Salman Odooli - Pharmaceutical Sciences Research Center, School of Pharmacy, Shiraz University of Medical Sciences, Shiraz, Iran.
Mohammad Kargar - Department of Microbiology, Jahrom Branch, Islamic Azad University, Jahrom, Iran.
Younes Ghasemi - Department of Pharmaceutical Biotechnology, School of Pharmacy, Shiraz University of Medical Sciences, Shiraz, Iran.

Introduction and Objectives: Development of easy, accurate, and rapid procedures for qualitative and quantitative control of probiotic products is necessary since probiotic bacteria require a minimum concentration of 106 CFU/ml to exert their beneficial effects. Traditional culture-dependent methods have some limitations and disadvantages. The majority of quantitative real-time PCR (qPCR) procedures, as an alternative culture-independent method, are based on the quantification of the 16S rRNA gene. However, the quantitative data obtained by 16S rRNA-based qPCR are ambiguous because the 16S rRNA genes are present in the bacterial genome in multiple copies.Materials and Methods: A new specific primer set targeting a highly conserved sequence of the single-copy tuf gene was designed for Bifidobacterium animalis subsp. lactis BB-12 and its specificity was evaluated through PCR reactions with DNAs extracted from prevalent probiotic Bifidobacterial and Lactobacilli strains. Tuf gene-based qPCR assay was developed for the quantification of BB-12. Finally, BB-12 was detected and enumerated through tuf gene-based PCR, tuf gene-based qPCR, and selective plate count during shelf life and after the expiry date of commercial probiotic yoghurts. Results: Designed tuf gene-based primer set was specific for BB-12. The obtained standard curve of tuf gene-based qPCR reactions from 104-109CFU/ml was linear (R2=0.98) with the efficiency of 90.4%. Significantly decrease in the BB-12 counts was observed through both selective plate count and qPCR methods during shelf-life. However, these counts were according to the CODEX standard (106 CFU/ml) until the expiry date. The BB-12 count decreased rapidly and fell below the CODEX standard after the expiry date. Totally, significant differences were observed between the BB-12 counts derived from the qPCR and selective plate count, so that the bacterial counts obtained with the qPCR were higher than selective plate count. Conclusion: Despite the fact that the new single-copy tuf gene-based qPCR assay developed here is a specific, rapid, and easy method for quantification of both cultivable and dormant BB-12 cells, it does not distinguish dead and viable cells. Moreover, selective plate count method doesn’t quantify dormant bacterial populations. We deduce that the choice of enumeration method for probiotic bacteria may have a significant effect on the results of the analysis. So, qPCR assessments can serve as a complementary procedure for culture-based methods, and traditional cultural methods must still be used as a complementary golden standard for molecular approaches. For the sake of equitable judgment, we propose the use of Propidium monoazide (a DNA-intercalating dye that can selectively enter dead cells, covalently bind to DNA, and inhibit the PCR) in combination with single-copy based qPCR (PMA-qPCR), and subsequently comparing obtained results with the respective single-copy based qPCR and selective plate count.

Bifdobacterium BB-12, Quantification, tuf gene, qPCR, Plate count, Probiotic yoghurt, Shelf-life.