Yakult Central Institute for Microbiological Research, Kunitachi, Tokyo, Japan.
J Appl Microbiol. 2011 Jan;110(1):209-17. doi: 10.1111/j.1365-2672.2010.04873.x. Epub 2010 Oct 13.
To develop a quick and accurate PCR-based method to evaluate viable Bifidobacterium breve strain Yakult (BbrY) in human faeces.
The number of BbrY in faeces was detected by using strain-specific quantitative real-time PCR (qPCR) derived from a randomly amplified polymorphic DNA analysis. And using propidium monoazide (PMA) treatment, which combined a DNA-intercalating dye for covalently linking DNA in dead cells and photoactivation, only viable BbrY in the faeces highly and significantly correlated with the number of viable BbrY added to faecal samples within the range of 10(5) -10(9) cells per g of faeces was enumerated. After 11 healthy subjects ingested 10·7 log CFU of BbrY daily for 10 days, 6·9 (± 1·5) log CFU g(-1) [mean (± SD)] of BbrY was detected in faeces by using strain-specific transgalactosylated oligosaccharide-carbenicillin (T-CBPC) selective agar medium. Viable BbrY detected by qPCR with PMA treatment was 7·5 (± 1·0) log cells per g and the total number (viable and dead) of BbrY detected by qPCR without PMA treatment was 8·1 (± 0·8) log cells per g.
Strain-specific qPCR with PMA treatment evaluated viable BbrY in faeces quickly and accurately.
Combination of strain-specific qPCR and PMA treatment is useful for evaluating viable probiotics and its availability in humans.
开发一种快速准确的基于 PCR 的方法，用于评估人类粪便中双歧杆菌短双歧杆菌 Yakult（BbrY）的活菌数。
使用源自随机扩增多态性 DNA 分析的菌株特异性定量实时 PCR（qPCR）检测粪便中的 BbrY 数量。并使用吖啶单脒（PMA）处理，该处理方法结合了一种 DNA 嵌入染料，用于共价连接死细胞中的 DNA 并进行光激活，只有粪便中活的 BbrY 与在粪便样本中添加的活的 BbrY 数量高度显著相关，范围为 10(5)-10(9)个细胞/g。在 11 名健康受试者每天摄入 10(7)log CFU 的 BbrY 10 天后，使用菌株特异性半乳糖基转移寡糖-羧苄青霉素（T-CBPC）选择性琼脂培养基在粪便中检测到 6.9(±1.5)log CFU/g 的 BbrY。经 PMA 处理的 qPCR 检测到的活菌数为 7.5(±1.0)log 个细胞/g，未经 PMA 处理的 qPCR 检测到的总活菌数（活菌和死菌）为 8.1(±0.8)log 个细胞/g。
经 PMA 处理的菌株特异性 qPCR 可快速准确地评估粪便中的活菌数。
菌株特异性 qPCR 与 PMA 处理相结合，可用于评估活益生菌及其在人体中的可用性。
