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Thus, the effective diagnosis of all human EVs infections and the monitoring of epidemiological and ecological dynamic changes are urgently needed.<\/jats:p>\n<\/jats:sec>\n<jats:sec id=\"sec002\">\n<jats:title>Methods<\/jats:title>\n<jats:p>Based on two comprehensive virological surveillance systems of hand, foot and mouth disease (HFMD), real-time PCR and nested RT-PCR (RT-snPCR) methods based on the enteroviral VP1, VP4-VP2 and VP4 regions were designed to directly detect all human EVs serotypes in clinical specimens.<\/jats:p>\n<\/jats:sec>\n<jats:sec id=\"sec003\">\n<jats:title>Results<\/jats:title>\n<jats:p>The results showed that the proposed serotyping strategy exhibit very high diagnostic efficiency (Study 1: 99.9%; Study 2: 89.5%), and the variance between the study was due to inclusion of the specific Coxsackie virus A6 (CVA6) real-time RT-PCR and VP4 RT-snPCR in Study 1 but not Study 2. Furthermore, only throat swabs were collected and analyzed in Study 2, whereas in Study 1, if a specific EV serotype was not identified in the primary stool sample, other sample types (rectal swab and throat swab) were further tested where available. During the study period from 2013 to 2018, CVA6 became one of the main HFMD causative agents, whereas the level of enterovirus A71 (EV-A71) declined in 2017.<\/jats:p>\n<\/jats:sec>\n<jats:sec id=\"sec004\">\n<jats:title>Conclusion<\/jats:title>\n<jats:p>The findings of this study demonstrate the appropriate application of PCR methods and the combination of biological sample types that are useful for etiological studies and propose a molecular strategy for the direct detection of human EVs in clinical specimens associated with HFMD.<\/jats:p>\n<\/jats:sec>","DOI":"10.1371\/journal.pone.0241614","type":"journal-article","created":{"date-parts":[[2020,11,9]],"date-time":"2020-11-09T18:45:42Z","timestamp":1604947542000},"page":"e0241614","update-policy":"https:\/\/doi.org\/10.1371\/journal.pone.corrections_policy","source":"Crossref","is-referenced-by-count":9,"title":["Molecular strategy for the direct detection and identification of human enteroviruses in clinical specimens associated 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