title: A Pan-Dengue Virus Reverse Transcription-Insulated Isothermal PCR Assay Intended for Point-of-Need Diagnosis of Dengue Virus Infection by Use of the POCKIT Nucleic Acid Analyzer.
abstract: Dengue virus (DENV) infection is considered a major public health problem in developing tropical countries where the virus is endemic and continues to cause major disease outbreaks every year. Here, we describe the development of a novel, inexpensive, and user-friendly diagnostic assay based on a reverse transcription-insulated isothermal PCR (RT-iiPCR) method for the detection of all four serotypes of DENV in clinical samples. The diagnostic performance of the newly established pan-DENV RT-iiPCR assay targeting a conserved 3' untranslated region of the viral genome was evaluated. The limit of detection with a 95% confidence was estimated to be 10 copies of in vitro-transcribed (IVT) RNA. Sensitivity analysis using RNA prepared from 10-fold serial dilutions of tissue culture fluid containing DENVs suggested that the RT-iiPCR assay was comparable to the multiplex real-time quantitative RT-PCR (qRT-PCR) assay for DENV-1, -3, and -4 detection but 10-fold less sensitive for DENV-2 detection. Subsequently, plasma collected from patients suspected of dengue virus infection (n = 220) and individuals not suspected of dengue virus infection (n = 45) were tested by the RT-iiPCR and compared to original test results using a DENV NS1 antigen rapid test and the qRT-PCR. The diagnostic agreement of the pan-DENV RT-iiPCR, NS1 antigen rapid test, and qRT-PCR tests was 93.9%, 84.5%, and 97.4%, respectively, compared to the composite reference results. This new RT-iiPCR assay along with the portable POCKIT nucleic acid analyzer could provide a highly reliable, sensitive, and specific point-of-need diagnostic assay for the diagnosis of DENV in clinics and hospitals in developing countries.
authors.affiliation: Virus Research and Testing Center, Bio and Drug Discovery Division, Korea Research Institute of Chemical Technology, Daejeon, Republic of Korea yygo@krict.re.kr ubalasuriya@uky.edu.
authors.forename: Yun Young
authors.initials: YY
authors.lastname: Go
authors.affiliation: Department of Veterinary Pathobiology, Faculty of Veterinary Medicine and Animal Science, University of Peradeniya, Peradeniya, Sri Lanka.
authors.forename: R P V Jayanthe
authors.initials: RPVJ
authors.lastname: Rajapakse
authors.affiliation: Faculty of Medicine, University of Peradeniya, Peradeniya, Sri Lanka.
authors.forename: Senanayake A M
authors.initials: SAM
authors.lastname: Kularatne
authors.affiliation: GeneReach USA, Lexington, Massachusetts, USA.
authors.forename: Pei-Yu Alison
authors.initials: PA
authors.lastname: Lee
authors.affiliation: Virus Research and Testing Center, Bio and Drug Discovery Division, Korea Research Institute of Chemical Technology, Daejeon, Republic of Korea.
authors.forename: Keun Bon
authors.initials: KB
authors.lastname: Ku
authors.affiliation: Virus Research and Testing Center, Bio and Drug Discovery Division, Korea Research Institute of Chemical Technology, Daejeon, Republic of Korea.
authors.forename: Sangwoo
authors.initials: S
authors.lastname: Nam
authors.affiliation: GeneReach USA, Lexington, Massachusetts, USA.
authors.forename: Pin-Hsing
authors.initials: PH
authors.lastname: Chou
authors.affiliation: GeneReach USA, Lexington, Massachusetts, USA.
authors.forename: Yun-Long
authors.initials: YL
authors.lastname: Tsai
authors.affiliation: GeneReach USA, Lexington, Massachusetts, USA.
authors.forename: Yu-Lun
authors.initials: YL
authors.lastname: Liu
authors.affiliation: GeneReach USA, Lexington, Massachusetts, USA.
authors.forename: Hsiao-Fen Grace
authors.initials: HG
authors.lastname: Chang
authors.affiliation: GeneReach USA, Lexington, Massachusetts, USA.
authors.forename: Hwa-Tang Thomas
authors.initials: HT
authors.lastname: Wang
authors.affiliation: Maxwell H. Gluck Equine Research Center, Department of Veterinary Science, College of Agriculture, Food and Environment and Department of Microbiology, Immunology and Molecular Genetics, College of Medicine, University of Kentucky, Lexington, Kentucky, USA yygo@krict.re.kr ubalasuriya@uky.edu.
authors.forename: Udeni B R
authors.initials: UBR
authors.lastname: Balasuriya
mesh_terms: D003715:Dengue / Q000175:diagnosis*; D003716:Dengue Virus / Q000235:genetics / Q000302:isolation & purification*; D006801:Humans; D025202:Molecular Diagnostic Techniques / Q000379:methods*; D019095:Point-of-Care Systems; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity