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A two\u2010tube multiplex real\u2010time reverse transcription\u2010polymerase chain reaction (RT\u2010PCR) assay for the simultaneous detection of Zika virus (ZIKV), chikungunya virus (CHIKV), dengue virus (DENV), yellow fever virus (YFV), West Nile virus (WNV), and Japanese encephalitis virus (JEV) was developed and optimized with high specificity and sensitivity. The detection limit for all the six\u00a0viruses could reach as low as five\u00a0genome equivalent copies and 2.8\u2009\u00d7\u200910<jats:sup>\u2212<\/jats:sup><jats:sup>3<\/jats:sup> tissue culture infectious doses (TCID<jats:sub>50<\/jats:sub>) for ZIKV, YFV, CHIKV and 2.8\u2009\u00d7\u200910<jats:sup>\u22122<\/jats:sup> TCID<jats:sub>50<\/jats:sub>\u00a0for JEV per reaction, with high accuracy and precision (<jats:italic>R<\/jats:italic><jats:sup>2<\/jats:sup>\u2009&gt;\u20090.99). The coefficient of variation of intra\u2010assay and inter\u2010assay for our quantitative reverse transcription\u2010polymerase chain reaction (qRT\u2010PCR) assay was low, and the obtained positive rates ad <jats:italic>C<\/jats:italic><jats:sub>t<\/jats:sub> values of this assay were comparable with singleplex commercial kits. Moreover, the multiplex qRT\u2010PCR assay was able to detect possible co\u2010infections without competitive inhibition of target viral genomes. In conclusion, our rapid, sensitive, cost\u2010effective multiplex qRT\u2010PCR will be of great use for differential diagnosis in a clinical setting and epidemiological investigation during surveillance.<\/jats:p>","DOI":"10.1002\/jmv.27658","type":"journal-article","created":{"date-parts":[[2022,2,11]],"date-time":"2022-02-11T09:27:45Z","timestamp":1644571665000},"page":"2528-2536","update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":24,"title":["Simultaneous detection of Zika, chikungunya, dengue, yellow fever, West Nile, and Japanese encephalitis viruses by a two\u2010tube multiplex real\u2010time RT\u2010PCR assay"],"prefix":"10.1002","volume":"94","author":[{"given":"Zhixiang","family":"Xu","sequence":"first","affiliation":[{"name":"Shenzhen Key Laboratory of Pathogen and Immunity, National Clinical Research Center for Infectious Disease, State Key Discipline of Infectious Disease, Shenzhen Third People's Hospital Second 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