Molecular Biotechnology

, Volume 45, Issue 1, pp 49–55 | Cite as

Development of a Quantitative Competitive Reverse Transcription Polymerase Chain Reaction (QC-RT–PCR) for Detection and Quantitation of Chikungunya Virus

  • Shashi Sharma
  • Paban Kumar Dash
  • S. R. Santhosh
  • Jyoti Shukla
  • Manmohan Parida
  • P. V. Lakshmana Rao
Research

Abstract

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, and 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 102 to 1010 copies. Specificity was confirmed using closely related alpha and 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 and accurate method and has the potential usefulness for clinical diagnosis due to simultaneous detection and quantification of CHIKV in acute-phase serum samples.

Keywords

QC-RT–PCR Molecular diagnosis Real-time PCR Chikungunya 

Notes

Acknowledgements

The authors are thankful to Director, Defence Research and Development Establishment, Jhansi Road, Gwalior, for his support, constant inspiration, and providing the necessary facilities for this study. This work is supported by an Institutional Build-up fund.

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Copyright information

© Springer Science+Business Media, LLC 2010

Authors and Affiliations

  • Shashi Sharma
    • 1
  • Paban Kumar Dash
    • 1
  • S. R. Santhosh
    • 1
  • Jyoti Shukla
    • 1
  • Manmohan Parida
    • 1
  • P. V. Lakshmana Rao
    • 1
  1. 1.Division of VirologyDefence Research & Development Establishment (DRDE)GwaliorIndia

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