Single-tube nested PCR using immobilized internal primers for the identification of dengue virus serotypes

Abstract

Molecular techniques based on the detection of genomic sequences by reverse transcription (RT)-PCR, nested PCR, or real-time PCR have made possible the rapid diagnosis of dengue virus (DENV) infections, and these approaches have been accepted by clinical laboratories as the new standard method for the detection of dengue virus in acute-phase serum samples. One of these PCR-based assays, the two-step RT nested PCR (RT-NPCR) technique is used routinely in laboratories worldwide. In the present study, the two-step RT-NPCR as described by Lanciotti and collaborators (1992) was adapted to a novel single-tube nested PCR (STNPCR) format, which is less prone to cross-contamination and reduces reaction cost and time. When standards for each dengue serotype were tested, the detection limit of the STNPCR was at least 10 copies for DENV-1 and 100 copies for DENV-2 and DENV-3, whereas the detection limit for the two-step RT-NPCR was 100 copies for each serotype. Sera from 22 patients with confirmed DENV-3 infections and from 14 healthy individuals were then tested in the STNPCR format using the system described by Lanciotti as the reference standard. The results indicated a sensitivity of 75.9% (CI 95%,) and a specificity of 100% for the RT-STNPCR. Athough RT-STNPCR was less sensitive than the conventional twostep RT-NPCR for the detection of virus in serum samples, it was still adequately sensitive, and the advantages associated with a single-tube format may outweigh the somewhat lower assay sensitivity, making it useful for diagnosis in the field. Keywords nested PCR; diagnosis; dengue; single-tube Dengue virus is a mosquito-borne flavivirus and the most prevalent arbovirus in tropical and subtropical regions of Asia, Africa and Central and South America (Harris et al., 2000). There are four distinct serotypes, DENV-1, DENV-2, DENV-3 and DENV-4, all of which may cause an asymptomatic infection or a spectrum of diseases ranging from dengue fever (DF) to more * Corresponding author. Tel.: +; fax: +449. E-mail address: emarques@cpqam.fiocruz.br (E. T. A Marques). This manuscript is dedicated to the family and former students of Dr. Abath.Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. severe clinical forms such as dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS) (Guzman and Kouri, 2004). In recent years, dengue virus infection has emerged as a major public health problem, with expanding geographical distribution and increasing epidemic activity (Guha-Sapir and Schimmer, 2005). NIH Public AccessAlthough dengue virus infection confers protective immunity to the ho...

Characterization of small RNA populations in non-transgenic and aflatoxin-reducing-transformed peanut

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Peritrophic membrane structure of Aedes aegypti (Diptera: Culicidae) mosquitoes after infection with dengue virus type 2 (D2-16681)

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