Short communication Evaluation of real-time PCR targeting the 16S rRNA and recA genes for the enumeration of bifidobacteria in probiotic products L. Masco a, ⁎ , T. Vanhoutte a , R. Temmerman c , J. Swings a,b , G. Huys a a Laboratory of Microbiology, Ghent University, K.L. Ledeganckstraat 35, B-9000 Gent, Belgium b BCCM™/LMG Bacteria Collection, Department of Biochemistry, Physiology and Microbiology, Ghent University, K.L. Ledeganckstraat 35, B-9000 Gent, Belgium c Laboratory of Microbial Ecology and Technology (LabMET), Faculty of Bioscience Engineering, Ghent University, Coupure Links 653, B-9000 Gent, Belgium Accepted 14 July 2006 Abstract The application of real-time PCR targeting the multicopy 16S rRNA gene and the single copy recA gene was evaluated for the enumeration of bifidobacteria in 29 probiotic products claimed to contain these organisms. Both assays relied on the use of genus-specific primers and the non- specific SYBR Green I chemistry. For both applications, the calibration curve was constructed using the type strain of Bifidobacterium animalis subsp. lactis. Upon correction with a factor corresponding to the 16S rRNA gene copy number, both assays generally produced comparable enumeration results. Only in exceptional cases, differences between both gene targets were found in probiotic products containing low amounts of bifidobacteria in which case the quantification of the multicopy 16S rRNA gene turned out to be more sensitive than the recA-based assay. On the other hand, the use of the latter single copy gene in real-time PCR quantification offers the advantage that no prior knowledge of bacterial content is required when using genus-specific primers, since no correction for multiple gene copies has to be performed. Only 11 of the analysed products (38%), including one dairy based product and ten dried products, contained a minimal Bifidobacterium concentration of 10 6 CFU per ml or g of product. Depending on the application, both assays proved to be rapid and reproducible alternatives for culture-based detection and quantification of bifidobacteria in probiotic products. © 2006 Elsevier B.V. All rights reserved. Keywords: Real-time PCR; 16S rRNA gene; recA gene; Bifidobacterium; Probiotic products 1. Introduction Bifidobacteria are natural inhabitants of the human gastro- intestinal tract and are known to contribute to a balanced intestinal microflora. Because of their probiotic potential, interest in the commercial exploitation of selected bifidobacter- ial strains in the functional food industry is growing rapidly. In parallel, this evolution has stimulated the need for advanced methods to perform qualitative and quantitative control measurements of newly developed probiotic products. In terms of quantification, traditional culture-dependent methods are still frequently used despite the fact that they are labour intensive and time consuming. In case of bifidobacteria, the reliability of many enumeration procedures is compromised by the lack of suitable media for the selective isolation of these organisms from probiotic products, which often also contain other lactic acid bacteria such as lactobacilli and Streptococcus thermophilus (Hamilton-Miller et al., 1999; Temmerman et al., 2003; Masco et al., 2005). Triggered by these shortcomings, culture- independent methods such as enzymatic-colorimetric assays (Bibiloni et al., 2000) and real-time PCR (Vitali et al., 2003) have been developed for the enumeration of bifidobacteria in probiotic products that overcome the limitations of conventional cultivation. Real-time PCR has been successfully applied for the detection and quantification of a variety of microorganisms in food, including pathogens (McKillip and Drake, 2004) and lactic acid bacteria (Pinzani et al., 2004; Furet et al., 2004). However, when it comes to enumerating bifidobacteria, most studies using real-time PCR have mainly focussed on the quantification of International Journal of Food Microbiology 113 (2007) 351 – 357 www.elsevier.com/locate/ijfoodmicro ⁎ Corresponding author. Tel.: +32 9 264 52 49; fax: +32 9 264 50 92. E-mail address: Liesbeth.Masco@UGent.be (L. Masco). 0168-1605/$ - see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.ijfoodmicro.2006.07.021