Development of a reverse transcription-loop-mediated isothermal amplification assay for detection of pandemic (H1N1) 2009 virus as a novel molecular method for diagnosis of pandemic influenza in resource-limited settings

Abstract

This paper reports on the development of a one-step, real-time reverse transcription-loop-mediated isothermal amplification (RT-LAMP) assay targeting the hemagglutinin (HA) gene for the rapid molecular-based detection of pandemic (H1N1) 2009 virus. The detection limit of the pandemic (H1N1) 2009 virus HA-specific RT-LAMP assay was same as that of the currently used real-time reverse transcription-PCR method. The assay detected the pandemic (H1N1) 2009 virus HA gene in 136 RNA samples extracted from nasopharyngeal swab specimens from Japanese and Vietnamese patients. No cross-reactive amplification with the RNA of other seasonal influenza viruses was observed, and the detection of specific viral genome targets in clinical specimens was achieved in less than 40 min. The sensitivity and specificity of the pandemic (H1N1) 2009 virus HA-specific RT-LAMP assay obtained in this study were 97.8% and 100%, respectively. Use of the (H1N1) 2009 virus HA-specific RT-LAMP assay will enable the faster and easier diagnosis of pandemic (H1N1) 2009 virus infection, especially in resource-limited situations in developing countries.

FIG. 1.

Real-time kinetics of pandemic (H1N1) 2009 virus HA-specific RT-LAMP assay (A) and TaqMan rRT-PCR with the SW H1 primer/probe set (B) performed in quadruplicate with 10-fold serial dilutions (from 10⁰ to 10⁴ copies) of in vitro-transcribed target RNA. RNA from all samples with more than 100 copies of the target RNA per reaction volume was amplified by both assays. Both assays showed the same detection limit, as RNA could be amplified from two of the four samples with 10 RNA copies per reaction volume. RNA from none of the samples with one RNA copy per reaction volume was amplified.

FIG. 2.

(A) Real-time kinetics of pandemic (H1N1) 2009 virus HA-specific RT-LAMP assay performed with 10 clinical specimens positive for pandemic (H1N1) 2009 virus and two pandemic (H1N1) 2009 virus-positive control RNA samples (samples 31783T and 31784T). (B) Agarose gel electrophoresis profile of pandemic (H1N1) 2009 virus HA-specific RT-LAMP assay product (lane 1). Lane M, 100-bp DNA marker (Sigma); lane 2, pandemic (H1N1) 2009 virus HA-specific RT-LAMP assay product digested with the HindIII restriction enzyme. (C) Direct visual detection of the pandemic (H1N1) 2009 virus HA-specific RT-LAMP assay product amplified in a 1.5-ml plastic tube after incubation at 60°C for 30 min on a heating block. Lane 1, amplification of pandemic (H1N1) 2009 virus RNA from sample 31844 in a reaction volume of 25 μl; lane 2, amplification of RNA from sample 31844 in a reaction volume of 12.5 μl; lane 3, no amplification of the negative control RNA in a reaction volume of 25 μl. Positive amplification is indicated by the detection of a change in the color of the FD to a bright fluorescent green under UV irradiation (lanes 1 and 2), and a negative reaction showed no fluorescence (lane 3).