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Detection and serotyping of dengue virus in serum samples by multiplex reverse transcriptase PCR-ligase detection reaction assay.

TitleDetection and serotyping of dengue virus in serum samples by multiplex reverse transcriptase PCR-ligase detection reaction assay.
Publication TypeJournal Article
Year of Publication2008
AuthorsDas S, Pingle MR, Muñoz-Jordán J, Rundell MS, Rondini S, Granger K, Chang G-JJ, Kelly E, Spier EG, Larone D, Spitzer E, Barany F, Golightly LM
JournalJ Clin Microbiol
Volume46
Issue10
Pagination3276-84
Date Published2008 Oct
ISSN1098-660X
KeywordsDengue, Dengue Virus, Humans, Reverse Transcriptase Polymerase Chain Reaction, Sensitivity and Specificity, Serotyping, Serum
Abstract

The detection and successful typing of dengue virus (DENV) from patients with suspected dengue fever is important both for the diagnosis of the disease and for the implementation of epidemiologic control measures. A technique for the multiplex detection and typing of DENV serotypes 1 to 4 (DENV-1 to DENV-4) from clinical samples by PCR-ligase detection reaction (LDR) has been developed. A serotype-specific PCR amplifies the regions of genes C and E simultaneously. The two amplicons are targeted in a multiplex LDR, and the resultant fluorescently labeled ligation products are detected on a universal array. The assay was optimized using 38 DENV strains and was evaluated with 350 archived acute-phase serum samples. The sensitivity of the assay was 98.7%, and its specificity was 98.4%, relative to the results of real-time PCR. The detection threshold was 0.017 PFU for DENV-1, 0.004 PFU for DENV-2, 0.8 PFU for DENV-3, and 0.7 PFU for DENV-4. The assay is specific; it does not cross-react with the other flaviviruses tested (West Nile virus, St. Louis encephalitis virus, Japanese encephalitis virus, Kunjin virus, Murray Valley virus, Powassan virus, and yellow fever virus). All but 1 of 26 genotypic variants of DENV serotypes in a global DENV panel from different geographic regions were successfully identified. The PCR-LDR assay is a rapid, sensitive, specific, and high-throughput technique for the simultaneous detection of all four serotypes of DENV.

DOI10.1128/JCM.00163-08
Alternate JournalJ. Clin. Microbiol.
PubMed ID18685000
PubMed Central IDPMC2566068
Grant ListUC1-AI062579 / AI / NIAID NIH HHS / United States

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