Development and Evaluation of Serotype- and Group-Specific Fluorogenic Reverse Transcriptase PCR (TaqMan) Assays for Dengue Virus

Abstract

Five fluorogenic probe hydrolysis (TaqMan) reverse transcriptase PCR (RT-PCR) assays were developed for serotypes 1 to 4 and group-specific detection of dengue virus. Serotype-and group-specific oligonucleotide primers and fluorogenic probes were designed against conserved regions of the dengue virus genome. The RT-PCR assay is a rapid single-tube method consisting of a 30-min RT step linked to a 45-cycle PCR at 95 and 60°C that generates a fluorogenic signal in positive samples. Assays were initially evaluated against cell culture-derived dengue stock viruses and then with 67 dengue viremic human sera received from Peru, Indonesia, and Taiwan. The TaqMan assays were compared to virus isolation using C6/36 cells followed by an immunofluorescence assay using serotype-specific monoclonal antibodies. Viral titers in sera were determined by plaque assay in Vero cells. The serotype-specific TaqMan RT-PCR assay detected 62 of 67 confirmed dengue virus-positive samples, for a sensitivity of 92.5%, while the group-specific assay detected 66 of 67 confirmed dengue virus-positive samples, for a sensitivity of 98.5%. The TaqMan RT-PCR assays have a specificity of 100% based on the serotype concordance of all assays compared to cell culture isolation and negative results obtained when 21 normal human sera and plasma samples were tested. Our results demonstrate that the dengue virus TaqMan RT-PCR assays may be utilized as rapid, sensitive, and specific screening and serotyping tools for epidemiological studies of dengue virus infections.

Temperature and Dengue Virus Infection in Mosquitoes: Independent Effects on the Immature and Adult Stages

“…The amount of viral RNA present in samples was determined using the Superscript III One-Step Quantitative RT-PCR System (qRT-PCR; Invitrogen, Carlsbad, CA) with a Light Cycler 480 system (Roche, Mannheim, Germany) using methods described elsewhere. 47 A standard curve method was used to relate the amount of DENV RNA present in samples to 10-fold serial dilutions of virus stock with known concentrations expressed in pfu per milliliter. 48,49 Mosquitoes were categorized based on status of infection: disseminated infections, positively infected bodies and legs; non-disseminated infections, infected bodies but the absence of virus in legs; and uninfected, absence of virus in the body.…”

Development of Multiplex Real-Time Reverse Transcriptase PCR Assays for Detecting Eight Medically Important Flaviviruses in Mosquitoes

“…The same fluorescence combination format has been applied for detection of DENV-1 to -4 in serum specimens from patients with dengue fever (4,14). The combination of universal primer pairs with four virus-specific probes simplifies the optimization step (4; this study); in contrast, other multiplex designs require four unique primer pairs and four different probes (2,14). Another significant improvement is the development of an in vitro-transcribed RNase-resistant RNA template for use as the copy number control.…”

Crimean–Congo hemorrhagic fever

“…The real-time PCR assay has many advantages over conventional RT-PCR methods, including lower contamination rate, higher sensitivity and specificity, and they are rapid, providing results in minutes instead of hours. Several investigators have reported the use of real-time PCR assays for detecting some viral causes of hemorrhagic fevers (Drosten et al, 2002a), including Ebola (Gibb et al, 2001;Towner et al, 2004;Weidmann et al, 2004), Rift Valley fever (Garcia et al, 2001), and dengue (Laue et al, 1999;Callahan et al, 2001;Houng et al, 2000) viruses. Drosten et al (2002a) developed a one-step real-time RT-PCR assay for detecting CCHFV using primers to the nucleoprotein gene; however, they used the DNA-intercalating dye, SybrGreen I, for detecting the PCR product because no conserved binding site for a 5 -nuclease probe could be found.…”