Evaluation of real-time PCR targeting the 16S rRNA and recA genes for the enumeration of bifidobacteria in probiotic products
Evaluation of real-time PCR targeting the 16S rRNA and recA genes for the enumeration of bifidobacteria in probiotic products
复制标题
DOI：
10.1016/j.ijfoodmicro.2006.07.021
复制
发表时间：
2007-02-15
影响因子：
5.4
通讯作者：
Huys, G.
中科院分区：
文献类型：
--
作者：
Masco, L.;Vanhoutte, T.;Huys, G.
实时PCR靶向多拷贝16S rRNA基因和单拷贝recA基因的应用进行了评估，用于计数声称含有这些生物体的29种益生菌产品中的双歧杆菌。两种检测方法均依赖于使用属特异性引物和非特异性SYBR绿色I化学。对于这两种应用，使用动物双歧杆菌亚种的模式菌株构建校准曲线。乳。在用对应于16S rRNA基因拷贝数的因子进行校正后，两种测定法通常产生相当的计数结果。仅在例外情况下，在含有少量双歧杆菌的益生菌产品中发现两种基因靶标之间的差异，在这种情况下，多拷贝16S rRNA基因的定量结果比基于recA的测定更灵敏。另一方面，在实时PCR定量中使用后一种单拷贝基因提供了这样的优点，即当使用属特异性引物时，不需要细菌含量的先验知识，因为不必进行多个基因拷贝的校正。仅11种分析的产品（38%），包括一种乳制品和十种干燥产品，含有每ml或g产品10（6）CFU的最小双歧杆菌浓度。根据不同的应用，这两种检测方法被证明是快速和可重复的替代方法，用于基于培养的检测和定量益生菌产品中的双歧杆菌。(c)2006 Elsevier B.V.保留所有权利。
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 nonspecific 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 I I of the analysed products (38%), including one dairy based product and ten dried products, contained a minimal Biflidobacterium 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. (c) 2006 Elsevier B.V. All rights reserved.