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首页> 美国卫生研究院文献>Applied and Environmental Microbiology >Improved Enumeration of Lactic Acid Bacteria in Mesophilic Dairy Starter Cultures by Using Multiplex Quantitative Real-Time PCR and Flow Cytometry-Fluorescence In Situ Hybridization
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Improved Enumeration of Lactic Acid Bacteria in Mesophilic Dairy Starter Cultures by Using Multiplex Quantitative Real-Time PCR and Flow Cytometry-Fluorescence In Situ Hybridization

机译：多重定量实时荧光定量PCR和流式细胞术-荧光原位杂交技术提高了嗜温乳品发酵剂中乳酸菌的计数

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Nucleic acid-based assays were developed to enumerate members of the three taxa Lactococcus lactis subsp. cremoris, L. lactis subsp. lactis, and Leuconostoc spp. in mesophilic starter cultures. To our knowledge the present is the first study to present a multiplex quantitative PCR (qPCR) strategy for the relative enumeration of bacteria. The multiplex qPCR strategy was designed to quantify the target DNA simultaneously relative to total bacterial DNA. The assay has a high discriminatory power and resolves concentration changes as low as 1.3-fold. The methodology was compared with flow cytometric fluorescence in situ hybridization (FLOW-FISH) and 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside (X-Gal)-calcium citrate agar-based plate counting. For enumeration by FLOW-FISH, three new probes having the same specificity as the qPCR assay were designed and established. A combination with flow cytometry greatly reduced the time consumed compared to manual enumeration. Both qPCR and FLOW-FISH yielded similar community compositions for 10 complex starter cultures, with all detected subpopulations being highly significantly correlated (P < 0.001). Correlations between X-Gal-calcium citrate agar-based CFU and qPCR-derived counts were highly significant (P < 0.01 and P < 0.001, respectively) for the number of acidifiers versus L. lactis subsp. cremoris and for Leuconostoc spp. as quantified by the two techniques, respectively. This confirmed that most acidifiers in the studied PROBAT cultures are members of L. lactis subsp. cremoris. Quantitative real-time PCR and FLOW-FISH were found to be effective and accurate tools for the bacterial community analysis of complex starter cultures.
机译：开发了基于核酸的测定法，以枚举三个乳类乳酸乳球菌亚种的成员。 cremoris，乳酸乳亚种。乳酸菌和亮粘菌属。在嗜温发酵剂中。就我们所知，本研究是第一个提出用于细菌相对计数的多重定量PCR（qPCR）策略的研究。设计了多重qPCR策略以相对于总细菌DNA同时定量目标DNA。该测定法具有很高的辨别力，可解决低至1.3倍的浓度变化。将该方法与流式细胞术荧光原位杂交（FLOW-FISH）和基于5-溴-4-氯-3-吲哚基-β-d-吡喃半乳糖苷（X-Gal）-柠檬酸钙琼脂的平板计数进行了比较。为了通过FLOW-FISH进行计数，设计并建立了三种与qPCR分析具有相同特异性的新探针。与手动计数相比，流式细胞仪的结合大大减少了所花费的时间。 qPCR和FLOW-FISH都为10种复杂的发酵剂培养物产生了相似的群落组成，所有检测到的亚群均具有高度显着相关性（P <0.001）。基于X-Gal柠檬酸钙琼脂的CFU与qPCR得出的计数之间的相关性非常高（分别为P <0.01和P <0.001）。 cremoris和Leuconostoc spp。分别由这两种技术量化。这证实了所研究的PROBAT培养物中的大多数酸化剂是乳酸乳球菌亚种的成员。 creemoris。发现实时定量PCR和FLOW-FISH是用于复杂发酵剂培养物中细菌群落分析的有效和准确的工具。

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