title: Development and evaluation of a 1-step duplex reverse transcription polymerase chain reaction for differential diagnosis of chikungunya and dengue infection.
abstract: Dengue (DEN) and chikungunya (CHIK) have emerged as the 2 most important arboviral infections of global significance. The similarities in clinical presentations, their circulation in the same geographic area, and the transmission through the same vector necessitate an urgent need for the differential diagnosis of these 2 infections. So far, no single assay is reported for differential diagnosis of these 2 infections. In this study, we report the development and evaluation of a 1-step single-tube duplex reverse transcription polymerase chain reaction (D-RT-PCR) assay by targeting E1 gene of CHIK and C-prM gene junction of DEN virus (DENV), respectively. The sensitivity of this assay was found to be better than conventional virus isolation and could detect as low as 100 copies of genomic RNA, which is equivalent to respective virus-specific RT-PCR. The evaluation was carried out with 360 clinical samples from recent CHIK and DEN outbreaks in India. This assay could also be able to detect dual infection of CHIK and DEN in 3 patients. The phylogenetic analysis based on the nucleotide sequencing of D-RT-PCR amplicon could precisely identify the genotypes of all the serotypes of DENV and CHIK viruses (CHIKV). These findings demonstrate the potential clinical and epidemiologic application of D-RT-PCR for rapid sensitive detection, differentiation, and genotyping of DENV and CHIKV in clinical samples.
authors.affiliation: Division of Virology, Defence Research and Development Establishment, Gwalior, MP 474 002, India. pabandash@rediffmail.com
authors.forename: Paban Kumar
authors.initials: PK
authors.lastname: Dash
authors.forename: Manmohan
authors.initials: M
authors.lastname: Parida
authors.forename: S R
authors.initials: SR
authors.lastname: Santhosh
authors.forename: Parag
authors.initials: P
authors.lastname: Saxena
authors.forename: Ambuj
authors.initials: A
authors.lastname: Srivastava
authors.forename: Mamidi
authors.initials: M
authors.lastname: Neeraja
authors.forename: V
authors.initials: V
authors.lastname: Lakshmi
authors.forename: P V Lakshmana
authors.initials: PV
authors.lastname: Rao
mesh_terms: D000818:Animals; D002646:Chikungunya virus / Q000145:classification / Q000235:genetics / Q000302:isolation & purification*; D017931:DNA Primers; D003715:Dengue / Q000175:diagnosis* / Q000821:virology; D003716:Dengue Virus / Q000145:classification / Q000235:genetics / Q000302:isolation & purification*; D003937:Diagnosis, Differential; D006801:Humans; D007194:India; D010802:Phylogeny; D012367:RNA, Viral / Q000302:isolation & purification; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA; D014036:Togaviridae Infections / Q000175:diagnosis* / Q000821:virology
chemical_list: D017931:DNA Primers; D012367:RNA, Viral