Rapid detection and differentiation of dengue virus serotypes by a real-time reverse transcription-loop-mediated isothermal amplification assay

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 degrees 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.

FIG. 1.

Schematic representation of primer design for RT-LAMP assay. The construction of two inner primers (FIP and BIP) with both sense and antisense sequences that help in loop formation is depicted. F1C and B2C are the complementary sequences of F1 and B2, respectively.

FIG. 2.

Agarose gel electrophoresis and restriction analysis of dengue virus serotype-specific RT-LAMP assay products on a 3% agarose gel. Lane M, 100-bp DNA ladder (Sigma Genosys, Japan); lane 1, DEN-1 RT-LAMP assay amplification; lane 2, RE (BanII) digestion of DEN-1 RT-LAMP assay product, 109 bp; lane 3, DEN-2 RT-LAMP assay amplification; lane 4, RE (BanII) digestion of DEN2 RT-LAMP product, 132 bp; lane 5, DEN-3 RT-LAMP assay amplification; lane 6, RE (BanII) digestion of DEN-3 RT-LAMP assay product, 172 bp; lane 7, DEN-4 RT-LAMP assay amplification; lane 8, RE (BanII) digestion of DEN-4 RT-LAMP assay product, 186 bp; lane 9, negative control without target RNA.

FIG. 3.

Real-time amplification of dengue virus by serotype-specific RT-LAMP assay depicting the kinetics of each serotype with regard to time of positivity. O.D., optical density

FIG. 4.

SYBR Green I fluorescent dye-mediated monitoring of dengue virus serotype-specific RT-LAMP assay amplification. (Left) Naked-eye inspection under normal light. The original orange color of the SYBR Green I changed to yellow in the case of positive amplification, whereas for a negative control with no amplification, the original orange color is retained. (Right) Visual observation of green fluorescence of DNA binding SYBR Green I under UV light.

FIG. 5.

Standard curves for four dengue virus serotype-specific RT-LAMP assays generated from the amplification plots between serial 10-fold dilutions of different virus concentrations (PFU) and the time of positivity by employing the serotype specific RT-LAMP assay.

FIG. 6.

Quantification of virus titer in acute-phase dengue patient serum samples, as determined from the time of positivity based on the RT-LAMP assay standard curve for each dengue virus serotype.