Development of a Multiplex PCR Assay for Efficient Detection of Two Potential Probiotic Strains Using Whole Genome-Based Primers

Abstract

Probiotics are microorganisms that exert strain-specific health-promoting effects on the host. Τhey are employed in the production of functional dairy or non-dairy food products; still, their detection in these complex matrices is a challenging task. Several culture-dependent and culture-independent methods have been developed in this direction; however, they present low discrimination at the strain level. Here, we developed a multiplex PCR assay for the detection of two potential probiotic lactic acid bacteria (LAB) strains, Lactiplantibacillus plantarum L125 and Lp. pentosus L33, in monocultures and yogurt samples. Unique genomic regions were identified via comparative genomic analysis and were used to produce strain-specific primers. Then, primer sets were selected that produced distinct electrophoretic DNA banding patterns in multiplex PCR for each target strain. This method was further implemented for the detection of the two strains in yogurt samples, highlighting its biotechnological applicability. Moreover, it can be applied with appropriate modifications to detect any bacterial strain with available WGS.

Dissecting the Genetic Basis of the Technological, Functional, and Safety Characteristics of Lacticaseibacillus paracasei SRX10

“…Whole-genome-based primers were designed for Lc. paracasei SRX10, following a recently published protocol [ 16 ]. Genome-wide comparative genomic analysis between the strain of interest and members of the same species was pursued to identify unique sequences.…”

“…NGS has facilitated the development of accurate methods for strain detection and quantification in fermented products. To this end, we previously developed a multiplex PCR protocol for strain-level detection based on genome-wide sequence polymorphisms [ 16 ]. In the present study, we followed the same pipeline to design strain-specific primers for single-step multiplex PCR detection of Lc.…”

“…Primer design based on unique regions of the WGS of Lc. paracasei SRX10 was performed following a previously published methodology [ 16 ]. Amplification reactions were performed at a final volume of 20 μL, consisting of 5 units Taq DNA polymerase (Minotech, Heraklion, Greece), 10 mM of each dNTP (Jena Bioscience, Jena, Germany), 1.5 mM MgCl 2 (Minotech), 1× Taq polymerase buffer (Minotech), and 10 ng of DNA template.…”

“…Importantly, genome-wide polymorphisms in strains of interest can be used for their efficient detection in complex multimicrobial matrices. Indeed, previous studies have exploited unique species- or strain-specific genetic regions to develop PCR-based detection protocols [ 15 , 16 ]. In particular, regulatory authorities require accurate labeling of functional foods, which includes proper identification and quantification of the strains present [ 17 ].…”