Optimizing Real-Time PCR for Accurate Identification and Quantification of Common Probiotic Bacteria in Iranian Probiotic Dairy Products

Abstract

The effectiveness of probiotics for health depends on the strain type and dosage. In this study, we endeavored to establish a robust quantitative Real-time PCR method for the evaluation probiotic strains in Iranian probiotic dairy products. The process was initiated by extracting DNA from selected bacterial strains and selecting well-matched primers. Then, we optimized the Real-time PCR method to efficiently identify and quantify three prevalent beneficial bacterial strains, including Lactobacillus acidophilus, Lactobacillus casei, and Bifidobacterium animalis subsp. lactis in four types of Iranian probiotic yogurt with different brands simultaneously and within 4 h. To overcome complications such as sample variability or interference with other common microorganisms in dairy products, we simultaneously used IPC and specific primers of each strain. The sensitivity and specificity obtained in the present study were 10⁶ CFU/mL for the targeted bacterial strains. The relative standard deviation (%RSD) was maintained at less than 2% regarding repeatability and reproducibility. The method designed and validated for identifying and counting these strains exhibits remarkable specificity, sensitivity, accuracy, optimal reaction efficiency, and minimal variance. Collectively, our method has proven to be specialized, cost-effective, and rapid, which makes it suitable for qualitative evaluation of probiotic dairy products in the industry.

Graphical Abstract