title: Detection of human Enterovirus 71 reverse transcription loop-mediated isothermal amplification (RT-LAMP).
abstract: OBJECTIVE
  In this study, a one-step, single-tube reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed and validated for the detection of human Enterovirus 71 (EV71), the major aetiological agent of hand, foot and mouth disease (HFMD).
  
  
  RESULTS
  Reverse transcription loop-mediated isothermal amplification assay was optimized to amplify VP1 gene in the presence of a specific primer set and Bst DNA polymerase at an isothermal temperature of 63°C for 1 h. Amplified products were evaluated by visual inspection and agarose gel electrophoresis. The detection limit of RT-LAMP assay was 10(-5) 100 TCID50 or 160 copies in samples after RNA extraction, which was 10-fold higher in sensitivity than traditional reverse transcription polymerase chain reaction (RT-PCR). The specific positive amplification was only observed in EV71 strains, while no amplification was detected in other tested viruses. Digestion with a specific Escherichia coli restriction enzymes V (EcoR V) demonstrated that the amplified product was unique. A good correlation between RT-LAMP and real-time RT-PCR was observed on the basis of the analysis of 33 clinical samples.
  
  
  CONCLUSIONS
  Reverse transcription loop-mediated isothermal amplification is a novel, alternative microbiological approach for rapid, sensitive and specific detection of EV71 in HFMD.
  
  
  CONCLUSIONS
  Reverse transcription loop-mediated isothermal amplification assay is suitable for the diagnosis of EV71 infection as a routine diagnostic tool for HFMD because of fewer requirements of experimental conditions such as private clinics, field applications as well as an epidemiological survey in epidemic areas. RT-LAMP can also be used as an alternative method for EV71 detection.
authors.affiliation: Department of Epidemiology, Southern Medical University, Guangzhou, China.
authors.forename: H
authors.initials: H
authors.lastname: Yaqing
authors.forename: Z
authors.initials: Z
authors.lastname: Wenping
authors.forename: Y
authors.initials: Y
authors.lastname: Zhiyi
authors.forename: W
authors.initials: W
authors.lastname: Xionghu
authors.forename: Y
authors.initials: Y
authors.lastname: Shouyi
authors.forename: Y
authors.initials: Y
authors.lastname: Hong
authors.forename: D
authors.initials: D
authors.lastname: Yingchun
authors.forename: H
authors.initials: H
authors.lastname: Guifang
mesh_terms: D036022:Capsid Proteins / Q000235:genetics; D017931:DNA Primers / Q000235:genetics; D004587:Electrophoresis, Agar Gel; D029821:Enterovirus A, Human / Q000235:genetics / Q000302:isolation & purification*; D006232:Hand, Foot and Mouth Disease / Q000175:diagnosis* / Q000821:virology; D006801:Humans; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D012367:RNA, Viral / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D012680:Sensitivity and Specificity
chemical_list: D036022:Capsid Proteins; D017931:DNA Primers; D012367:RNA, Viral