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

Despoina E Kiousi; Dimitrios M Karadedos; Anastasia Sykoudi; Panagiotis Repanas; Christina S Kamarinou; Anthoula A Argyri; Alex Galanis

doi:10.3390/microorganisms11102553

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

Microorganisms. 2023 Oct 13;11(10):2553. doi: 10.3390/microorganisms11102553.

Authors

Despoina E Kiousi¹, Dimitrios M Karadedos¹, Anastasia Sykoudi¹, Panagiotis Repanas¹, Christina S Kamarinou^{1

2}, Anthoula A Argyri², Alex Galanis¹

Affiliations

¹ Department of Molecular Biology and Genetics, Faculty of Health Sciences, Democritus University of Thrace, 68100 Alexandroupolis, Greece.
² Institute of Technology of Agricultural Products, Hellenic Agricultural Organization-DIMITRA, 14123 Lycovrissi, Greece.

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.

Keywords: bacterial identification; comparative genomics; lactic acid bacteria; multiplex PCR; probiotics; strain-specific; whole genome sequencing.

Grants and funding

This research was financed by the project “InTechThrace: Integrated Technologies in biomedical research: multi-level biomarker analysis in Thrace” (MIS Code 5047285) under the Operational Program “Competitiveness, Entrepreneurship and Innovation” (EPAnEK), co-funded by the European Regional Development Fund (ERDF) and national resources (Partnership Agreement 2014–2020).