title: Visual detection of human enterovirus 71 subgenotype C4 and Coxsackievirus A16 by reverse transcription loop-mediated isothermal amplification with the hydroxynaphthol blue dye.
abstract: A sensitive reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed for rapid visual detection of human enterovirus 71 subgenotype C4 (EV71-C4) and Coxsackievirus A16 (CVA16) infection, respectively. The reaction was performed in one step in a single tube at 65°C for 60 min with the addition of the hydroxynaphthol blue (HNB) dye prior to amplification. The detection limits of the RT-LAMP assay were 0.33 and 1.58 of a 50% tissue culture infective dose (TCID(50)) per reaction based on 10-fold dilutions of a titrated EV71 or CVA16 strain, respectively. No cross-reaction was observed with Coxsackievirus A (CVA) viruses (CVA2, 4, 5, 7, 9, 10, 14, and 24), Coxsackievirus B (CVB) viruses (CVB1,2,3,4, and 5) or ECHO viruses (ECHO3, 6, 11, and 19). The assay was further evaluated with 47 clinical stool specimens diagnosed previously with EV71, CVA16 or other human enterovirus infections. Virus isolates from stool samples were confirmed by virus neutralization testing and sequencing. RT-LAMP with HNB dye was demonstrated to be a sensitive and cost-effective assay for rapid visual detection of human EV71-C4 and CVA16.
authors.affiliation: State Key Laboratory for Molecular Virology and Genetic Engineering, National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Changbai Rd 155, Changping district, Beijing 102206, China.
authors.forename: Kai
authors.initials: K
authors.lastname: Nie
authors.forename: Yong
authors.initials: Y
authors.lastname: Zhang
authors.forename: Le
authors.initials: L
authors.lastname: Luo
authors.forename: Meng-Jie
authors.initials: MJ
authors.lastname: Yang
authors.forename: Xiu-Mei
authors.initials: XM
authors.lastname: Hu
authors.forename: Miao
authors.initials: M
authors.lastname: Wang
authors.forename: Shuang-Li
authors.initials: SL
authors.lastname: Zhu
authors.forename: Feng
authors.initials: F
authors.lastname: Han
authors.forename: Wen-Bo
authors.initials: WB
authors.lastname: Xu
authors.forename: Xue-Jun
authors.initials: XJ
authors.lastname: Ma
mesh_terms: D003384:Coxsackievirus Infections / Q000175:diagnosis*; D004770:Enterovirus / Q000235:genetics / Q000302:isolation & purification*; D004769:Enterovirus Infections / Q000175:diagnosis*; D005243:Feces / Q000821:virology; D006801:Humans; D025202:Molecular Diagnostic Techniques / Q000191:economics / Q000379:methods*; D009282:Naphthalenesulfonates / Q000378:metabolism; D021141:Nucleic Acid Amplification Techniques / Q000191:economics / Q000379:methods*; D012680:Sensitivity and Specificity; D013194:Staining and Labeling / Q000191:economics / Q000379:methods*
chemical_list: D009282:Naphthalenesulfonates; C540250:trisodium 3-hydroxy-4-((2Z)-2-(2-oxo-4-sulfonatonaphthalen-1-ylidene)hydrazinyl)naphthalene-2,7-disulfonate