Development of a quantitative competitive reverse transcription polymerase chain reaction (QC-RT-PCR) for detection and quantitation of Chikungunya virus
Autor(es): Sharma Shashi, Dash Paban Kumar, Santhosh S R, Shukla Jyoti, Parida Manmohan, Rao P V Lakshmana
Resumo: Chikungunya is one of the most important emerging arboviral infections of public health significance. Due to lack of a licensed vaccine, rapid diagnosis plays an important role in early management of patients. In this study, a QC-RT-PCR assay was developed to quantify Chikungunya virus (CHIKV) RNA by targeting the conserved region of E1 gene. A competitor molecule containing an internal insertion was generated, which provided a stringent control of the quantification process. The introduction of 10-fold serially diluted competitor in each reaction was further used to determine sensitivity. The applicability of this assay for quantification of CHIKV RNA was evaluated with human clinical samples, - the results were compared with real-time quantitative RT-PCR. The sensitivity of this assay was estimated to be 100 RNA copies per reaction with a dynamic detection range of 10(2) to 10(10) copies. Specificity was confirmed using closely related alpha - flaviviruses. The comparison of QC-RT-PCR result with real-time RT-PCR revealed 100% concordance for the detection of CHIKV in clinical samples. These findings demonstrated that the reported assay is convenient, sensitive - accurate method - has the potential usefulness for clinical diagnosis due to simultaneous detection - quantification of CHIKV in acute-phase serum samples.
Palavras-Chave: QC-RT-PCR; Molecular diagnosis; Real-time PCR; Chikungunya
Imprenta: Molecular Biotechnology, v. 45, n. 1, p. 49-55, 2010
Identificador do objeto digital: 10.1007/s12033-009-9238-9
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 - Vaccine ; Chikungunya virus - Public health
Data de publicação: 2010
