A Novel Multiplex RT-PCR Assay for Simultaneous Detection of Dengue and Chikungunya Viruses.
The goal of the study was to develop a specific, sensitive, and cost-effective molecular RT-PCR diagnostic assay for the rapid and simultaneous detection of the serotypes of dengue virus (DENV) and Chikungunya virus (CHIKV) from sera of suspected febrile patients. A single-tube, single-step multiplex RT-PCR (mRT-PCR) assay was designed for the detection of viral genomes from clinical and field samples. Specificity and sensitivity of the mRT-PCR assay were evaluated against six different combinations using two reverse transcriptases (AMV-RT and RT-Ace) and three DNA polymerases (LA-Taq, rTaq, and Tth). Among the six combinations, the AMV-RT and LA-Taq combination was more specific and sensitive than other enzyme combinations for detecting viral genomes of DENV-1, DENV-2, DENV-3, and DENV-4 (p < 0.01), and for detecting viral genomes of CHIKV (p < 0.05). The detection limits of the mRT-PCR were 10 focus forming units (FFU) for CHIKV and 1 FFU, 20 FFU, 0.1 FFU, and 10 FFU for DENV-1, DENV-2, DENV-3, and DENV-4, respectively. The primers used for the mRT-PCR did not show any cross-reactivity among the serotypes of DENV or CHIKV. Specificity and sensitivity of the newly developed mRT-PCR were validated using serum samples collected from febrile patients during dengue outbreaks in Bangladesh. The sensitivity for serotype detection of DENV and CHIKV was superior to the virus isolation method and the antigen detection method using the Dengue NS1-Ag assay. This novel mRT-PCR method can be used for molecular epidemiological surveillance of DENV and CHIKV in epidemic and endemic countries.
['Islam|Mohammed Alimul|MA|', 'El Zowalaty|Mohamed E|ME|', 'Islam|Sumaiya|S|', 'Sharif|Mohiuddin|M|0000-0002-5580-2823', 'Rahman|Md Rajibur|MR|', 'Amin|Mohammad Robed|MR|0000-0002-5500-5103', 'Ali|Md Mortuza|MM|', 'Rahman|Md Tanvir|MT|0000-0001-5432-480X', 'Morita|Kouichi|K|', 'Ashour|Hossam M|HM|']
D000330:Aedes D000818:Animals D001459:Bangladesh D002478:Cells, Cultured D065632:Chikungunya Fever D002646:Chikungunya virus D006224:Cricetinae D003715:Dengue D003716:Dengue Virus D006801:Humans D025202:Molecular Diagnostic Techniques D060885:Multiplex Polymerase Chain Reaction D012367:RNA, Viral D020133:Reverse Transcriptase Polymerase Chain Reaction D012680:Sensitivity and Specificity D065288:Serogroup D014773:Virology
specificity sensitivity dengue virus chikungunya virus mRT-PCR Flaviviridae Togaviridae arboviruses