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qPCR quantification of Carnobacterium maltaromaticum, Brochothrix thermosphacta, and Serratia liquefaciens growth kinetics in mixed culture.
Journal of Microbiological Methods ( IF 2.2 ) Pub Date : 2020-05-29 , DOI: 10.1016/j.mimet.2020.105961
Kaniz Mohsina 1 , Mandeep Kaur 1 , John P Bowman 1 , Shane Powell 1 , Mark L Tamplin 1
Affiliation  

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).



中文翻译:

混合培养中麦芽糖克氏杆菌,嗜热芽孢杆菌和液态粘质沙雷氏菌生长动力学的定量PCR定量。

需要描述混合培养中特定腐败微生物的生长动力学,以描述食物微生物组的进化。开发了一种qPCR方法,以在设计为模拟牛肉成分的肉汤培养基中选择性扩增单个肉变质细菌,麦芽糖克氏杆菌,嗜热性肉芽孢杆菌液化沙雷氏菌。为标准曲线构建生产了优化的DNA提取方法。方法的特异性由熔解曲线中的单个单个峰确定。麦芽糖梭菌,嗜热芽孢杆菌液化链球菌标准曲线的反应效率很高(R 2 = 0.98–0.99),并且线性定量在5 log CFU / ml范围内实现。同时考虑阈值循环(C t)和细菌浓度来计算变异系数。对于任何一种方法,在批间或批内的值不超过14%。比较来自平板计数和qPCR的生长动力学参数,显示 生长速率(GR)和最大种群密度(MPD)没有显着变化(P > .05);由于先天变异性高,滞后阶段持续时间(LPD)未包括在此比较中。在所有三个物种的混合培养实验中,通过qPCR和板数差异小于0.3 log CFU / ml(平均0.10 log CFU / ml,R 2  = 0.98)验证了每种分离物的对数定量。

更新日期:2020-05-29
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