Castillo Marisol, Martín-Orúe Susana M, Manzanilla Edgar G, Badiola Ignacio, Martín Marga, Gasa Josep
Departament de Ciència Animal i dels Aliments, Universitat Autònoma de Barcelona 08193, Bellaterra, Barcelona, Spain.
Vet Microbiol. 2006 Apr 16;114(1-2):165-70. doi: 10.1016/j.vetmic.2005.11.055. Epub 2005 Dec 27.
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.
为了评估实时荧光定量聚合酶链反应（qPCR）作为一种定量猪肠道细菌的方法，从断奶仔猪中采集空肠消化物样本。通过qPCR对总细菌、乳酸杆菌和肠杆菌进行定量，并将结果与传统方法获得的结果进行比较：用4',6-二脒基-2-苯基吲哚（DAPI染色）计数总细菌，用选择性培养法计数乳酸杆菌和肠杆菌。实时荧光定量聚合酶链反应显示，16S rRNA基因拷贝数高于DAPI计数或菌落形成单位。尽管存在差异，但两种方法的乳酸杆菌与肠杆菌比例相似（qPCR为2.5±0.58，选择性培养为3.1±0.71，P = 0.39）。考虑到通过对死菌或游离DNA进行定量的PCR高估以及传统方法的低估，讨论了PCR计数较高的可能原因。样本预处理的固有差异可能部分解释了观察到的差异。无论方法之间的数值差异如何，qPCR和传统方法获得的值在乳酸杆菌和总细菌方面均显示出显著相关性。鉴于这些结果，实时荧光定量聚合酶链反应似乎是一种定量胃肠道微生物变化的有效方法。
