Diagnostic potential of the peptide-mediated magnetic separation (PMS)-phage assay and PMS-culture to detect<i>Mycobacterium avium</i>subsp.<i>paratuberculosis</i>in bovine milk samples

O'Brien, L. M.; McAloon, Conor G.; Stewart, Linda; Strain, Sam; Grant, Irene R.

doi:10.1111/tbed.12794

Cited by 10 publications

(4 citation statements)

References 25 publications

(36 reference statements)

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“…At Queen’s University Belfast (QUB), we have been using the PMS-phage assay with only a few minor tweaks, in terms of milk sample preparation mainly (Foddai and Grant 2015 ), for many years now, chiefly to test for viable MAP in milk (Foddai and Grant 2017 ; O’Brien et al 2018 ) and calf milk replacer (Grant et al 2017 ). Our studies have consistently shown that the PMS-phage assay is a very sensitive test for detecting viable MAP, and a promising rapid alternative to MAP culture, which takes a long time to return results but is still considered the gold standard method for demonstrating the presence of viable MAP in veterinary samples.…”

Section: Introductionmentioning

confidence: 99%

A novel one-day phage-based test for rapid detection and enumeration of viable Mycobacterium avium subsp. paratuberculosis in cows’ milk

Foddai

Grant

2020

Appl Microbiol Biotechnol

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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% (LOD50%) of the optimised PhMS-qPCR assay was 10.00 MAP cells/50 ml milk (95% CI 1.20–82.83). Finally, in order to demonstrate the new assay’s ability to detect viable MAP in naturally contaminated milk, bulk tank milk samples from 100 dairy farms were tested. Forty-nine (49%) of these tested PhMS-qPCR-positive, with viable MAP numbers detected ranging from 3–126 MAP/50 ml. The novel PhMS-qPCR assay is a sensitive, specific and easy-to-apply phage-based assay for viable MAP, with potential application for milk surveillance or diagnosis of Johne’s disease. Key points • Phage-coated magnetic beads could capture, concentrate and lyse MAP cells from milk. • PhMS-qPCR assay proved to be a rapid, sensitive and specific test for viable MAP. • A potential application of PhMS-qPCR assay for milk surveillance was demonstrated.

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Section: Introductionmentioning

confidence: 99%

A novel one-day phage-based test for rapid detection and enumeration of viable Mycobacterium avium subsp. paratuberculosis in cows’ milk

Foddai

Grant

2020

Appl Microbiol Biotechnol

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“…rather than sensitivity (32.5% and 25% resp. ), since the level of concordance between the two assays was only 8% 37 .…”

Section: Discussionmentioning

confidence: 96%

A rapid phage assay for detection of viable Mycobacterium avium subsp. paratuberculosis in milk

Hosseiniporgham

Rebechesu

Pintore

et al. 2022

Sci Rep

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Paratuberculosis is an incurable gastroenteritis among ruminants that is promoted by Mycobacterium avium subsp. paratuberculosis (MAP), an acid-fast mycobacterium. To accelerate the detection of viable pathogen, a conventional (peptide mediated magnetic separation: PMS) and novel (phage-bead qPCR: PBQ) phage based assay was optimized. A superior limit of detection (LOD) of 10 MAP per 10 mL milk was suggested for PBQ compared to 100 cells/10 mL for PMS-phage assay. Via PBQ, viable MAP was found in 48.78% out 41 unpasteurized sheep and goat milk samples. Sheep milk samples (n = 29) that were tested by PMS-phage assay contained no viable MAP. The absence of viable MAP in milk collected from 21 of the recent sheep animals was also confirmed by PBQ after a 2-week gap. Although, the two phage assays comparably detected no viable MAP in the milk samples, MAP DNA and antibodies against MAP were recognized in milk and sera of some of these animals within two instances of sampling representing that some sheep animals were MAP shedders. In conclusion, PBQ and PMS-phage could be promising methods for the assessment of MAP viability in milk samples. However, PBQ was privileged over the PMS-phage assay due to the lower LOD, rapidity, higher sensitivity, lack of need to M. smegmatis and consequent virucidal treatment that are essential in PMS-phage assay for making lawn and inactivation of exogenous mycobacteriophages respectively.

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“…PMS plus phage amplification was found to have a sensitivity of 32% and 100% specificity, an improvement on culture (Table 1), but not sensitive enough for practical use. 35 Phage capture has been used to detect MAP in BMT by fusing D29 phage to tosylactivated paramagnetic beads combined with qPCR testing; the limit of detection is reported as 10 MAP cells/50 mL of milk. 36 Magnetic separation was also incorporated into the phage amplification assay to isolate MAP cells from peripheral blood mononuclear cells 25,37 (Table 1).…”

Section: Mycobacteriophage Amplificationmentioning

confidence: 99%

The Application of Bacteriophage Diagnostics for Bacterial Pathogens in the Agricultural Supply Chain: From Farm-to-Fork

Jones

Shield

Swift

2020

PHAGE

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Bacteriophages (phages) have great potential not only as therapeutics but as diagnostics. Indeed, they have been developed and used to diagnose and detect bacterial infections, primarily in human clinical settings. The ability to rapidly detect and control bacterial pathogens in agriculture is of primary importance to maintain food security, improve animal health, and prevent the passage of zoonotic pathogens into the human population. Culture-based detection methods are often labor-intensive, and require further confirmatory tests, increasing costs and processing times needed for diagnostics. Molecular detection methods such as polymerase chain reaction are commonly used to determine the safety of food, however, a major drawback is their inability to differentiate between viable and nonviable bacterial pathogens in food. Phage diagnostics have been proven to be rapid, capable of identifying viable pathogens and do not require cultivation to detect bacteria. Phage detection takes advantage of the specificity of interaction between phage and their hosts. Furthermore, phage detection is cost effective, which is vitally important in agricultural supply chains where there is a drive to keep costs down to ensure that the cost of food does not increase. The full potential of phage detection/diagnostics is not wholly realized or commercialized. This review explores the current use and potential future scope of phage diagnostics and their application to various bacterial pathogens across agriculture and food supply chains.

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Diagnostic potential of the peptide-mediated magnetic separation (PMS)-phage assay and PMS-culture to detectMycobacterium aviumsubsp.paratuberculosisin bovine milk samples

Cited by 10 publications

References 25 publications

A novel one-day phage-based test for rapid detection and enumeration of viable Mycobacterium avium subsp. paratuberculosis in cows’ milk

A novel one-day phage-based test for rapid detection and enumeration of viable Mycobacterium avium subsp. paratuberculosis in cows’ milk

A rapid phage assay for detection of viable Mycobacterium avium subsp. paratuberculosis in milk

The Application of Bacteriophage Diagnostics for Bacterial Pathogens in the Agricultural Supply Chain: From Farm-to-Fork

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