Rapid and Specific Detection and Quantitation of Probiotic Strains from Fecal Samples by Combining MLST Marker Q-PCR Amplification with Nanopore Sequencing

To precisely quantify the target probiotic strain in human fecal samples, the MLST marker gene sequences of Lactobacillus strains present in Probiotic Formulan were analyzed. Marker genes exhibiting high interspecific sequence specificity were selected as amplification targets. Using extracted fecal DNA, quantitative real-time PCR (qPCR) was performed to amplify these marker genes. The resulting amplicons were then subjected to nanopore sequencing. By determining the proportion of the strain-specific reference sequence within the sequenced amplicons, the absolute copy number of the probiotic strain in the fecal sample was accurately calculated.