Development of a PCR assay for the strain-specific identification of probiotic strain Lactobacillus paracasei IMPC2.1

Abstract

Recent investigations clearly indicate that the probiotic bacterium Lactobacillus paracasei IMPC2.1 can be incorporated into vegetables to obtain innovative probiotic foods whose marketing has been authorized by the Italian Ministry of Health. In this study, strain IMPC2.1 was characterized at a molecular level in order to define its taxonomic position and to develop a PCR test for strain-specific identification. Molecular methods, such as 16S rRNA gene sequencing and multiplex PCR, have provided evidence that strain IMPC2.1 indeed belongs to the L. paracasei species. In addition, a cluster analysis of fluorescent amplified fragment length polymorphism (f-AFLP) data strongly indicated that strain IMPC2.1 and nine other L. paracasei strains (including strain ATCC 334) belong to the same species and are definitely differentiated from the type strain L. casei ATCC 393. The f-AFLP technique was also used to identify a strain-specific DNA fragment of L. paracasei IMPC2.1 — encoding an amino acid sequence similar to a glycosyltransferase of probiotic strain L actobacillus rhamnosus HN001 — which enabled us to develop a rapid PCR test for strain-specific identification. The strain-specificity of the PCR test was assessed by comparison with a total of 73 bacterial strains mainly isolated from vegetable products that did not produce any amplified fragment. These strains belonged to the L. paracasei species, to 6 additional species of Lactobacillus and to Weissella cibaria, W. confusa, Lactococcus lactis, Leuconostoc mesenteroides and Pediococcus pentosaceus. A method similar to the one used in this study can be adopted to develop easy, rapid detection techniques for monitoring other bacteria in complex microbiota.