One-Step Rapid Reverse Transcription–PCR Assay for Detecting and Typing Dengue Viruses with GC Tail and Induced Fluorescence Resonance Energy Transfer Techniques for Melting Temperature and Color Multiplexing

Abstract

Background: Dengue fever is an arthropod-borne infection caused by dengue viruses (DVs; DEN-1 to DEN-4).Early diagnosis is critical to prevent severe disease progression and the spreading of DV because no vaccine or specific treatment is available; therefore, a rapid and specific diagnostic assay capable of detecting and typing all serotypes would be ideal.

Global Epidemiology of Dengue Outbreaks in 1990–2015: A Systematic Review and Meta-Analysis

“…Membrane blood feeding of antisera against mosGCTLs has been found to efficiently reduce DENV-2 infections among mosquitoes, suggesting that immunization against mosGCTLs may serve as a feasible approach for preventing dengue infection (Liu et al, 2014). Additionally, to reduce the rate of mortality in dengue patients, the application of early clinical and laboratory diagnosis, intravenous rehydration, staff training and hospital reorganization should be ensured during outbreaks (Lo et al, 2007; WHO, 2012). …”

RNA and DNA Diagnostics

“…These include melting curve analysis on traditional SYBR Green reaction (Jothikumar and Griffiths 2002), parallel real-time reactions in multiple tubes combined with melt-curve analysis to identify multiple viruses (Sugita et al 2008) or PCR primer tail extensions followed by melt-curve analysis to distinguish between viral serotypes, combined with fluorophore detection (Lo et al 2007) (Fig. 1).…”

Expecting the unexpected: nucleic acid-based diagnosis and discovery of emerging viruses

“…Real-time PCR has rapidly become the favored method of choice in clinical virology laboratories. It is routinely used in targetfocused tests for many viruses, including HIV-1 [53], parvovirus [54], BTV [55], WNV [56], dengue virus [57], influenza A virus, influenza B virus and at least eight human herpes viruses. Multiplexing is utilized in some of these tests, both to enable coamplification of internal and external controls along with the target viral nucleic acid (e.g., [55]), as well as for reasons of economy and work flow, to include several of the detectable targets in one or two reaction tubes.…”

“…Another strategy to increase the level of multiplexing is to utilize GC-rich extensions on the tails of PCR primers, allowing discrimination of amplified targets by melt-curve ana lysis. Lo et al used this approach, combined with the use of two fluorophores and the intercalating dye SYBR ® Green [57]. Using this hybrid strategy, the authors were able to detect and differentiate dengue virus serotypes 1, 2, 3 and 4.…”

“…Next season, the prevalent strain of influenza A virus may change, necessitating redesign of the assay and renegotiation of the regulatory process. Review Barnard, Hall & Gould Although sophisticated assays have been designed to increase the breadth of analytes detected in real-time assays, such as the use of extensions on PCR primers and melt-curve analysis [57], fluorescence systems are dependent on labels and remain limited by the number of optical detection channels. They are therefore limited in the number of target analytes that can be selected, necessitating redesign to accommodate the emergence of novel, recombinant, reassorted or genetic variants.…”