title: Simultaneous detection and differentiation of dengue virus serotypes 1-4, Japanese encephalitis virus, and West Nile virus by a combined reverse-transcription loop-mediated isothermal amplification assay.
abstract: BACKGROUND
  Rapid identification and differentiation of mosquito-transmitted flaviviruses in acute-phase sera of patients and field-caught vector mosquitoes are important for the prediction and prevention of large-scale epidemics.
  
  
  RESULTS
  We developed a flexible reverse-transcription loop-mediated isothermal amplification (RT-LAMP) unit for the detection and differentiation of dengue virus serotypes 1-4 (DENV1-4), Japanese encephalitis virus (JEV), and West Nile virus (WNV). The unit efficiently amplified the viral genomes specifically at wide ranges of viral template concentrations, and exhibited similar amplification curves as monitored by a real-time PCR engine. The detection limits of the RT-LAMP unit were 100-fold higher than that of RT-PCR in 5 of the six flaviviruses. The results on specificity indicated that the six viruses in the assay had no cross-reactions with each other. By examining 66 viral strains of DENV1-4 and JEV, the unit identified the viruses with 100% accuracy and did not cross-react with influenza viruses and hantaviruses. By screening a panel of specimens containing sera of 168 patients and 279 pools of field-caught blood sucked mosquitoes, results showed that this unit is high feasible in clinical settings and epidemiologic field, and it obtained results 100% correlated with real-time RT-PCR.
  
  
  CONCLUSIONS
  The RT-LAMP unit developed in this study is able to quickly detect and accurately differentiate the six kinds of flaviviruses, which makes it extremely feasible for screening these viruses in acute-phase sera of the patients and in vector mosquitoes without the need of high-precision instruments.
authors.affiliation: Department of Epidemiology, Shanghai Key Laboratory of Medical Biodefense, Second Military Medical University, Shanghai, China.
authors.forename: Shuhua
authors.initials: S
authors.lastname: Li
authors.forename: Meiyu
authors.initials: M
authors.lastname: Fang
authors.forename: Bin
authors.initials: B
authors.lastname: Zhou
authors.forename: Hongxia
authors.initials: H
authors.lastname: Ni
authors.forename: Qiuxia
authors.initials: Q
authors.lastname: Shen
authors.forename: Hongwei
authors.initials: H
authors.lastname: Zhang
authors.forename: Yifang
authors.initials: Y
authors.lastname: Han
authors.forename: Jianhua
authors.initials: J
authors.lastname: Yin
authors.forename: Wenjun
authors.initials: W
authors.lastname: Chang
authors.forename: Guozhang
authors.initials: G
authors.lastname: Xu
authors.forename: Guangwen
authors.initials: G
authors.lastname: Cao
mesh_terms: D000818:Animals; D009033:Culicidae / Q000821:virology*; D003716:Dengue Virus / Q000145:classification / Q000235:genetics / Q000302:isolation & purification*; D004664:Encephalitis Virus, Japanese / Q000145:classification / Q000235:genetics / Q000302:isolation & purification*; D005260:Female; D006801:Humans; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D012680:Sensitivity and Specificity; D044967:Serum / Q000821:virology*; D014773:Virology / Q000379:methods*; D014902:West Nile virus / Q000145:classification / Q000235:genetics / Q000302:isolation & purification*