Development and evaluation of SYBR Green I-based one-step real-time RT-PCR assay for detection and quantification of Chikungunya virus

• Published in: Journal of clinical virology 2007-07, Vol.39 (3), p.188-193
• Main Authors: 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
• Format: Article
• Language: English
• Subjects: Allergy and Immunology; Alphavirus Infections - diagnosis; Alphavirus Infections - virology; Biological and medical sciences; Chikungunya virus; Chikungunya virus - genetics; Chikungunya virus - isolation & purification; DNA Primers; Fluorescent Dyes - metabolism; Fundamental and applied biological sciences. Psychology; Human viral diseases; Humans; Infectious Disease; Infectious diseases; Medical sciences; Microbiology; Miscellaneous; Organic Chemicals - metabolism; Real-time PCR; Reverse Transcriptase Polymerase Chain Reaction - methods; RNA, Viral - blood; RNA, Viral - isolation & purification; Sensitivity and Specificity; Species Specificity; SYBR Green I; Viral diseases; Viral Envelope Proteins - genetics; Viral Load; Virology
• ISSN: 1386-6532; 1873-5967
• DOI: 10.1016/j.jcv.2007.04.015

Abstract The development of a one-step SYBR Green I-based real-time RT-PCR assay is reported for detection and 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 and cycle threshold ( Ct ) value over a range of 107 –0.1 PFU/ml. The reported assay was found to be 10-fold more sensitive compared to conventional RT-PCR with a detection limit of 0.1 PFU/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 standard curve, which varies from 0.1 to 107 PFU/ml. These findings demonstrated that the reported assay has the potential usefulness for clinical diagnosis due to simultaneous detection and quantification of Chikungunya virus in acute-phase patient serum samples.