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

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 ϳ40 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 >10

A Mouse Model of Chikungunya Virus–Induced Musculoskeletal Inflammatory Disease

“…RNA was reverse transcribed with superscript III (Invitrogen) and analyzed by 25 cycles of PCR for the presence of ␤-actin or 30 cycles of PCR for the presence of CHIKV using previously published primers specific for the E1 region of the genome. 23 The ic-CHIKVSL15649 plasmid was used as a positive control. RNA samples that had not been subjected to reverse transcription were used as a negative control.…”

Persistent Chronic Inflammation and Infection by Chikungunya Arthritogenic Alphavirus in Spite of a Robust Host Immune Response

“…High viral load is more likely to be detected in newborns and elderly CHIKV disease patients, but to what extent can this contribute to increased pathogenesis remains to be addressed (21). Interestingly, and in contrast to Dengue virus, the apparition of IgG anti-CHIKV Abs was detected in the first week (even at day 2 in some patients) following infection and illustrating the rapid seroconversion and robust adaptive immune response (8).…”

Development of Field-Based Real-Time Reverse Transcription–Polymerase Chain Reaction Assays for Detection of Chikungunya and O’nyong-nyong Viruses in Mosquitoes

“…The molecular diagnosis of CHIKV has only been evaluated for human samples [16][17][18][19][20][21][22][23] and no real-time RT-PCR assay exists for detecting ONNV present in human or mosquito F igure 1. Detection of CHIKV in infected mosquitoes using real-time reverse transcription-polymerase chain reaction.…”

“…Several groups have developed real-time RT-PCR assays for the molecular diagnosis of CHIK involving human samples. [16][17][18][19][20][21][22][23] However, these assays have not been evaluated for detecting CHIKV RNA in infected mosquitoes. Additionally, no real-time RT-PCR assay exists for detecting ONNV RNA in human or mosquito samples.…”