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

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 fro...

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Vydáno v:	PLoS neglected tropical diseases Ročník 12; číslo 5; s. e0006381
Hlavní autoři:	Lopez-Jimena, Benjamin, Bekaert, Michaël, Bakheit, Mohammed, Frischmann, Sieghard, Patel, Pranav, Simon-Loriere, Etienne, Lambrechts, Louis, Duong, Veasna, Dussart, Philippe, Harold, Graham, Fall, Cheikh, Faye, Oumar, Sall, Amadou Alpha, Weidmann, Manfred
Médium:	Journal Article
Jazyk:	angličtina
Vydáno:	United States Public Library of Science 29.05.2018 Public Library of Science (PLoS)
Témata:	Antigens Aquaculture Assaying Bioassay Biology and life sciences Blood Culicidae Dengue Dengue - virology Dengue fever Dengue Virus - classification Dengue Virus - genetics Dengue Virus - isolation & purification Dengue Virus - metabolism Detection Diagnosis DNA DNA Primers - genetics Ebola virus Ebolavirus Gene sequencing Genetic aspects Genomes Human diseases Humans Infections Infectious diseases Laboratories Lava Life Sciences Medicine and Health Sciences Microbiology and Parasitology Molecular chains Molecules Nucleic Acid Amplification Techniques - instrumentation Nucleic Acid Amplification Techniques - methods Nucleic acids Nucleotide sequence PCR People and Places Phylogeny Physical Sciences Primers Principal components analysis Probability theory Quality Quality assessment Quality control Real time Reproducibility Research and analysis methods Reverse transcriptase Reverse Transcription Ribonucleic acid RNA RNA, Viral - genetics Samples Sequencing Serotypes Serum Software Supervision Tanzania Transcription Tropical diseases Vector-borne diseases Viral diseases Virology Viruses Tanzania Cambodia United Kingdom > UK Senegal Scotland France Germany India
ISSN:	1935-2735, 1935-2727, 1935-2735
On-line přístup:	Získat plný text
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Popis
Shrnutí:	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. 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. 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.
Bibliografie:	ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 14 ObjectType-Article-2 ObjectType-Undefined-1 ObjectType-Feature-3 content type line 23 PMCID: PMC5973574 I have read the journal's policy and the authors of this manuscript have the following competing interests: MBa and SF are employed by a commercial company, MAST Diagnostica GmbH, an industrial partner included in the EU-funded DiscoGnosis project.
ISSN:	1935-2735 1935-2727 1935-2735
DOI:	10.1371/journal.pntd.0006381