Journal
JOURNAL OF VIROLOGICAL METHODS
Volume 162, Issue 1-2, Pages 1-7Publisher
ELSEVIER
DOI: 10.1016/j.jviromet.2009.03.006
Keywords
Multiplex real-time RT-PCR; Arboviruses; Absolute quantitation; Internal control; Dengue virus typing; High Resolution Melting
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Chikungunya (CHIKV), Dengue (DENY) and West Nile (WNV) viruses are arthropod-borne viruses that are able to emerge or re-emerge in many regions due to climatic changes and increase in travel. Since these viruses produce similar clinical signs it is important for physicians and epidemiologists to differentiate them rapidly. A molecular method was developed for their detection and quantitation in plasma samples and a DENY typing technique were developed. The method consisted in performing two multiplex real-time one-step RT-PCR assays, to detect and quantify the three viruses. Both assays were conducted in a single run, from a single RNA extract containing a unique coextracted and coamplified composite internal control. The quantitation results were close to the best detection thresholds obtained with simplex RT-PCR techniques. The differentiation of DENV types was performed using a High Resolution Melting technique. The assays enable the early diagnosis of the three arboviruses during viremia, including cases of coinfection. The method is rapid, specific and highly sensitive with a potential for clinical diagnosis and epidemiological surveillance. A DENV positive sample can be typed conveniently using the High Resolution Melting technique using the same apparatus (C) 2009 Elsevier B.V. All rights reserved.
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