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

• Published in: Journal of Clinical Microbiology Vol. 45; no. 2; pp. 351 - 357
• Main Authors: Parida, M.M, Santhosh, S.R, Dash, P.K, Tripathi, N.K, Lakshmi, V, Mamidi, N, Shrivastva, A, Gupta, N, Saxena, P, Babu, J. Pradeep, Rao, P.V. Lakshmana, Morita, Kouichi
• Format: Journal Article
• Language: English
• Published: Washington, DC American Society for Microbiology 01.02.2007
• Subjects: Acute Disease; Alphavirus Infections - diagnosis; Alphavirus Infections - virology; Animals; Biological and medical sciences; Cell Line; Chikungunya virus; Chikungunya virus - genetics; Chikungunya virus - isolation & purification; Cricetinae; Fundamental and applied biological sciences. Psychology; Humans; Infectious diseases; Medical sciences; Microbiology; Miscellaneous; Nucleic Acid Amplification Techniques - methods; Reverse Transcriptase Polymerase Chain Reaction - methods; RNA, Viral - analysis; RNA, Viral - blood; RNA, Viral - isolation & purification; Sensitivity and Specificity; Time Factors; Virology; Virus; Amplification; Microbiology; Transcription; Chikungunya virus; Togaviridae; Alphavirus; Detection; Real time
• ISSN: 0095-1137; 1098-660X; 1098-5530
• DOI: 10.1128/jcm.01734-06

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.