RNA sequencing reveals stable reference genes for reverse-transcription quantitative PCR monitoring of Lactiplantibacillus plantarum in fermented milk

Lactiplantibacillus plantarum is a widely studied lactic acid bacterium with important roles in gut health and fermented food production. Accurate quantification of viable populations is crucial for evaluating the efficacy of probiotics and ensuring product quality. In this study, RNA sequencing was applied for the first time to systematically screen stable reference genes (RG) in L. plantarum for reverse-transcription quantitative PCR (RT-qPCR) under 4 distinct fermentation conditions. Among 3,092 genes analyzed, 22 candidates exhibiting CV values below 0.1 were identified. Primer validation demonstrated acceptable amplification efficiencies (90.95%-134.98%), and stability ranking by geNorm, NormFinder, BestKeeper, and RefFinder consistently identified tagO and dnaG as the most stable RG, outperforming conventionally used RG such as GAPDH. Storage validation experiments of fermented skim milk demonstrated significant improvements in quantification accuracy. Functional validation during fermented skim milk storage further demonstrated that tagO- and dnaG-based normalization provided viable counts closely aligned with plate counting, whereas GAPDH significantly overestimated bacterial abundance. These findings highlight tagO- and dnaG as robust RG for L. plantarum, establishing a novel framework for precise RT-qPCR-based quantification in dairy fermentations and advancing quality control of probiotic products.