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

e 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. Dengue virus (DENV) is a mosquito-borne human pathogen that belongs to the genus Flavivirus in the family Flaviviridae (1). DENV is an enveloped virus with a single-stranded, positivesense RNA genome approximately 10.7 kb in length (2). The genomic RNA includes 5= and 3= untranslated regions (UTRs) and a single open reading frame that encodes a single polyprotein that is cleaved into three structural proteins (capsid [C], premembrane/membrane [prM/M], and envelope [E]) and seven nonstructural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5) (3). DENV is comprised of four distinct serotypes (DENV-1, -2, -3, and -4), and all four serotypes are currently circulating in the Pacific Islands, Americas, Asia, and Africa. In addition to this, distinct genotypes have been identified within each serotype (4, 5), highlighting the extensive genetic variability of the DENV serotypes.DENV infection is considered a major public health problem in developing tropical countries where the virus is endemic, and it is continuously spreading to new geographical areas around the world (6, 7). Moreover, frequent international travel to regions where dengue is endemic or epidemic increases the risk of DENV infection for travelers (8,9), and travel contributes to the escalating numbers of imported dengue cases in temperate regions. More than 2.5 billion people ...

Field-Deployable Reverse Transcription-Insulated Isothermal PCR (RT-iiPCR) Assay for Rapid and Sensitive Detection of Foot-and-Mouth Disease Virus

“…The POCKIT ™ analyser is a relatively inexpensive (~US$ 5000.00) compared with currently available thermocyclers. It is a compact (28 × 25 × 8.5 cm), lightweight (~2.1 kg) and user‐friendly instrument specially designed to perform PCR and RT‐PCR assays based on Rayleigh–Bénard convection (Chang et al., ; Tsai et al., ; Balasuriya, ; Ambagala et al., ; Chua et al., ; Go et al., ). It measures the fluorescence intensity of each sample once prior to and once after the PCR or RT‐PCR amplification is complete, automatically calculates the ratio between initial and final fluorescence intensities or signal‐to‐noise ratio (R1) and therefore requires no data interpretation.…”

Validation of the Pockit Dengue Virus Reagent Set for Rapid Detection of Dengue Virus in Human Serum on a Field-Deployable PCR System

“…Hence, the index assay can serve as a relatively inexpensive, rapid, and simple point-of-need (PON) tool for early detection of viremic dengue patients in routine dengue diagnosis, improving clinical management in underserved communities. The pan-DENV RT-iiPCR described previously also had excellent analytical sensitivity, specificity, and reproducibility (43).…”

“…The POTKIT dengue virus reagent set, targeting the 3= untranslated region of the DENV RNA genome, was derived from the pan-DENV RT-iiPCR system reported previously (43). In this study, clinical performance of the assay was assessed with serum samples collected from dengue-suspected patients in Taiwan.…”

Neutralization of Acidic Intracellular Vesicles by Niclosamide Inhibits Multiple Steps of the Dengue Virus Life Cycle In Vitro

“…TCF was clarified by centrifugation, and 1-ml aliquots were stored at −80 °C until further use. Viral titres were quantified by focus-forming assay on Vero76 cells as described previously 54 . Zika virus, an MR766 strain (ATCC ® VR-1838 TM ) purchased from ATCC, was amplified, and viral titres were measured by plaque assay on Vero76 cells.…”

“…Viral RNA in the TCF samples was extracted using a QIAamp ® Viral RNA Mini kit (Qiagen) according to the manufacturer's instructions. RT-qPCR was performed using a SuperScript III one-step RT-PCR system with Platinum Taq polymerase (Invitrogen), primers/probe sets targeting NS5 or E genes and a QuantStudio 6 real-time PCR system (Applied Biosystems, Foster City, CA, USA) as described previously 54 . The relative viral RNA expression levels were calculated by the ΔΔC T method, and β-actin was used as an endogenous control.…”