Development of reverse-transcription loop-mediated isothermal amplification assay for rapid detection and differentiation of dengue virus serotypes 1-4

Abstract

Background: Dengue virus (DENV), the most widely prevalent arbovirus, continues to be a threat to human health in the tropics and subtropics. Early and rapid detection of DENV infection during the acute phase of illness is crucial for proper clinical patient management and preventing the spread of infection. The aim of the current study was to develop a specific, sensitive, and robust reverse transcriptase loop-mediated isothermal amplification (RT-LAMP) assay for detection and differentiation of DENV1-4 serotypes.

Results: The method detection primers, which were designed to target the different DENV serotypes, were identified by inspection of multiple sequence alignments of the non-structural protein (NS) 2A of DENV1, NS4B of DENV2, NS4A of DENV3 and the 3' untranslated region of the NS protein of DENV4. No cross-reactions of the four serotypes were observed during the tests. The detection limits of the DENV1-4-specific RT-LAMP assays were approximately 10-copy templates per reaction. The RT-LAMP assays were ten-fold more sensitive than RT-PCR or real-time PCR. The diagnostic rate was 100% for clinical strains of DENV, and 98.9% of the DENV-infected patients whose samples were tested were detected by RT-LAMP. Importantly, no false-positives were detected with the new equipment and methodology that was used to avoid aerosol contamination of the samples.

Conclusion: The RT-LAMP method used in our study is specific, sensitive, and suitable for further investigation as a useful alternative to the current methods used for clinical diagnosis of DENV1-4, especially in hospitals and laboratories that lack sophisticated diagnostic systems.

Fig. 1

The DENV genome and the regions of the RT-LAMP primers of DENV1-4, respectively. a, the region of the RT-LAMP primer for DENV1; b, the region of the RT-LAMP primer for DENV2; c, the region of the RT-LAMP primer for DENV3; d, the region of the RT-LAMP primer for DENV4

Fig. 2

Agarose gel electrophoresis and restriction analysis of DENV serotype-specific RT-LAMP assay products on a 2 % agarose gel. M, DL1000 DNA ladder (TAKARA, Japan); 1, DENV1 RT-LAMP amplification; 2, XbaI restriction enzyme digestion of DENV1 RT-LAMP product, 190,235,280 bp respectively; 3, DENV2 RT-LAMP amplification; 4, SpeI restriction enzyme digestion of DENV2 RT-LAMP product, 190,205,220 bp respectively; 5, DENV3 RT-LAMP assay amplification; 6, BglII restriction enzyme digestion of DENV3 RT-LAMP product, 145,185,210 bp respectively; 7, DENV4 RT-LAMP assay amplification; 8, AbaI restriction enzyme digestion of DENV4 RT-LAMP product, 180,210 bp respectively

Fig. 3

Specificity of RT-LAMP assay for the detection of DENV1. a Agarose gel electrophoresis analysis of the DENV1 RT-LAMP amplification product, showing the specificity of the primers. b The real-time monitoring over time for the DENV1 RT-LAMP reaction. c Visual inspection of the RT-LAMP specificity assay with SYBR Green I corresponding to the agarose gel electrophoresis analysis. 1, negative [43]; 2–3, DNA of HSV and EBV, respectively; 4–9, RNA of JEV, YFV and DENV1-4, respectively; M, DL1000 DNA Marker

Fig. 4

Specificity of RT-LAMP assay for the detection of DENV2. a Agarose gel electrophoresis analysis of the DENV2 RT-LAMP amplification product, showing the specificity of the primers. b The real-time monitoring over time for the DENV2 RT-LAMP reaction. c Visual inspection of the RT-LAMP specificity assay with SYBR Green I corresponding to the agarose gel electrophoresis analysis. 1, negative (water); 2–3, DNA of HSV and EBV, respectively; 4–9, RNA of JEV, YFV and DENV1-4, respectively; M, DL1000 DNA Marker

Fig. 5

Specificity of RT-LAMP assay for the detection of DENV3. a Agarose gel electrophoresis analysis of the DENV3 RT-LAMP amplification product, showing the specificity of the primers. b The real-time monitoring over time for the DENV3 RT-LAMP reaction. c Visual inspection of the RT-LAMP specificity assay with SYBR Green I corresponding to the agarose gel electrophoresis analysis. 1, negative [43]; 2–3, DNA of HSV and EBV, respectively; 4–9, RNA of JEV, YFV and DENV1-4, respectively; M, DL1000 DNA Marker

Fig. 6

Specificity of RT-LAMP assay for the detection of DENV4. a Agarose gel electrophoresis analysis of the DENV4 RT-LAMP amplification product, showing the specificity of the primers. b The real-time monitoring over time for the DENV4 RT-LAMP reaction. c Visual inspection of the RT-LAMP specificity assay with SYBR Green I corresponding to the agarose gel electrophoresis analysis. 1, negative (water); 2–3, DNA of HSV and EBV, respectively; 4–9, RNA of JEV, YFV and DENV1-4, respectively; M, DL1000 DNA Marker

Fig. 7

Specificity of RT-PCR assays for the detection of DENV1-4. Agarose gel electrophoresis analysis of the DENV1-4 RT-PCR amplification product, showing the specificity of the primers, DENV1-4-F3 and B3. (A-D) DENV1-4. 1, negative (water); 2–3, DNA of HSV and EBV, respectively; 4–9, RNA of JEV, YFV and DENV1-4, respectively; M, DL1000 DNA Marker

Fig. 8

Comparison of the sensitivity of RT-PCR, real-time PCR and RT-LAMP for detection of DENV1. a Agarose gel electrophoresis analysis of detection limit of the RT-PCR assay for the detection of DENV1 RNA. b The real-time monitoring over time for detection limit of the real-time PCR assay for the detection of the DENV1 cDNAs. c Agarose gel electrophoresis analysis of the detection limit of the RT-LAMP assay for the detection of DENV1 RNA d Visual inspection of the RT-LAMP assay corresponding to agarose gel electrophoresis analysis. e The real-time monitoring of detection limit of the RT-LAMP assay. 1, negative [43]; 2–7, correspond to serial ten-fold dilutions of DENV1 RNA templates from 1 to 10⁵copies; M, DL1000 DNA Marker

Fig. 9

Comparison of the sensitivity of RT-PCR, real-time PCR and RT-LAMP for detection of DENV2. a Agarose gel electrophoresis analysis of detection limit of the RT-PCR assay for the detection of DENV2 RNA. b The real-time monitoring over time for detection limit of the real-time PCR assay for the detection of the DENV2 cDNAs. c Agarose gel electrophoresis analysis of the detection limit of the RT-LAMP assay for the detection of DENV2 RNA d Visual inspection of the RT-LAMP assay corresponding to agarose gel electrophoresis analysis. e The real-time monitoring of detection limit of the RT-LAMP assay. 1, negative (water); 2–7, correspond to serial ten-fold dilutions of DENV2 RNA templates from 1 to 10⁵copies; M, DL1000 DNA Marker

Fig. 10

Comparison of the sensitivity of RT-PCR, real-time PCR and RT-LAMP for detection of DENV3. a Agarose gel electrophoresis analysis of detection limit of the RT-PCR assay for the detection of DENV3 RNA. b The real-time monitoring over time for detection limit of the real-time PCR assay for the detection of the DENV3 cDNAs. c Agarose gel electrophoresis analysis of the detection limit of the RT-LAMP assay for the detection of DENV3 RNA d Visual inspection of the RT-LAMP assay corresponding to agarose gel electrophoresis analysis. e The real-time monitoring of detection limit of the RT-LAMP assay. 1, negative [43]; 2–7, correspond to serial ten-fold dilutions of DENV3 RNA templates from 1 to 10⁵copies; M, DL1000 DNA Marker

Fig. 11

Comparison of the sensitivity of RT-PCR, real-time PCR and RT-LAMP for detection of DENV4. a Agarose gel electrophoresis analysis of detection limit of the RT-PCR assay for the detection of DENV4 RNA. b The real-time monitoring over time for detection limit of the real-time PCR assay for the detection of the DENV4 cDNAs. c Agarose gel electrophoresis analysis of the detection limit of the RT-LAMP assay for the detection of DENV4 RNA d Visual inspection of the RT-LAMP assay corresponding to agarose gel electrophoresis analysis. e The real-time monitoring of detection limit of the RT-LAMP assay. 1, negative (water); 2–7, correspond to serial ten-fold dilutions of DENV4 RNA templates from 1 to 10⁵copies; M, DL1000 DNA Marker

Fig. 12

Comparison of the amplification efficiency and time of real-time PCR and RT-LAMP. a Standard curves generated by linear regression analysis of TTP of LAMP and Ct of real-time PCR measured for each amplification versus the log₁₀ number of DENV-1 RNA or cDNA copies for each standard dilution. b Standard curves generated by linear regression analysis of the time for each amplification versus the log₁₀ number of DENV-1 RNA or cDNA copies each standard dilution. Data are shown as mean ± standard error of the mean (S.E.M) at least three independent experiments. Ct, cycle threshold; TTP, time-to-positive