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


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.


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



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.


  1. 1.
    Schuffenecker, I., Iteman, I., Michault, A., Murri, S., Frangeul, L., Vaney, M.C., & Lavenir, R. (2006). Genome microevolution of Chikungunya viruses causing the Indian Ocean outbreak. PLoS Medicine, 3(7), e263.Google Scholar
  2. 2.
    Ravi, V. (2006). Re-emergence of Chikungunya virus in India. Indian Journal of Medical Microbiology, 24, 83–84.CrossRefGoogle Scholar
  3. 3.
    Jupp, P. G., & McIntosh, B. M. (1988). Chikungunya virus disease. In T. P. Monath (Ed.), The arboviruses: Epidemiology and ecology (pp. 137–157). Boca Raton, FL: CRC Press.Google Scholar
  4. 4.
    Carey, D. E. (1971). Chikungunya and dengue: A case of mistaken identity? Journal of the History of Medicine and Allied Sciences, 26, 243–262.CrossRefGoogle Scholar
  5. 5.
    Griffin, D. E. (2001). Alpha viruses. In P. M. Howley (Ed.), Field’s virology (pp. 917–962). New York: Lippincott Williams and Wilikins.Google Scholar
  6. 6.
    Khan, A. H., Morita, K., Mdel, M. D., Parquet, M. D., Mdel, C., Hasebe, F., et al. (2002). Complete nucleotide sequence of Chikungunya virus and evidence for an internal polyadenylation site. Journal of General Virology, 83, 3075–3084.Google Scholar
  7. 7.
    Strauss, J. H., & Strauss, E. G. (1994). The alphaviruses: Gene expression, replication, and evolution. Microbiological Reviews, 58, 491–562.Google Scholar
  8. 8.
    Powers, A. M., Brault, A. C., Tesh, R. B., & Weaver, S. C. (2000). Re-emergence of Chikungunya and O’nyong-nyong viruses: Evidence for distinct geographical lineages and distant evolutionary relationships. Journal of General Virology, 81, 471–479.Google Scholar
  9. 9.
    Bronzoni, R. V., Moreli, M. L., Cruz, A. C., & Figueiredo, L. T. (2004). Multiplex nested PCR for Brazilian Alphavirus diagnosis. Transactions of the Royal Society of Tropical Medicine and Hygiene, 98, 456–461.CrossRefGoogle Scholar
  10. 10.
    Greiser-Wilke, I. M., Moennig, V., Kaaden, O. R., & Shope, R. E. (1991). Detection of alphaviruses in a genus-specific antigen capture enzyme immunoassay using monoclonal antibodies. Journal of Clinical Microbiology, 29, 131–137.Google Scholar
  11. 11.
    Hasebe, F., Parquet, M. C., Pandey, B. D., Mathenge, E. G., Morita, K., & Balasubramaniam, V. (2002). Combined detection and genotyping of Chikungunya virus by specific reverse transcription-polymerase chain reaction. Journal of Medical Virology, 67, 370–374.CrossRefGoogle Scholar
  12. 12.
    Pfeffer, M., Proebster, B., Kinney, R. M., & Kaaden, O. S. (1997). Genus-specific detection of alphaviruses by a semi-nested reverse transcription-polymerase chain reaction. American Journal of Tropical Medicine and Hygiene, 57, 709–718.Google Scholar
  13. 13.
    Santhosh, S. R., Parida, M. M., Dash, P. K., & Rao, P. V. L. (2007). Development and evaluation of SYBR Green I-based one-step real-time RT-PCR assay for detection and quantification of Chikungunya virus. Journal of Clinical Virology, 39, 188–193.CrossRefGoogle Scholar
  14. 14.
    Pastorino, B., Bessaud, M., Grandadam, M., Murri, S., Tolou, H. J., & Peyrefitte, C. N. (2005). Development of a TaqMan RT-PCR assay without RNA extraction step for detection and quantification of African Chikungunya viruses. Journal of Virological Methods, 124, 65–71.CrossRefGoogle Scholar
  15. 15.
    Wang, W. K., Lee, C. N., Kao, C. L., Lin, Y. L., & King, C. C. (2000). Quantitative competitive reverse transcription-PCR for quantification of dengue virus RNA. Journal of Clinical Microbiology, 38, 3306–3310.Google Scholar
  16. 16.
    Zerlauth, G. (1996). IQ-PCR: A quality-assured and validated viral genome assay system. Haemostaseologie, 16, 279–281.Google Scholar
  17. 17.
    Drosten, C., Kummerer, B. M., Schmitz, H., & Gunthar, S. (2003). Molecular diagnostics of viral hemorrhagic fevers. Antiviral Research, 57, 61–87.CrossRefGoogle Scholar
  18. 18.
    Santhosh, S. R., Dash, P. K., Parida, M. M., Khan, M., Tiwari, M., & Rao, P. V. L. (2008). Comparative full genome analysis revealed A226V shift in 2007 Indian Chikungunya virus isolates. Virus Research, 135, 36–41.CrossRefGoogle Scholar
  19. 19.
    Dash, P. K., Parida, M. M., Santhosh, S. R., Saxena, P., Srivastava, A., Neeraja, M., et al. (2008). Development and evaluation of a 1-step duplex reverse transcription polymerase chain reaction for differential diagnosis of chikungunya and dengue infection. Diagnostic Microbiology and Infectious Disease, 62, 52–74.CrossRefGoogle Scholar
  20. 20.
    Krieg, P. (1991). Improved synthesis of full length RNA probe at reduced incubation temperatures. Nucleic Acids Research, 18, 6463.CrossRefGoogle Scholar
  21. 21.
    Kit, L. S. (2002). Emerging and re-emerging diseases in Malaysia. Asia-Pacific Journal of Public Health, 14, 6–8.Google Scholar
  22. 22.
    Telenti, A., Imboden, P., & Germann, D. (1992). Competitive polymerase chain reaction using an internal standard: Application to the quantitation of viral DNA. Journal of Virological Methods, 239, 259–268.CrossRefGoogle Scholar
  23. 23.
    Hazari, S., Acharya, S. K., & Panda, S. K. (2004). Development and evaluation of a quantitative competitive reverse transcription polymerase chin reaction (RT-PCR) for hepatitis C virus RNA in serum using transcribed thio-RNA as internal control. Journal of Virological Methods, 116, 45–54.CrossRefGoogle Scholar
  24. 24.
    Vaughan, G., Hernandez, G., Gudino, J., Olivera, H., Piedra, A., & Gutierrez, A. (2008). An alternative method for the synthesis of competitor RNA transcripts useful for specific detection and quantitation of dengue virus serotype 2 genome and replicative intermediate RNA. Journal of Virological Methods, 152, 72–76.CrossRefGoogle Scholar

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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