Development of Quantitative PCR Method for Quantifying Streptococcus thermophilus Growth During Batch Culture
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Abstract
Streptococcus thermophilus is a thermophilic lactic acid bacterium that is commonly used in food fermentation. However, quantifying bacterial populations using conventional culture-dependent assays is laborious. The genome-based method has been recognized as an alternative rapid method. In this study, we developed a method to use a specific gene of S. thermophilus, the glucokinase gene (GlcK), by quantitative PCR (qPCR). A 139-bp PCR product was successfully cloned and used to generate a DNA standard curve by plotting the threshold cycle (Cq) versus log DNA concentration of plasmid DNA, with an amplification efficiency of 97.2%. Coefficient of variation was calculated by considering both Cq and bacterial cells enumerated by plate counts, which indicated a log CFU/mL (1.69-6.56) and log DNA copies (2.07-6.03). This linear relationship revealed a quantitative curve (R2 = 0.989) with a detection limit of range from 2.07 to 6.03 log copies per reaction. In terms of efficiency and repeatability, the relative standard deviations (RSDs) were in the range of 92-110% and 0.01%, respectively. Lastly, we used the developed qPCR method to determine the growth curves of bacterial cells and the specific growth rate (m) during batch culture for 24 h. The established method facilitated the determination of specific growth rates, showing a specific growth rate of 0.57 h-1 with lactose supplementation and 0.30 h-1 in the absence of lactose. Hence, qPCR-based methods facilitated reliable quantification of S. thermophilus during fermentation.
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