title: Development and evaluation of reverse transcription-loop-mediated isothermal amplification assay for rapid detection of enterovirus 71.
abstract: BACKGROUND
  Hand, foot, and mouth disease (HFMD) caused by enterovirus 71 (EV71) is very common in China. It is difficult to distinguish between EV71 and coxsackievirus A16 (CVA16) infections in clinical HFMD patients. Routine laboratory diagnosis of EV71 infection is time-consuming and requires expensive instruments. In this study, we have developed a one-step, single tube, reverse transcription-loop-mediated isothermal amplification (RT-LAMP) assay for rapid and sensitive detection of EV71.
  
  
  METHODS
  Six primers that can recognize 6 distinct regions on the VP2 gene of EV71 were designed for RT-LAMP assay. The amplification was completed by incubating all reagents in a single tube with reverse transcriptase and Bst DNA polymerase under the isothermal condition (60°C) for 60 min, and could be evaluated by using GoldView staining under a handheld ultraviolet torch lamp or electrophoresis analysis.
  
  
  RESULTS
  A total of 123 specimens collected from suspicious patients with HFMD were simultaneously detected by RT-LAMP and PCR fluorescence probing assay. The RT-LAMP amplified products containing EV71 were digested by HinfI and TaqI restriction endonucleases; in contrast, non-specific products with CVA16, coxsackievirus A4 and coxsackievirus B3 could not be detected in RT-LAMP assay. Meanwhile, RT-LAMP assay could amplify EV71 virus with a detection limit of 1 PFU/ml within 60 min. Compared with PCR fluorescence probing assay, RT-LAMP assay exhibited 98.4% identity during the detection of EV71 viral RNA without the missing of positive samples.
  
  
  CONCLUSIONS
  Our results indicated that RT-LAMP is a rapid, sensitive, specific and accurate method for the detection of EV71 in clinical specimens. Therefore, this developed method has potential application for rapid and comprehensive surveillance for EV71 infection, especially in developing country.
authors.affiliation: Department of Clinical Laboratory, The Third Affiliated Hospital of Suzhou University, No, 185 Juqian street, Changzhou, Jiangsu 213003, PR China. shiweifeng67@163.com
authors.forename: Weifeng
authors.initials: W
authors.lastname: Shi
authors.forename: Kun
authors.initials: K
authors.lastname: Li
authors.forename: Yun
authors.initials: Y
authors.lastname: Ji
authors.forename: Qingbo
authors.initials: Q
authors.lastname: Jiang
authors.forename: Mei
authors.initials: M
authors.lastname: Shi
authors.forename: Zuhuang
authors.initials: Z
authors.lastname: Mi
mesh_terms: D036022:Capsid Proteins / Q000235:genetics; D016009:Chi-Square Distribution; D002675:Child, Preschool; D003937:Diagnosis, Differential; D004587:Electrophoresis, Agar Gel; D004770:Enterovirus; D029821:Enterovirus A, Human / Q000235:genetics / Q000302:isolation & purification*; D005456:Fluorescent Dyes; D006232:Hand, Foot and Mouth Disease / Q000175:diagnosis / Q000821:virology*; D006801:Humans; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D012313:RNA; D012194:RNA-Directed DNA Polymerase / Q000378:metabolism; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
chemical_list: D036022:Capsid Proteins; D005456:Fluorescent Dyes; C034477:RNA primers; D012313:RNA; D012194:RNA-Directed DNA Polymerase