Development of a sensitive real-time reverse transcriptase PCR assay with an internal control to detect and quantify chikungunya virus

Autor(es): Laurent Philippe, Le Roux Karin, Grivard Philippe, Bertil Gérard, Naze Florence, Picard Miguel, Staikowsky Frédérik, Barau Georges, Schuffenecker Isabelle, Michault Alain


Resumo: The chikungunya virus (CHIKV; Alphavirus, Togaviridae) has emerged in the south Western Indian Ocean since early 2005. A major outbreak of CHIKV infection occurred in Réunion Isl-, where the virus is transmitted by Aedes albopictus mosquitoes. Facing an outbreak of unprecedented magnitude, we developed a rapid, sensitive, - reliable assay for the detection - quantification of CHIKV in plasma samples. A dual-color TaqMan 1-step reverse transcriptase PCR assay was developed in a LightCycler 2.0 system. A coextracted - coamplified chimerical RNA sequence was used as an internal control (IC) to eliminate false-negative results. The CHIKV-specific - IC probes were labeled with 6-carboxyfluorescein (530 nm) - the wide span dye DYXL (705 nm), respectively, eliminating the need for color compensation. A synthetic RNA was used as an external calibrator for CHIKV absolute quantification. The detection limit was 350 copies/mL (3 copies/capillary). A further improvement to approximately 40 copies/mL was obtained by use of a larger volume of plasma. The assay specificity was confirmed in vitro - in silico. CHIKV in 343 patients was present at viral loads >10(8) copies/mL, mainly in newborns - seniors >60 years old. Long viremic phases of up to 12 days were seen in 6 patients. The assay is rapid, CHIKV-specific, - highly sensitive, - it includes an IC. It proved useful to detect - quantify CHIKV during the Réunion Isl- epidemic. The assay might be applicable to other CHIKV epidemics, especially in the Indian subcontinent, where an extensive outbreak is ongoing.


Imprenta: Clinical Chemistry, v. 53, n. 8, p. 1408-1414, 2007


Identificador do objeto digital: 10.1373/clinchem.2007.086595


Descritores: Chikungunya virus - Pathogenesis ; Chikungunya virus - RNA ; Chikungunya virus - Viral infections ; Chikungunya virus - PCR detection ; Chikungunya virus - Real Time PCR ; Chikungunya virus - RT-PCR ; Chikungunya Virus - Virus ; Chikungunya virus - Epidemic ; Chikungunya virus - Public health


Data de publicação: 2007