Laboratory of Food Biotechnology, Institute of Food Science and Nutrition, Swiss Federal Institute of Technology, Schmelzbergstrasse. 7 8092 Zurich, Switzerland.
Food Microbiol. 2010 Apr;27(2):236-42. doi: 10.1016/j.fm.2009.10.010. Epub 2009 Oct 21.
Viability of probiotic bacteria is traditionally assessed by plate counting which has several limitations, including underestimation of cells in aggregates or chains morphology. We describe a quantitative PCR (qPCR)-based method for an accurate enumeration of viable cells of Bifidobacterium longum NCC2705 exhibiting different morphologies by measuring the mRNA levels of cysB and purB, two constitutively expressed housekeeping genes. Three primer-sets targeting short fragments of 57-bp of cysS and purB and one 400-bp fragment of purB were used. Cell quantification of serially diluted samples showed a good correlation coefficient of R(2) 0.984 +/- 0.003 between plate counts and qRT-PCR for all tested primer sets. Loss of viable cells exposed to a lethal heat stress (56 degrees C, 10, 20 and 30 min) was estimated by qRT-PCR and plate counts. No significant difference was observed using qRT-PCR targeting the 400-bp fragment of purB compared to plate counts indicating that this fragment is a suitable marker of cell viability. In contrast, the use of the 57-bp fragments led to a significant overestimation of viable cell counts (18 +/- 3 and 7 +/- 2 fold for cysB and purB, respectively). Decay of the mRNA fragments was studied by treatment of growing cells with rifampicin prior qRT-PCR. The 400-bp fragment of purB was faster degraded than the 57-bp fragments of cysB and purB. The 400-bp fragment of purB was further used to enumerate viable cells in aggregate state. Cell counts were more than 2 log(10) higher using the qRT-PCR method compared to plate counts. Growing interest in probiotic characteristics of aggregating bacteria cells make this technique a valuable tool to accurately quantify viable probiotic bacteria exhibiting heterogeneous morphology.
传统上，益生菌细菌的活力是通过平板计数来评估的，这种方法有几个局限性，包括低估了聚集或链状形态的细胞。我们描述了一种基于定量 PCR（qPCR）的方法，通过测量两个组成型表达的管家基因 cysB 和 purB 的 mRNA 水平，准确计数具有不同形态的长双歧杆菌 NCC2705 的活菌数。使用针对 cysS 和 purB 的短片段（57-bp）和 purB 的一个 400-bp 片段的三个引物对。对连续稀释样品的细胞定量显示，所有测试的引物对的平板计数和 qRT-PCR 之间的相关系数 R(2)为 0.984 +/- 0.003。通过 qRT-PCR 和平板计数估计了暴露于致死热应激（56°C，10、20 和 30 分钟）的活菌数的损失。使用针对 purB 的 400-bp 片段的 qRT-PCR 与平板计数相比没有观察到显着差异，表明该片段是细胞活力的合适标志物。相比之下，使用 57-bp 片段导致活菌计数的显着高估（分别为 cysB 和 purB 的 18 +/- 3 和 7 +/- 2 倍）。在进行 qRT-PCR 之前，用利福平处理生长细胞以研究 mRNA 片段的衰减。与 cysB 和 purB 的 57-bp 片段相比，purB 的 400-bp 片段降解得更快。进一步使用 purB 的 400-bp 片段对聚集状态下的活菌进行计数。与平板计数相比，qRT-PCR 方法的细胞计数高 2 个对数（10）。对聚集细菌细胞的益生菌特性的日益关注使该技术成为准确量化具有异质形态的活菌的有价值工具。
