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. 2013 Aug 21:13:387.
doi: 10.1186/1471-2334-13-387.

Detection of dengue viruses using reverse transcription-loop-mediated isothermal amplification

Boon-Teong Teoh  1 , Sing-Sin Sam, Kim-Kee Tan, Jefree Johari, Mohammed Bashar Danlami, Poh-Sim Hooi, Rafi Md-Esa, Sazaly AbuBakar
Affiliations

Detection of dengue viruses using reverse transcription-loop-mediated isothermal amplification

Boon-Teong Teoh et al. BMC Infect Dis. .

Abstract

Background: Early and rapid detection of dengue virus (DENV) infection during the febrile period is crucial for proper patient management and prevention of disease spread. An easy to perform and highly sensitive method is needed for routine implementation especially in the resource-limited rural healthcare settings where dengue is endemic.

Methods: A single-tube reverse transcription-loop-mediated isothermal amplification (RT-LAMP) assay with a set of nine primers was developed for the detection of all four DENV serotypes and their different genotypes. The sensitivity and specificity of the RT-LAMP were evaluated. The clinical applicability of RT-LAMP assay for detection of DENV RNA was assessed in a total of 305 sera of clinically-suspected dengue patients. The test results of RT-LAMP were statistically compared to those of quantitative reverse transcription-polymerase chain reaction (qRT-PCR), IgM- and IgG-capture enzyme-linked immunosorbent assays (ELISA).

Results: Acute DENV infection was confirmed in 171 samples (n = 305); 43.3% (74/171) and 46.8% (80/171) of the samples were positive for DENV using RT-LAMP and qRT-PCR, respectively. The combination of RT-LAMP with the dengue IgM and IgG ELISA increased detection of acute DENV infection to 97.7% (167/171), in comparison to only 70.8% (121/171) when dengue IgM and IgG ELISA alone were used. The RT-LAMP assays showed high concordance (κ = 0.939) with the qRT-PCR. The RT-LAMP assay detected up to 10 copies of virus RNA within an hour but 100% reproducibility (12/12) was achieved with 100 copies. There was no cross reactivity of RT-LAMP with other closely related arboviruses.

Conclusion: The RT-LAMP assay developed in this study is sensitive, specific and simple to perform. The assay improved the detection of dengue when used in combination with serological methods.

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Figures

Figure 1
Figure 1
Visual observation of fluorescence in RT-LAMP reactions under UV light. Tube 1, negative control; tube 2–5, DENV-1 genotype I, II, III and sylvatic, respectively; tube 6–7, DENV-2 Asian I and cosmopolitan, respectively; tube 8–10, DENV-3 genotype I, II and III, respectively; tube 11–12, DENV-4 subgenotype IIa and IIb, respectively; tube 13–15, JEV, CHIKV and SINV, respectively.
Figure 2
Figure 2
Confirmation of RT-LAMP amplification of DENV. Restriction enzyme digestion of DENV-specific RT-LAMP-amplified DNA fragments were performed using BanII. The DNA fragments were separated on a 2% agarose gel. Lane M, 100-bp plus DNA ladder; lane 1, DENV-1 RT-LAMP assay amplification; lane 2, BanII digestion of DENV-1 RT-LAMP-amplified DNA fragments, 129 bp and 233 bp; lane 3, DENV-2 RT-LAMP assay amplification; lane 4, BanII digestion of DENV-2 RT-LAMP-amplified DNA fragments, 135 bp and 231 bp; lane 5, DENV-3 RT-LAMP assay amplification; lane 6, BanII digestion of DENV-3 RT-LAMP-amplified DNA fragments, 129 bp and 231 bp; lane 7, DENV-4 RT-LAMP assay amplification; lane 8, BanII digestion of DENV-4 RT-LAMP-amplified DNA fragments, 141 bp and 231 bp.
Figure 3
Figure 3
Time threshold of positivity for RT-LAMP assays of serially diluted DENV RNA. The mean of Tt-values was calculated with available positive results out of twelve replicates. Error bars indicate the standard deviations of Tt-values from the mean.
Figure 4
Figure 4
Number of clinical samples positive by RT-LAMP and qRT-PCR. Six samples were tested positive for DENV RNA by qRT-PCR but negative by RT-LAMP. One sample positive by RT-LAMP but negative by qRT-PCR was excluded.
Figure 5
Figure 5
Sensitivity of dengue diagnostic methods against laboratory-confirmed dengue samples (n = 171). Sensitivity between groups was compared using McNemar’s exact test (two-tailed).
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