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qPCR quantification of Carnobacterium maltaromaticum, Brochothrix thermosphacta, and Serratia liquefaciens growth kinetics in mixed culture

Mohsina, K, Kaur, M ORCID: 0000-0003-0731-6166, Bowman, JP ORCID: 0000-0002-4528-9333, Powell, S ORCID: 0000-0001-5082-1630 and Tamplin, ML ORCID: 0000-0003-2652-2408 2020 , 'qPCR quantification of Carnobacterium maltaromaticum, Brochothrix thermosphacta, and Serratia liquefaciens growth kinetics in mixed culture' , Journal of Microbiological Methods, vol. 175 , pp. 1-7 , doi: 10.1016/j.mimet.2020.105961.

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Abstract

Quantifying growth kinetics of specific spoilage microorganisms in mixed culture is required to describe the evolution of food microbiomes. A qPCR method was developed to selectively amplify individual meat spoilage bacteria, Carnobacterium maltaromaticum, Brochothrix thermosphacta and Serratia liquefaciens, within a broth medium designed to simulate the composition of beef. An optimized method of DNA extraction was produced for standard curve construction. Method specificity was determined by individual single peaks in melt curves. Reaction efficiency for standard curves of C. maltaromaticum, B. thermosphacta and S. liquefaciens was high (R2 = 0.98–0.99), and linear quantification was achieved over a 5 log CFU/ml range. Coefficient of variation was calculated considering both threshold cycle (Ct) and bacterial concentration; the value did not exceed 14% for inter- or intra-runs for either method. Comparison of growth kinetic parameters derived from plate count and qPCR showed no significant variation (P > .05) for growth rate (GR) and maximum population density (MPD); lag phase duration (LPD) was not included in this comparison due to high innate variability. Log quantification of each isolate was validated in a mixed-culture experiment for all three species with qPCR and plate count differing less than 0.3 log CFU/ml (average 0.10 log CFU/ml, R2 = 0.98).

Item Type: Article
Authors/Creators:Mohsina, K and Kaur, M and Bowman, JP and Powell, S and Tamplin, ML
Keywords: growth kinetics, spoilage bacteria, qPCR, mixed culture, meat, predictive microbiology, growth modelling, spoilage
Journal or Publication Title: Journal of Microbiological Methods
Publisher: Elsevier Science Bv
ISSN: 0167-7012
DOI / ID Number: 10.1016/j.mimet.2020.105961
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© 2020 Elsevier B.V. All rights reserved.

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