Rapid Detection and Differentiation of Dengue Virus Serotypes by a Real-Time Reverse Transcription-Loop-Mediated Isothermal Amplification Assay

Author:

Parida Manmohan1,Horioke Kouhei1,Ishida Hiroyuki1,Dash Paban Kumar2,Saxena Parag2,Jana Asha Mukul2,Islam Mohammed Alimul1,Inoue Shingo1,Hosaka Norimitsu3,Morita Kouichi14

Affiliation:

1. Department of Virology, Institute of Tropical Medicine, Nagasaki University, 1-12-4, Sakamoto, Nagasaki 852-8523, Japan

2. Department of Virology, Defense R & D Establishment, Jhansi Road, Gwalior-474002, M. P., India

3. Eiken Chemical Co. Ltd., 1381-3 Shimoishigami, Ohtawara, Tochigi 324-0036, Japan

4. CREST, Japan Science and Technology Corporation, Saitama 332-0012, Japan

Abstract

ABSTRACT The development and validation of a one-step, real-time, and quantitative dengue virus serotype-specific reverse transcription-loop-mediated isothermal amplification (RT-LAMP) assay targeting the 3′ noncoding region for the rapid detection and differentiation of dengue virus serotypes are reported. The RT-LAMP assay is very simple and rapid, wherein the amplification can be obtained in 30 min under isothermal conditions at 63°C by employing a set of four serotype-specific primer mixtures through real-time monitoring in an inexpensive turbidimeter. The evaluation of the RT-LAMP assay for use for clinical diagnosis with a limited number of patient serum samples, confirmed to be infected with each serotype, revealed a higher sensitivity by picking up 100% samples as positive, whereas 87% and 81% of the samples were positive by reverse transcription-PCR and virus isolation, respectively. The sensitivity and specificity of the RT-LAMP assay for the detection of viral RNA in patient serum samples with reference to virus isolation were 100% and 93%, respectively. The optimal assay conditions with zero background and no cross-reaction with other closely related members of the Flavivirus family (Japanese encephalitis, West Nile, and St. Louis encephalitis viruses) as well as within the four serotypes of dengue virus were established. None of the serum samples from healthy individuals screened in this study showed any cross-reaction with the four dengue virus serotype-specific RT-LAMP assay primers. These findings demonstrate that RT-LAMP assay has the potential clinical application for detection and differentiation of dengue virus serotypes, especially in developing countries.

Publisher

American Society for Microbiology

Subject

Microbiology (medical)

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