Loop-mediated isothermal amplification: Development, validation and application of simple and rapid assays for quantitative detection of species of Arcobacteraceae family- and species-specific Aliarcobacter faecis and Aliarcobacter lanthieri.
Agriculture
Animals
Arcobacter
/ classification
Campylobacteraceae
/ classification
DNA Primers
DNA, Bacterial
/ analysis
Feces
/ microbiology
Humans
Molecular Diagnostic Techniques
Nucleic Acid Amplification Techniques
RNA, Ribosomal, 16S
Reproducibility of Results
Sensitivity and Specificity
Species Specificity
Water Microbiology
Aliarcobacter faecis
Aliarcobacter lanthieri
Arcobacteraceae
loop-mediated isothermal amplification
quantitation
Journal
Journal of applied microbiology
ISSN: 1365-2672
Titre abrégé: J Appl Microbiol
Pays: England
ID NLM: 9706280
Informations de publication
Date de publication:
Jul 2021
Jul 2021
Historique:
revised:
04
11
2020
received:
20
01
2020
accepted:
05
11
2020
pubmed:
12
11
2020
medline:
6
7
2021
entrez:
11
11
2020
Statut:
ppublish
Résumé
The family Arcobacteraceae formerly genus Arcobacter has recently been reclassified into six genera. Among nine species of the genus Aliarcobacter, Aliarcobacter faecis and Aliarcobacter lanthieri have been identified as emerging pathogens potentially cause health risks to humans and animals. This study was designed to develop/optimize, validate and apply Arcobacteraceae family- and two species-specific (A. faecis and A. lanthieri) loop-mediated isothermal amplification (LAMP) assays to rapidly detect and quantify total number of cells in various environmental niches. Three sets of LAMP primers were designed from conserved and variable regions of 16S rRNA (family-specific) and gyrB (species-specific) genes. Optimized Arcobacteraceae family-specific LAMP assay correctly amplified and detected 24 species, whereas species-specific LAMP assays detected A. faecis and A. lanthieri reference strains as well as 91 pure and mixed culture isolates recovered from aquatic and faecal sources. The specificity of LAMP amplification of A. faecis and A. lanthieri was further confirmed by restriction fragment length polymorphism analysis. Assay sensitivities were tested using variable DNA concentrations extracted from simulated target species cells in an autoclaved agricultural water sample by achieving a minimum detection limit of 10 cells mL Overall, these three DNA-based rapid and cost-effective novel LAMP assays are sensitive and can be completed in less than 40 min. They have potential for on-site quantitative detection of species of family Arcobacteraceae, A. faecis and A. lanthieri in food, environmental and clinical matrices. The newly developed LAMP assays are specific, sensitive, accurate with higher reproducibility that have potential to facilitate in a less equipped lab setting and can help in early quantitative detection and rate of prevalence in environmental niches. The assays can be adopted in the diagnostic labs and epidemiological studies.
Identifiants
pubmed: 33174331
doi: 10.1111/jam.14926
pmc: PMC8359143
doi:
Substances chimiques
DNA Primers
0
DNA, Bacterial
0
RNA, Ribosomal, 16S
0
Types de publication
Journal Article
Validation Study
Langues
eng
Sous-ensembles de citation
IM
Pagination
288-299Subventions
Organisme : Agriculture and Agri-Food Canada
ID : J-001609
Organisme : Agriculture and Agri-Food Canada
ID : J-000157
Informations de copyright
© 2020 The Authors. Journal of Applied Microbiology published by John Wiley & Sons Ltd on behalf of Society for Applied Microbiology.
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