Detection of Dengue Virus RNA in Patients after Primary or Secondary Dengue Infection by Using the TaqMan Automated Amplification System

Abstract

In consecutive serum samples from 25 tourists with acute dengue fever, virus-specific RNA was detected by using fully automated TaqMan reverse transcriptase PCR. For this amplification technique new primers and special fluorochrome-labeled probes had to be synthesized. During amplification the increasing amount of viral DNA could simultaneously be measured in the tightly sealed tubes. Dengue virus RNA was found in almost all patients (17 of 18), if the samples had been taken soon after the onset of symptoms and before anti-dengue virus antibody had been produced. RNA was detectable in only one of five persons who had anti-dengue virus immunoglobulin M (IgM) antibodies but not yet IgG antibodies. In 30 late samples with both IgG and IgM antibodies viral RNA was no longer demonstrable. In two early samples from two frequent travelers obtained 1 and 2 days after the onset of symptoms significant IgG antibody titers were present but there were no anti-dengue virus IgM antibodies. In these samples a viral load of >5 × 106 dengue virus RNA copies (dengue types 1 and 2) was detectable. These findings of a high viral load in the presence of anti-dengue virus IgG antibody are suggestive of a secondary dengue virus infection. In the 20 tourists (17 plus 1 plus 2) in whom viral RNA was found, the dengue virus serotype could be related to the area where the infection had taken place. Most of our patients came from southeast Asia and most frequently had dengue virus type 1 infections (8 of 20).

Recent advancements for the evaluation of anti-viral activities of natural products

“…Mathematical analysis of the data, and comparison to control reactions containing known amounts of template, allows one to calculate the amount of input DNA in the initial reaction. The severity of some diseases has been shown to correlate with the viral load, making real-time PCR quantitation useful to study, not simply the presence of a virus but the role of viral reactivation or persistence in the progression of the disease [27][28][29]. This property has been successfully adapted to screen potential anti-viral test compounds in vitro.…”

A novel coding-region RNA element modulates infectious dengue virus particle production in both mammalian and mosquito cells and regulates viral replication in Aedes aegypti mosquitoes

“…qRT-PCR was performed using a modification to the method of Laue et al (1999), which uses a FAM-TAMRA labeled probe and primer set that is directed to the viral NS5 gene. The TaqMan One-Step Master Mix (Applied Biosystems) system was used with the following modifications to the protocol: the RT step was conducted at 48 °C for 30 min, the initial denaturation step was performed at 95 °C for 10 min, the annealing temperature was set at 60 °C, and the reaction volume was fixed at 20 μL.…”

Real-time Fluorescent PCR Techniques to Study Microbial–Host Interactions

“…More commonly, real-time PCR studies simply describe the presence of viruses in their host with a long-term view of improving clinical management of the affected patients. Viruses studied to date include the bunyaviruses (Stram et al, 2004), caliciviruses (Song et al, 1997), flaviviruses (Laue et al, 1999;Kilpatrick et al, 1996;White et al, 2002;Callahan et al, 2001;Ratge et al, 2002;Ishiguro et al, 1995;Komurian-Pradel et al, 2001;Lanciotti et al, 2000;Beames et al, 2000), hepadnaviruses (Weinberger et al, 2000;Chen et al, 2001;Cane et al, 1999;Brechtbuehl et al, 2001), herpesviruses Takaya et al, 1996;Song et al, 2002;Whiley et al, 2003b;Fan and Gulley, 2001;Schalasta et al, 2000b;Kearns et al, 2001a;Stevens et al, 2002;Loparev et al, 2000;Gallagher et al, 1999;Kennedy et al, 1997b;Fernandez et al, 2002;Biggar et al, 2000;Tanaka et al, 2000b;Locatelli et al, 2000;Ohyashiki et al, 2000;Kearns et al, 2001c;Lallemand et al, 2000;White and Campbell, 2000;Najioullah et al, 2001;Schaade et al, 2000;Capone et al, 2001;Lo et al, 1999;Niesters et al, 2000;…”

“…Quantitative real-time PCR has similarly examined the interaction between virus and host and monitored changes in viral load resulting from antiviral therapy ultimately impacting on the treatment regimen selected (Nitsche et al, 1999;Clementi, 2000;Limaye et al, 2000). Because disease severity and viral load are linked, microbial quantitation by real-time PCR has proven beneficial when studying the impact of viral reactivation, persistence or isolated gene expression on the progression of disease (Ohyashiki et al, 2000;Chen et al, 2003;Kearns et al, 2001c;Furuta et al, 2001;Limaye et al, 2001;Tanaka et al, 2000a,b;Lallemand et al, Real-time Fluorescent PCR Techniques to Study 2000;Laue et al, 1999;Kimura et al, 1999;Chang et al, 1999;Lo et al, 1999;Hawrami and Breur, 1999;Hoshino et al, 2000;Nitsche et al, 2000;Machida et al, 2000;Limaye et al, 2001;Najioullah et al, 2001;Gault et al, 2001). Altered microbial tropism or replication and the effect of these changes on the host cell can also be followed using real-time PCR (Kennedy et al, 1998a(Kennedy et al, , 1999a.…”