Quantitative PCR combined with propidium monoazide treatment for real-time and rapid determination of five viable Lactobacillus

Abstract:

[Background] Lactobacillus is commonly found in fermented foods which associated with food quality and safety. Quantifying the real-time dynamic and tracking the composition of viable Lactobacillus is substantial to explore metabolic function in fermentation and even intestinal microorganism system. [Objective] To establish and employ a real-time and rapid approach to detect 5 kinds of viable Lactobacillus at species level by quantitative PCR combined with propidium monoazide (PMA) treatment, and then assess its applicability. [Methods] Using Lactobacillu plantarum, Lactobacillus fermentum, Lactobacillus brevis, Lactobacillus acidophilus and Lactobacillus casei which are the common Lactobacillus strains in fermented foods as the target, the specific primers for quantitative PCR (qPCR) analysis were searched and screened. We also optimized PMA conditions and ascertained the specificity, sensitivity and reliability of the method. Then 5 kinds of viable Lactobacillus during Chinese rice wine fermentation were quantified by PMA-qPCR. [Results] The optimal process included that 20 μmol/L PMA for 15 min incubation followed by 15 min photoactivation can eliminate 99.89% amplified signal of non-viable bacteria. This method exhibited good specificity to accurately identify 5 kinds of Lactobacillus, and possessed strong linear relationship (R2>0.98) and high sensitivity (limit of detection=101.8? 103.2 CFU/mL) and the variation coefficients of Cq values were less than 1%. Additionally, it had no statistically significant (p>0.05) difference compared with plate count. Furthermore, the PMA-qPCR method was applied during Chinese rice wine fermentation, indicating that Lactobacillus fermentum, Lactobacillus casei and Lactobacillus brevis were the dominant Lactobacillus (59%?89%), which was consistent with known Lactobacillus composition in Chinese rice wine brewing system. [Conclusion] The established PMA-qPCR method could quickly and accurately quantify 5 kinds of viable Lactobacillus, which provided a feasible approach on tracking the real-time composition of viable Lactobacillus and detecting viable but nonculturable Lactobacillus in samples.