Development and preliminary evaluation of real-time PCR assays for six lactic acid bacteria

This study developed new real-time PCR assays for six lactic acid bacteria (LAB): Ligilactobacillus agilis, Limosilactobacillus fermentum, Lactobacillus johnsonii, Ligilactobacillus salivarius, Pediococcus pentosaceus, and Weissella cibaria for their application in the food industry. For each target bacterium, a novel primer/probe set was designed in a conserved and species-specific genomic region, with proper length (13 ~ 30nt), Tm (probes: 68 ~ 70℃; primers: 58 ~ 60℃), GC content (30 ~ 80%), and amplicon length (50 ~ 150 bp). The Gibbs free energy (ΔG, an indicator for stability of secondary structures) of potential hairpins, self-dimers, and cross-dimers of the primers and probes adhered largely to the recommended values with minor deviations. BLAST analysis verified the conservation and specificity of each target sequence. After establishing the reaction mixture and the thermal procedure, each new assay was preliminarily evaluated in inclusivity, specificity, amplification efficiency, and precision. All tested samples of each target bacterium generated positive results. All tested non-target bacterial samples yielded negative results. Amplification efficiency of each assay, measured using 10-fold serial dilutions of a positive sample, was 95 ~ 100%. Precision (repeatability and reproducibility) of each assay generated relative standard deviations (RSDs) < 2%. Collectively, these newly developed assays predictably have high inclusivity, specificity, amplification efficiency, and precision, which need more comprehensive validation before industrial application.