Development and evaluation of SYBR Green I-based one-step real-time RT-PCR assay for detection and quantification of Chikungunya virus
Autor(es): Santhosh S R, Parida M M, Dash P K, Pateriya A, Pattnaik B, Pradhan H K, Tripathi N K, Ambuj S, Gupta N, Saxena P, Lakshmana Rao P V
Resumo: The development of a one-step SYBR Green I-based real-time RT-PCR assay is reported for detection - quantification of Chikungunya virus (CHIKV) in acute-phase patient serum samples by targeting the E1 structural gene. A linear relationship was obtained between the virus concentration - cycle threshold (C(t)) value over a range of 10(7)-0.1PFU/ml. The reported assay was found to be 10-fold more sensitive compared to conventional RT-PCR with a detection limit of 0.1PFU/ml. The feasibility of this reported assay system for clinical diagnosis was validated with 51 suspected acute-phase serum samples of the recent CHIKV epidemic in southern India, 2006. The comparative evaluation with acute-phase patient serum samples revealed the higher sensitivity of real-time RT-PCR assay by picking up six additional samples with low copy number of template. None of the healthy serum samples analyzed in this study showed amplification. The quantification of the viral load in the acute-phase serum samples was also determined employing the st-ard curve, which varies from 0.1 to 10(7)PFU/ml. These findings demonstrated that the reported assay has the potential usefulness for clinical diagnosis due to simultaneous detection - quantification of Chikungunya virus in acute-phase patient serum samples.
Palavras-Chave: SYBR Green I; Real-time PCR; Chikungunya virus
Imprenta: Journal of Clinical Virology, v. 39, n. 3, p. 188-193, 2007
Identificador do objeto digital: 10.1016/j.jcv.2007.04.015
Descritores: Chikungunya virus - Biosynthesis ; Chikungunya virus - DNA ; Chikungunya virus - Pathogenesis ; Chikungunya virus - Proteins ; 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
