Rapid detection and typing of dengue viruses from clinical samples by using reverse transcriptase-polymerase chain reaction

Abstract

We report on the development and application of a rapid assay for detecting and typing dengue viruses. Oligonucleotide consensus primers were designed to anneal to any of the four dengue virus types and amplify a 511-bp product in a reverse transcriptase-polymerase chain reaction (PCR). First, we produced a cDNA copy of a portion of the viral genome in a reverse transcriptase reaction in the presence of primer D2 and then carried out a standard PCR (35 cycles of heat denaturation, annealing, and primer extension) with the addition of primer Dl. The resulting double-stranded DNA product of the RT-PCR was typed by two methods: dot blot hybridization of the 511-bp amplified product to dengue virus type-specific probes or a second round of PCR amplification (nested PCR) with type-specific primers, yielding DNA products the unique sizes of which were diagnostic for each dengue virus serotype. The accumulated data demonstrated that dengue viruses can be accurately detected and typed from viremic human serum samples.

Rapid detection and differentiation of dengue virus serotypes by NS1 specific reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay in patients presenting to a tertiary care hospital in Hyderabad, India

“…Although, WHO (World Health Organization, 2009) recommends the detection of DENV RNA, as the most effective diagnostic method in the acute phase of the illness, its use is limited due to lack of infrastructure and technical expertise. More recently, molecular techniques to detect virus genomic RNA sequence by the reverse transcription-polymerase chain reaction (RT-PCR) and the real-time quantitative RT-PCR (qRT-PCR) are gradually being accepted as new standards over virus isolation for the detection of DENV in the acute sera (Lanciotti et al, 1992;Shu et al, 2003). These PCR-based methods require either high-precision instruments for the amplification or elaborate methods for detection of the amplified products.…”

“…In addition, a panel of 50 samples collected from healthy individuals was included as negative controls. Before performing the RT-LAMP assay, all the 350 samples were also screened for DENVspecific RNA by RT-PCR (Lanciotti et al, 1992;Neeraja et al, 2013) and NS1 antigen by Panbio Dengue Early ELISA assay (Inverness Medical Innovations, Australia), Dengue IgG and IgM capture ELISA (Pan Bio, Queensland, Australia).…”

Covert COVID-19 and false-positive dengue serology in Singapore

“…A chest radiograph led to testing for SARS-CoV-2 by RT-PCR (in-house laboratory-developed test detecting the N and ORF1ab genes) from a nasopharyngeal swab, which returned positive. The original seropositive sample and additional urine and blood samples tested negative for dengue, chikungunya, and Zika viruses by RT-PCR, [3][4][5] and a repeat dengue rapid test (SD Bioline) was also negative. Thus, the initial dengue seroconversion result was deemed a false positive.…”

Computational prediction and identification of dengue virus-specific CD4+ T-cell epitopes

“…We also included three flavivirusnaïve Guangdong residues. DEN-1 infection was confirmed by virus isolation in C6/36 cell culture and subsequent serotype identification with DEN serotype-specific RT-PCR during the viremia-phase (Lanciotti et al, 1992). In addition, DEN infection was also confirmed by using commercial capture-immunoglobulin M (IgM) and IgG enzyme-linked immunosorbent assay (ELISA) during the convalescent-phase as described previously (Kuno et al, 1985).…”