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Journal of Clinical Microbiology, February 2007, p. 351-357, Vol. 45, No. 2
0095-1137/07/$08.00+0     doi:10.1128/JCM.01734-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Rapid and Real-Time Detection of Chikungunya Virus by Reverse Transcription Loop-Mediated Isothermal Amplification Assay{triangledown}

M. M. Parida,1* S. R. Santhosh,1 P. K. Dash,1 N. K. Tripathi,1 V. Lakshmi,2 N. Mamidi,2 A. Shrivastva,1 N. Gupta,1 P. Saxena,1 J. Pradeep Babu,1 P. V. Lakshmana Rao,1 and Kouichi Morita3

Division of Virology, Defence Research and Development Establishment, Gwalior 474002 M. P., India,1 Department of Microbiology, Nizam's Institute of Medical Sciences, Hyderabad 500 082 A. P., India,2 Department of Virology, Institute of Tropical Medicine, Nagasaki University, 1-12-4 Sakamoto, Nagasaki 852-8523, Japan3

Received 22 August 2006/ Returned for modification 23 October 2006/ Accepted 8 November 2006

The standardization and validation of a one-step, single-tube, accelerated, quantitative reverse transcription (RT) loop-mediated isothermal amplification (RT-LAMP) assay targeting the E1 gene for the rapid and real-time detection of Chikungunya virus (CHIKV) are reported. A linear relationship between the amount of template and time of positivity value over a range of 2 x 108 to 2 x 102 copies was obtained. The feasibility of CHIKV RT-LAMP for clinical diagnosis was validated with patient serum samples from an ongoing epidemic in Southern India. Optimal assay conditions with zero background were established for the detection of low levels of CHIKV in acute-phase patient serum samples. The comparative evaluation of the RT-LAMP assay with acute-phase patient serum samples demonstrated exceptionally higher sensitivity by correctly identifying 21 additional positive borderline cases that were missed by conventional RT-PCR (P < 0.0001) with a detection limit of 20 copies. The quantification of virus load in patient serum samples was also determined from the standard curve based on their time of positivity and was found to be in the range of 2 x 108 to 2 x 101 copies. In addition, the field applicability of the RT-LAMP assay was also demonstrated by standardizing SYBR Green I-based RT-LAMP wherein the amplification was carried out in a water bath at 63°C for 60 min, which was followed by monitoring gene amplification with the naked eye through color changes. These findings demonstrated that the RT-LAMP assay is a valuable tool for rapid, real-time detection as well as quantification of CHIKV in acute-phase serum samples without requiring any sophisticated equipment and has potential usefulness for clinical diagnosis and surveillance of CHIKV in developing countries.

* Corresponding author. Mailing address: Division of Virology, Defence R & D Establishment, Jhansi Road, Gwalior 474002 M. P., India. Phone: 91-751-2233495. Fax: 91-751-2351148. E-mail: paridamm{at}rediffmail.com.

{triangledown} Published ahead of print on 29 November 2006.

Journal of Clinical Microbiology, February 2007, p. 351-357, Vol. 45, No. 2
0095-1137/07/$08.00+0     doi:10.1128/JCM.01734-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.



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