Evaluation of real-time PCR targeting the 16S rRNA and recA genes for the enumeration of bifidobacteria in probiotic products.
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.
Masco
L
L
Laboratory of Microbiology, Ghent University, K.L. Ledeganckstraat 35, B-9000 Gent, Belgium. Liesbeth.Masco@UGent.be
Vanhoutte
T
T
Temmerman
R
R
Swings
J
J
Huys
G
G
D052160:Benzothiazoles; D001644:Bifidobacterium / Q000302:isolation & purification*; D002138:Calibration; D015169:Colony Count, Microbial / Q000379:methods* / Q000592:standards; D004269:DNA, Bacterial / Q000032:analysis; D003959:Diamines; D005798:Genes, Bacterial; D009930:Organic Chemicals; D016133:Polymerase Chain Reaction / Q000379:methods*; D019936:Probiotics; D011804:Quinolines; D012336:RNA, Ribosomal, 16S / Q000235:genetics*; D011938:Rec A Recombinases / Q000235:genetics*; D013045:Species Specificity
D052160:Benzothiazoles; D004269:DNA, Bacterial; D003959:Diamines; D009930:Organic Chemicals; D011804:Quinolines; D012336:RNA, Ribosomal, 16S; C098022:SYBR Green I; D011938:Rec A Recombinases