Quantification of total bacteria, enterobacteria and lactobacilli populations in pig digesta by real-time PCR
Quantification of total bacteria, enterobacteria and lactobacilli populations in pig digesta by real-time PCR
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DOI：
10.1016/j.vetmic.2005.11.055
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发表时间：
2006-04-16
影响因子：
3.3
通讯作者：
Gasa, J
中科院分区：
文献类型：
--
作者：
Castillo, M;Martín-Orúe, SM;Gasa, J
从断奶猪中采集空肠空肠样品，以评价实时PCR（qPCR）作为定量猪肠细菌的方法。通过qPCR定量总细菌、乳酸杆菌和肠杆菌，并将结果与传统方法获得的结果进行比较：4 '，6-二脒基-2-苯基吲哚（DAPI染色）用于总细菌，选择性培养用于乳酸杆菌和肠杆菌。实时PCR显示16 S rRNA基因拷贝数高于DAPI计数或CFU。尽管存在差异，但两种方法之间的乳酸杆菌：肠杆菌比率相似（qPCR为2.5 +/- 0.58，选择性培养为3.1 +/- 0.71，P = 0.39）。较高的PCR计数的可能原因进行了讨论，考虑到高估与PCR的死细菌或游离DNA的定量，也低估与传统的方法。样品预处理的固有差异可以部分解释观察到的差异。无论方法之间的数值差异如何，通过qPCR和传统方法获得的值显示出乳酸杆菌和总细菌的显著相关性。根据这些结果，实时PCR似乎是一种有效的方法来量化胃肠道中的微生物变化。(c)2005 Elsevier B. V.保留所有权利。
Jejunum digesta samples were taken from weaning pigs in order to evaluate real-time PCR (qPCR) as a method for quantifying pig gut bacteria. Total bacteria, lactobacilli and enterobacteria were quantified by qPCR and the results were compared with those obtained with traditional methods: 4',6-diamidino-2-phenylindole (DAPI staining) for total bacteria, selective culture for lactobacilli and enterobacteria. Real-time PCR showed higher values in terms of 16S rRNA gene copies than DAPI counts or CFU. Despite the differences, the lactobacilli:enterobacteria ratio was similar between methods (2.5 +/- 0.58 for qPCR and 3.1 +/- 0.71 for selective culture, P = 0.39). Possible reasons for the higher PCR counts are discussed considering both an overestimation with PCR by quantification of dead bacteria or free DNA and also an underestimation with conventional methods. Inherent differences in the pre-treatment of the samples could partially explain the discrepancies observed. Regardless of the numerical differences between methods, values obtained by qPCR and traditional methods showed a significant correlation for lactobacilli and total bacteria. In the light of these results, real-time PCR seems a valid method to quantify microbial shifts in the gastrointestinal tract. (c) 2005 Elsevier B.V. All rights reserved.