Proceedings of the 10th International Colloquium on Paratuberculosis

#98 An inter-laboratory ring-trial evaluating molecular and culture detection <strong>of</strong> Mycobacterium
avium subsp. paratuberculosis in dairy products: Outcome <strong>of</strong> an EU ParaTBTools
workpackage
John Anthony Donaghy, Michael Rowe, Anne Fisher, Jan Rademaker, Pat Murphy, Beatrice Blanchard, Geertrui
Vlaemynck, Hans-Georg Walte, Philipp Hammer
Food Microbiology Branch, Agri-Food & Biosciences Institute, Belfast, N. Ireland, United Kingdom; NIZO food
research, Ede, The Ne<strong>the</strong>rlands; Moorepark Food Research Centre, Ireland; Adiagene, France; ILVO, Belgium; Max
Rubner-Institute, Federal Institute <strong>of</strong> Nutrition and Food, Kiel, Germany
There is a need for standardization and harmonization <strong>of</strong> culture and molecular methods for <strong>the</strong> detection/isolation/recovery
<strong>of</strong> Mycobacterium avium subsp. paratuberculosis (Map) from dairy matrices. The objective <strong>of</strong>
this study was to perform a dairy products ring-trial <strong>of</strong> culture and molecular methods across European laboratories.
The prescribed molecular method used was a Real Time IS900-based TaqMan assay in combination
with <strong>the</strong> commercial Map DNA extraction kit (Adiapure ® ). The prescribed culture method used was HEYM agar
supplemented with PANTA, nisin and penicillin.
The ‘blind’ ring-trial was completed according to <strong>the</strong> NordVal validation method: eleven participant
laboratories, one Map strain, three contamination levels, duplicates for each level and five dairy matrices. The
dairy matrices analysed were Map-infected faecally-contaminated milk, artificially-contaminated Gouda, Danish
Blue and Munster cheeses and yogurt. The contamination levels <strong>of</strong> cheeses and yogurt were 5 x 103 cfu/
g(high) and 50 cfu/g (low) and 1.8 x 103 cfu/g(high) and 18 cfu/g(low) for faecally-contaminated milk. In addition
to <strong>the</strong> prescribed methods assessed, 12 alternate molecular and 14 culture ‘own laboratory’ detection protocols
were employed on test samples.
For <strong>the</strong> prescribed molecular method, average sensitivities across all laboratories, for combined high and
low contamination levels, were 95%, 96%, 100%, 84% and 96% for faecally-contaminated milk, yogurt, Gouda,
Danish Blue and Munster cheese respectively. Specificities for all matrices were in <strong>the</strong> range 95-100%. Overall
accordance (repeatability) and concordance (reproducibility) values were 0.975 and 91.7% respectively for <strong>the</strong>
molecular method assessed. Culture methods employed showed lower sensitivities than molecular methods,
Map was recovered more frequently at ‘high’ inoculum levels.
The reproducibility <strong>of</strong> a commercial Map DNA extraction kit used in combination with a Real Time TaqMan
assay was demonstrated in this ring-trial. The extraction and assay protocol was applicable to a range <strong>of</strong> dairy
matrices whereas <strong>the</strong> trial demonstrated <strong>the</strong> idiosyncrasies associated with Map culture methods.
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