Development of a Sensitive Real-Time Reverse Transcriptase PCR Assay with an Internal Control to Detect and Quantify Chikungunya Virus

Abstract

Abstract Background: 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 Island, where the virus is transmitted by Aedes albopictus mosquitoes. Facing an outbreak of unprecedented magnitude, we developed a rapid, sensitive, and reliable assay for the detection and quantification of CHIKV in plasma samples. Methods: A dual-color TaqMan 1-step reverse transcriptase PCR assay was developed in a LightCycler 2.0 system. A coextracted and coamplified chimerical RNA sequence was used as an internal control (IC) to eliminate false-negative results. The CHIKV-specific and IC probes were labeled with 6-carboxyfluorescein (530 nm) and 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. Results: The detection limit was 350 copies/mL (3 copies/capillary). A further improvement to ∼40 copies/mL was obtained by use of a larger volume of plasma. The assay specificity was confirmed in vitro and in silico. CHIKV in 343 patients was present at viral loads >108 copies/mL, mainly in newborns and seniors >60 years old. Long viremic phases of up to 12 days were seen in 6 patients. Conclusions: The assay is rapid, CHIKV-specific, and highly sensitive, and it includes an IC. It proved useful to detect and quantify CHIKV during the Réunion Island epidemic. The assay might be applicable to other CHIKV epidemics, especially in the Indian subcontinent, where an extensive outbreak is ongoing.