Development and validation of four one-step real-time RT-LAMP assays for specific detection of each dengue virus serotype

Background: 4 one-step, real-time, reverse transcription loop-mediated isothermal amplification (RT-LAMP) assays were developed for the detection of dengue virus (DENV) serotypes by considering 2,056 full genome DENV sequences. DENV1 and DENV2 RT-LAMP assays were validated with 31 blood and 11 serum samples from Tanzania, Senegal, Sudan and Mauritania. DENV3 and DENV4 RT-LAMP assays were validated with 25 serum samples from Cambodia Methodology/Principal findings: 4 final reaction primer mixes were obtained by using a combination of Principal Component Analysis of the full DENV genome sequences, and LAMP primer design based on sequence alignments using the LAVA software. These mixes contained 14 (DENV1), 12 (DENV2), 8 (DENV3) and 3 (DENV4) LAMP primer sets. The assays were evaluated with an External Quality Assessment panel from Quality Control for Molecular Diagnostics. The assays were serotype-specific and did not cross-detect with other flaviviruses. The limits of detection, with 95% probability, were 22 (DENV1), 542 (DENV2), 197 (DENV3) and 641 (DENV4) RNA molecules, and 100% reproducibility in the assays was obtained with up to 102 (DENV1) and 103 RNA molecules (DENV2, DENV3 and DENV4). Validation of the DENV2 assay with blood samples from Tanzania resulted in 23 samples detected by RT-LAMP, demonstrating that the assay is 100% specific and 95.8% sensitive (positive predictive value of 100% and a negative predictive value of 85.7%). All serum samples from Senegal, Sudan and Mauritania were detected and 3 untyped as DENV1. The sensitivity of RT-LAMP for DENV4 samples from Cambodia did not quite match qRT-PCR. Conclusions/Significance: We have shown a novel approach to design LAMP primers that makes use of fast growing sequence databases. The DENV1 and DENV2 assays were validated with viral RNA extracted clinical samples, showing very good performance parameters. Author summary: The co-existence of several dengue virus (DENV) serotypes within the same location and/or individuals as well as a single mosquito being able to carry multiple DENV serotypes highlight the necessity of specific diagnostic tools capable of detect and serotype DENV strains circulating worldwide. In addition, these methodologies must be highly sensitive in order to detect the genome at low levels (i.e., before the onset of clinical symptoms) and not cross-detect other flaviviruses. Isothermal amplification methods (such as reverse transcription loop-mediated isothermal amplification, RT-LAMP) are affordable for laboratories with limited resources and do not need expensive equipment. Because of the increasing number of publicly available full DENV genome sequences, traditional primer design tools are not able to handle such huge amount of information. Therefore, to be able to cover all the diversity documented, we developed 4 complicated oligonucleotide mixes for the individual detection of DENV1-4 serotypes by RT-LAMP. This approach combined Principal Component Analysis, phylogenetic analysis and LAVA algorithm. Our assays are specific and do not cross-react with other arboviruses and DNA pathogens included in this study, they are sensitive and have been validated with samples from Tanzania, Senegal, Sudan, Mauritania and Cambodia, showing very good performance parameters.