Detection of Dengue Viral RNA Using a Nucleic Acid Sequence-Based Amplification Assay

Wu, Shuenn-Jue; Lee, Eun Mi; Putvatana, Ravithat; Shurtliff, Roxanne N.; Porter, Kevin R.; Wuryadi, Suharyono; Watts, Douglas M.; King, Chwan-Chuen; Murphy, Gerald S.; Hayes, Curtis G.; Romano, Joseph

doi:10.1128/jcm.39.8.2794-2798.2001

Cited by 103 publications

(70 citation statements)

References 18 publications

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“…Results were obtained in less than two hours. 159 Wu et al, 2001, applied an isothermal nucleic acid sequence-based amplification (NASBA) assay to amplify the four dengue serotypes using a set of universal primers and serotype-specific capture probes for typing. Nucleic acid was amplified without thermo cycling and the product was detected by probe hybridization using electrochemiluminescence.…”

Section: Genome Detectionmentioning

confidence: 99%

Dengue diagnosis, advances and challenges

Guzmán

Kourı́

2004

International Journal of Infectious Diseases

277

225

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Dengue diagnosis was one of the topics discussed at the symposium 'The Global Threat of Dengue - Desperately Seeking Solutions' organized during the 10th International Congress of Infectious Diseases held in Singapore in 2002. In this paper, a review is presented focusing on the main advances, problems and challenges of dengue diagnosis.IgM capture ELISA, virus isolation in mosquito cell lines and live mosquitoes, dengue specific monoclonal antibodies and PCR have all represented major advances in dengue diagnosis. However, an appropriate rapid, early and accessible diagnostic method useful both for epidemiological surveillance and clinical diagnosis is still needed. Also, tools that suggest a prognosis allowing for better management are also needed. Finally, laboratory infrastructure, technical expertise and research capacity must be improved in endemic countries in order to positively influence dengue surveillance, clinical case management and the development of new approaches to dengue control.

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Section: Genome Detectionmentioning

confidence: 99%

Dengue diagnosis, advances and challenges

Guzmán

Kourı́

2004

International Journal of Infectious Diseases

277

225

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“…The plaque reduction neutralization tests were performed using modified protocols from Russell et al, Kochel et al, and Wu et al (9,14,18). Vero cells were seeded in six-well plates at a density of 2.5 ϫ 10 5 cells/well and incubated overnight.…”

Section: Methodsmentioning

confidence: 99%

Flow Cytometry-Based Assay for Titrating Dengue Virus

et al. 2005

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Plaque assays for titrating dengue virus (DENV) are time-consuming and not suitable for strains that do not plaque. Fluorescence-activated cell sorting (FACS) has been used to detect DENV-infected cells. Here we describe a FACS-based assay for titrating DENV. We determined that at 24 h postinfection, the number of infected cells detected by FACS represented the first round of infection and therefore could be used as a readout of the number of infectious particles in the inoculum. When the titers of different laboratory and clinical strains of DENV were compared using FACS, plaque, and endpoint dilution assays, for most strains the FACS titers were comparable to titers obtained by plaque or endpoint dilution assays. The FACS assay is an improvement over the plaque assay because the infection period is reduced from 5 to 7 days to 24 h and the assay can be used to titrate clinical isolates that frequently do not form clear plaques on cell monolayers. The novel FACS-based methods described here will facilitate laboratory studies of dengue.Dengue is a mosquito-borne viral disease of global public health significance. Throughout the world there are more than 2.5 billion people at risk of dengue virus (DENV) infection. Each year there are an estimated 100 million cases of dengue viral infection worldwide, with 250,000 people developing the more severe dengue hemorrhagic fever/dengue shock syndrome, which is often fatal (5). DENV is a positive-polarity RNA virus in the family Flaviviridae, and the DENV complex consists of four distinct serotypes designated DENV1 through DENV4 (10).Laboratory studies of dengue virus are difficult because the virus does not grow to high titers in cell culture and assays for titrating virus and measuring virus neutralization are timeconsuming. These problems are exacerbated when working with primary clinical isolates of virus. Standard methods for titrating DENV and measuring the ability of antisera to neutralize the virus are based on plaque assays which require 5 to 7 days to complete. Some isolates, especially among primary clinical isolates, do not form clear plaques on cell monolayers. Better methods for titrating the virus and measuring the ability of antisera to neutralize dengue need to be developed. Within the past 10 years, fluorescence-activated cell sorter (FACS)-based methods have been developed to follow infection and determine titers of viruses such as human immunodeficiency virus, herpesvirus, measles virus, influenza virus, Epstein-Barr virus, and rabies virus (1,4,12,13,16). FACS has also been used to detect DENV in clinical samples and to measure the ability of the virus to infect a variety of cells (2,3,7,8,11,17). Here we report on a FACS-based assay for titrating DENVs and for characterizing the ability of antisera to neutralize the virus. MATERIALS AND METHODSCells, viruses, and antisera. Aedes albopictus C6/36 mosquito cells were obtained from the Centers for Disease Control and Prevention, Fort Collins. Vero 76 (African Green monkey kidney) cells were purchased fro...

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“…These results clearly demonstrated that the proposed assay is capable of detecting DV in a serotype-specific manner with high sensitivity. Unlike the two-step NASBA amplification and hybridization and ECL detection procedure (Wu et al, 2001), the amplification, detection, and serotyping of DVs were accomplished in one step in a single tube within a shorter period of time. In principle, DV detection could be achieved with a very simple device, for example a battery-powered UV lamp since the QDs can be excited at any wavelength beyond the shortest absorption wavelength of the QDs.…”

Section: Selectivitymentioning

confidence: 99%