A novel one-day phage-based test for rapid detection and enumeration of viable Mycobacterium avium subsp. paratuberculosis in cows' milk.
Milk, Mycobacterium avium subsp. paratuberculosis (MAP), Phagomagnetic separation, Quantitative PCR (qPCR), Viability assay
Journal
Applied microbiology and biotechnology
ISSN: 1432-0614
Titre abrégé: Appl Microbiol Biotechnol
Pays: Germany
ID NLM: 8406612
Informations de publication
Date de publication:
Nov 2020
Nov 2020
Historique:
received:
29
07
2020
accepted:
13
09
2020
revised:
10
09
2020
pubmed:
25
9
2020
medline:
15
5
2021
entrez:
24
9
2020
Statut:
ppublish
Résumé
Bacteriophage-based methods for the rapid detection of viable Mycobacterium avium subsp. paratuberculosis (MAP) in veterinary specimens are a recent addition to the Johne's disease diagnostic toolbox. Here, we report the use of D29 mycobacteriophage-coated tosylactivated paramagnetic beads to capture and concentrate MAP cells from samples (termed phagomagnetic separation, PhMS) and then naturally lyse viable MAP cells (from the inside out) to provide DNA for IS900 qPCR purposes. Transmission electron microscopy confirmed that D29 phages had bound to beads in the correct orientation and that the phage-coated beads captured MAP cells from a suspension. During test optimization, conventional IS900 PCR results were used to subjectively assess the effect of different phage:bead coating ratios, differing amounts of coated beads during PhMS, optimal incubation time post-PhMS to obtain maximal MAP DNA, and the potential benefit of a brief heat shock (55 °C/1 min) prior to IS900 TaqMan qPCR. The limit of detection 50% (LOD
Identifiants
pubmed: 32970181
doi: 10.1007/s00253-020-10909-0
pii: 10.1007/s00253-020-10909-0
pmc: PMC7567713
doi:
Substances chimiques
DNA, Bacterial
0
Types de publication
Journal Article
Langues
eng
Sous-ensembles de citation
IM
Pagination
9399-9412Subventions
Organisme : Invest Northern Ireland
ID : AFQCC 4540
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