Comparison of rpoA and pheS Gene Sequencing to 16S rRNA Gene Sequencing in Identification and Phylogenetic Analysis of LAB from Probiotic Food Products and Supplements

Abstract

An investigation of the relevance of sequenced protein-coding genes in comparison with 16S rRNA gene sequences to correctly delineate and discriminate between closely related Lactobacillus isolates is presented. These reportedly probiotic Lactobacillus strains were obtained from probiotic supplements and food products. Probiotic Lactobacillus species have been found to have several potential health benefits and technological properties which may be strain-dependent. It was deemed necessary to investigate molecular techniques for the identification and discrimination between closely related isolates. The 16S rRNA, pheS, and rpoA genes were amplified in a polymerase chain reaction using the oligonucleotide pairs 27F/1492R, pheS-21-F/pheS-23-R, and rpoA-21-F/rpoA-23-R. The PCR amplicons were separated using gel agarose electrophoresis and observed by the Bio-Rad Gel Doc^TM XR+ Imaging system transilluminator. Sequencing of the PCR amplicons was done in an ABI PRISM^TM 3100 genetic analyzer. MEGA 5.05 software was employed in doing the phylogenetic analysis. Identification by 16S rRNA gene sequencing was confirmed by pheS and rpoA gene sequencing. Subsequent phylogenetic analysis and concatenation showed that interspecies and intraspecies degrees of diversity and similarity were better enabled by pheS and rpoA primers. Overall, findings revealed cases of inaccurate identification of Lactobacillus strains in probiotic supplements on the market, highlighting the relevance of the multigenic approach in more accurately identifying Lactobacillus strains. Supplemental materials are available for this article; see the publisher's online edition of Food Biotechnology to view the supplemental file.

Keywords:

• protein-coding genes
• housekeeping genes
• phylogenetic analysis
• concatenation
• probiotics
• evolutionary divergence
• split decomposition analysis

ACKNOWLEDGMENT

Tshwane University of Technology is acknowledged for providing funds that enabled this investigation to be completed.