease, School of Life Science, Xiamen University, Xiamen, Fujian, China.
authors.forename: Shiyin
authors.initials: S
authors.lastname: Zhang
authors.affiliation: Xiamen Innovax Biotech Co., LTD, Xiamen, Fujian, China.
authors.forename: Jin
authors.initials: J
authors.lastname: Wang
authors.affiliation: Xiamen Innovax Biotech Co., LTD, Xiamen, Fujian, China.
authors.forename: Qiang
authors.initials: Q
authors.lastname: Yan
authors.affiliation: Xiamen Center for Disease Control and Prevention, Fujian, China.
authors.forename: Shuizhen
authors.initials: S
authors.lastname: He
authors.affiliation: Xiamen Innovax Biotech Co., LTD, Xiamen, Fujian, China.
authors.forename: Wenbin
authors.initials: W
authors.lastname: Zhou
authors.affiliation: National Institute of Diagnostics and Vaccine Development in Infectious Disease, School of Life Science, Xiamen University, Xiamen, Fujian, China; School of Public Health, Xiamen University, Xiamen, China.
authors.forename: Shengxiang
authors.initials: S
authors.lastname: Ge
authors.affiliation: National Institute of Diagnostics and Vaccine Development in Infectious Disease, School of Life Science, Xiamen University, Xiamen, Fujian, China; School of Public Health, Xiamen University, Xiamen, China.
authors.forename: Ningshao
authors.initials: N
authors.lastname: Xia
mesh_terms: D002681:China; D017931:DNA Primers / Q000235:genetics; D004770:Enterovirus / Q000302:isolation & purification*; D029821:Enterovirus A, Human / Q000302:isolation & purification*; D006232:Hand, Foot and Mouth Disease / Q000175:diagnosis* / Q000821:virology*; D006801:Humans; D025202:Molecular Diagnostic Techniques / Q000379:methods*; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*
chemical_list: D017931:DNA Primers