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. 2004 Jan;42(1):257-63.
doi: 10.1128/JCM.42.1.257-263.2004.

Real-time reverse transcription loop-mediated isothermal amplification for rapid detection of West Nile virus

Manmohan Parida  1 , Guillermo Posadas, Shingo Inoue, Futoshi Hasebe, Kouichi Morita
Affiliations

Real-time reverse transcription loop-mediated isothermal amplification for rapid detection of West Nile virus

Manmohan Parida et al. J Clin Microbiol. 2004 Jan.

Abstract

A one-step, single tube, real-time accelerated reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed for detecting the envelope gene of West Nile (WN) virus. The RT-LAMP assay is a novel method of gene amplification that amplifies nucleic acid with high specificity, efficiency, and rapidity under isothermal conditions with a set of six specially designed primers that recognize eight distinct sequences of the _target. The whole procedure is very simple and rapid, and amplification can be obtained in less than 1 h by incubating all of the reagents in a single tube with reverse transcriptase and Bst DNA polymerase at 63 degrees C. Detection of gene amplification could be accomplished by agarose gel electrophoresis, as well as by real-time monitoring in an inexpensive turbidimeter. When the sensitivity of the RT-LAMP assay was compared to that of conventional RT-PCR, it was found that the RT-LAMP assay demonstrated 10-fold higher sensitivity compared to RT-PCR, with a detection limit of 0.1 PFU of virus. By using real-time monitoring, 10(4) PFU of virus could be detected in as little as 17 min. The specificity of the RT-LAMP assay was validated by the absence of any cross-reaction with other, closely related, members of the Flavivirus group, followed by restriction digestion and nucleotide sequencing of the amplified product. These results indicate that the RT-LAMP assay is extremely rapid, cost-effective, highly sensitive, and specific and has potential usefulness for rapid, comprehensive WN virus surveillance along with virus isolation and/or serology.

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Figures

FIG. 1.
FIG. 1.
(A) Agarose gel electrophoresis and restriction analysis of the RT-LAMP product of the E gene of WN virus. Lanes: M, 100-bp DNA ladder (Sigma Genosys); 1, RT-LAMP with WN virus strain NY99; 2, RT-LAMP products digested with AluI (175 bp); 3, RT-LAMP without _target RNA. (B and C) Comparative sensitivities of RT-LAMP and RT-PCR for detection of WN virus strain NY99 RNA. The amplification by RT LAMP (B) shows a ladder-like pattern, whereas the RT-PCR (C) shows a 201-bp amplification product. Lanes: M, 100-bp DNA ladder (Sigma Genosys); 1, 10,000 PFU; 2, 1,000 PFU; 3, 100 PFU; 4, 10 PFU; 5, 1 PFU; 6, 0.1 PFU; 7, 0.01 PFU; 8, 0.001 PFU; 9, 0.0001 PFU; 10, 0 PFU (negative control without _target RNA).
FIG. 2.
FIG. 2.
(A) Effect of temperature on the time kinetics of the RT-LAMP reaction of WN virus strain NY99 as monitored by measurement of turbidity in a Loopamp real-time turbidimeter (LA-200; Teramecs). (B) Kinetics of RT-LAMP amplification of WN virus strain NY99 RNA with and without the loop primers as monitored by real-time measurement of turbidity in a Loopamp real-time turbidimeter (LA-200; Teramecs). (C) Sensitivity of the RT-LAMP assay for detection of WN virus RNA as monitored by real-time measurement of turbidity (LA-200, Teramecs). Serial 10-fold dilutions of virus strain NY99 ranging from 10,000 plaque-forming units to 0.0001 plaque-forming unit were tested.
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