Development of a single-tube one-step RT-LAMP assay to detect the Chikungunya virus genome

A single-tube one-step real-time reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay for rapid detection of chikungunya virus (CHIKV) targeting the conserved 6K-E1 target region was developed. The assay was validated with sera collected from a CHIKV outbreak in Senegal in 20...

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Bibliographic Details
Published in:PLoS neglected tropical diseases Vol. 12; no. 5; p. e0006448
Main Authors: Lopez-Jimena, Benjamin, Wehner, Stefanie, Harold, Graham, Bakheit, Mohammed, Frischmann, Sieghard, Bekaert, Michaël, Faye, Oumar, Sall, Amadou Alpha, Weidmann, Manfred
Format: Journal Article
Language:English
Published: United States Public Library of Science 29.05.2018
Public Library of Science (PLoS)
Subjects:
Aquaculture
Assaying
Bioassay
Bioinformatics
Biology and life sciences
Chikungunya fever
Chikungunya Fever - diagnosis
Chikungunya Fever - virology
Chikungunya virus
Chikungunya virus - classification
Chikungunya virus - genetics
Chikungunya virus - isolation & purification
Dengue fever
Detection
Diagnosis
DNA
DNA Primers - genetics
Genetic aspects
Genome, Viral
Genomes
Genomics
Humans
Infections
Laboratories
Lava
Medicine and Health Sciences
Methods
Molecular chains
Molecules
Nucleic Acid Amplification Techniques - methods
Nucleic acids
Nucleotide sequence
Oligonucleotides
Outbreaks
PCR
Phylogeny
Physical sciences
Polymerase chain reaction
Principal components analysis
Quality assessment
Quality control
Reproducibility
Research and analysis methods
Reverse transcriptase
Reverse transcription
Ribonucleic acid
RNA
Sensitivity and Specificity
Software
Supervision
Transcription
Tropical diseases
Vector-borne diseases
Viral diseases
Viroses
Viruses
Senegal
Scotland
United Kingdom > UK
Germany
ISSN:1935-2735, 1935-2727, 1935-2735
Online Access:Get full text
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Description
Summary:A single-tube one-step real-time reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay for rapid detection of chikungunya virus (CHIKV) targeting the conserved 6K-E1 target region was developed. The assay was validated with sera collected from a CHIKV outbreak in Senegal in 2015. A novel design approach by combining Principal Component Analysis and phylogenetic analysis of 110 available CHIKV sequences and the LAMP oligonucleotide design software LAVA was used. The assay was evaluated with an External Quality Assessment panel from the European Network for Diagnostics of "Imported" Viral Diseases and was shown to be sensitive and specific and did not cross-detect other arboviruses. The limit of detection as determined by probit analysis, was 163 molecules, and 100% reproducibility in the assays was obtained for 103 molecules (7/8 repetitions were positive for 102 molecules). The assay was validated using 35 RNA samples extracted from sera, and results were compared with those obtained by quantitative RT-PCR carried out at the Institut Pasteur Dakar, demonstrating that the RT-LAMP is 100% sensitive and 80% specific, with a positive predictive value of 97% and negative predictive value of 100%. The RT-LAMP appeared to show superior performance with material stored for months compared to qRT-PCR and can be therefore recommended for use in infrastructures with poor settings.
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All authors have read and approved this manuscript and declare that there are no conflicts of interest (both financial and personal). MBa and SF belong to MAST Diagnostica GmbH, an industry partner included in the EU-funded DiscoGnosis project.
ISSN:1935-2735
1935-2727
1935-2735
DOI:10.1371/journal.pntd.0006448