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

journal contribution
posted on 2023-05-20, 15:27 authored by Mohsina, K, Mandeep KaurMandeep Kaur, John BowmanJohn Bowman, Shane PowellShane Powell, Mark TamplinMark Tamplin
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).

History

Publication title

Journal of Microbiological Methods

Volume

175

Article number

105961

Number

105961

Pagination

1-7

ISSN

0167-7012

Department/School

Tasmanian Institute of Agriculture (TIA)

Publisher

Elsevier Science Bv

Place of publication

Po Box 211, Amsterdam, Netherlands, 1000 Ae

Rights statement

© 2020 Elsevier B.V. All rights reserved.

Repository Status

  • Restricted

Socio-economic Objectives

Carcass meat (incl. fish and seafood)

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