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

Research output: Journal Publications and Reviews (RGC: 21, 22, 62)21_Publication in refereed journalpeer-review

22 Scopus Citations
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Author(s)

  • R.P.V. Jayanthe Rajapakse
  • Senanayake A.M. Kularatne
  • Pei-Yu Alison Lee
  • Keun Bon Ku
  • Sangwoo Nam
  • Pin-Hsing Chou
  • Yun-Long Tsai
  • Yu-Lun Liu
  • Hsiao-Fen Grace Chang
  • Hwa-Tang Thomas Wang
  • Udeni B. R. Balasuriya

Detail(s)

Original languageEnglish
Pages (from-to)1528-1535
Journal / PublicationJournal of Clinical Microbiology
Volume54
Issue number6
Online published23 May 2016
Publication statusPublished - Jun 2016
Externally publishedYes

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.

Citation Format(s)

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. / Go, Yun Young; Rajapakse, R.P.V. Jayanthe; Kularatne, Senanayake A.M.; Lee, Pei-Yu Alison; Ku, Keun Bon; Nam, Sangwoo; Chou, Pin-Hsing; Tsai, Yun-Long; Liu, Yu-Lun; Chang, Hsiao-Fen Grace; Wang, Hwa-Tang Thomas; Balasuriya, Udeni B. R.

In: Journal of Clinical Microbiology, Vol. 54, No. 6, 06.2016, p. 1528-1535.

Research output: Journal Publications and Reviews (RGC: 21, 22, 62)21_Publication in refereed journalpeer-review