title: [Colorimetric detection of coxsackievirus A6 by reverse transcription loop mediated isothermal amplification].
abstract: OBJECTIVE
  To develop a simple, rapid and sensitive colorimetric reverse-transcription loop-mediated isothermal amplification (RT-LAMP) for rapid detection of coxsackievirus A6 (CV-A6) based on the colour chang of hydroxy naphthol blue (HNB).
  
  
  METHODS
  The method employed a set of six primers that recognized sequences of VP1 gene for amplification of nucleic acid under isothermal conditions at 63 °C for 50 min. The products were detected through visual inspection of color change by the pre-addition of HNB dye. The specificity was validated by detecting a collection of different human enteroviruses. The sensitivity of this assay was evaluated by serial dilutions of RNA molecules from in vitro transcription of CV-A6 VP1 gene, and compared with real-time RT-PCR (rRT-PCR) in parallel. This assay was evaluated with 92 clinical specimens from patients with hand-foot-mouth disease.
  
  
  RESULTS
  A positive color (sky blue) was only observed in the preparation of CV-A6, whereas none of the other 23 kinds of human enteroviruses showed a color change. The HNB based RT-LAMP showed a sensitivity of 100 copies/reaction, which was at the same level as that of the rRT-PCR. The result of RT-LAMP in analysis of 92 clinical specimens was consistent with that of the rRT-PCR. The kappa correlation between the two methods was 1 and both of the sensitivity and specificity of the RT-LAMP assay were 100%.
  
  
  CONCLUSIONS
  The established RT-LAMP assay had good specificity and sensitivity and thus demonstrated to be a promising screening tool for CV-A6 infection. It also has the potential to be used in resource-limited clinical sites and field study.
authors.affiliation: National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing 102206, China.
authors.forename: Li
authors.initials: L
authors.lastname: Guan
authors.forename: Songtao
authors.initials: S
authors.lastname: Xu
authors.forename: Kai
authors.initials: K
authors.lastname: Nie
authors.forename: Dan
authors.initials: D
authors.lastname: Zhang
authors.forename: Xinna
authors.initials: X
authors.lastname: Li
authors.forename: Wenbo
authors.initials: W
authors.lastname: Xu
authors.affiliation: Email: maxj@ivdc.chinacdc.cn.
authors.forename: Xuejun
authors.initials: X
authors.lastname: Ma
mesh_terms: D003124:Colorimetry; D004396:Coloring Agents / Q000737:chemistry; D017931:DNA Primers; D004770:Enterovirus / Q000302:isolation & purification*; D006232:Hand, Foot and Mouth Disease / Q000821:virology; D006801:Humans; D007202:Indicators and Reagents / Q000737:chemistry; D009282:Naphthalenesulfonates / Q000737:chemistry; D021141:Nucleic Acid Amplification Techniques; D060888:Real-Time Polymerase Chain Reaction; D048348:Reverse Transcription; D012680:Sensitivity and Specificity
chemical_list: D004396:Coloring Agents; D017931:DNA Primers; D007202:Indicators and Reagents; D009282:Naphthalenesulfonates; C540250:trisodium 3-hydroxy-4-((2Z)-2-(2-oxo-4-sulfonatonaphthalen-1-ylidene)hydrazinyl)naphthalene-2,7-disulfonate