Abstract
Background4 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 CambodiaMethodology/Principal findings4 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/SignificanceWe 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.
Highlights
Dengue is a worldwide public health concern annually affecting more than 100 million people in tropical and subtropical areas [1, 2]
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
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
Summary
Dengue is a worldwide public health concern annually affecting more than 100 million people in tropical and subtropical areas [1, 2]. It is caused by dengue virus (DENV), the most common vector-borne viral pathogen of humans, transmitted by mosquitoes of the Aedes genus (primarily A. aegypti and to a lesser extent A. albopictus), as previously reviewed [3]. The DENV genome encodes three structural proteins (C, capsid; prM, pre-membrane, and E, envelope) at the N terminus and seven non-structural (NS) proteins (NS1, NS2a, NS2b, NS3, NS4a, NS4b and NS5) [5, 6] This virus is classified into four phylogenetically related and loosely antigenically distinct serotypes (DENV1, DENV2, DENV3 and DENV4), each of which contains phylogenetically different genotypes [7,8,9]. DENV3 and DENV4 RT-LAMP assays were validated with 25 serum samples from Cambodia
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