Enumeration of Mycobacterium avium subsp. paratuberculosis by quantitative real-time PCR, culture on solid media and optical densitometry.
Enumeration of Mycobacterium avium subsp. paratuberculosis by quantitative real-time PCR, culture on solid media and optical densitometry.
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DOI：
10.1186/1756-0500-5-114
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发表时间：
2012-02-22
影响因子：
1.8
通讯作者：
Pavlik, Ivo
中科院分区：
文献类型：
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作者：
Kralik, Petr;Beran, Vladimir;Pavlik, Ivo
背景：不同的方法用于确定鸟分枝杆菌亚种的数量。副结核杆菌（MAP）细胞悬浮液。其中大多数基于培养（CFU测定）或MAP细胞的目视/仪器直接计数。在这项研究中，我们已经比较了培养方法与以前发表的F57为基础的定量实时PCR（F57 qPCR）方法，以确定他们的相对能力，以计数悬浮液中具有相同的光密度（OD）的三种不同的MAP分离株的数量。麦克法兰浊度标准也进行了比较，F57 qPCR和文化，由于其频繁的列入和使用的MAP studies.FINDINGS：MAP的数量在2倍系列稀释的分离株与各自的OD测量确定F57 qPCR和文化。发现与F57 qPCR相比，培养物提供了较低的MAP CFU计数约2 log 10。麦克法兰标准（如E.结论：在所有后续计数均采用相同方法的实验中，建议使用培养和/或qPCR方法估计MAP数。当然不建议使用培养物作为qPCR实验的标准品，反之亦然。
BACKGROUND: Different approaches are used for determining the number of Mycobacterium avium subsp. paratuberculosis (MAP) cells in a suspension. The majority of them are based upon culture (determination of CFU) or visual/instrumental direct counting of MAP cells. In this study, we have compared the culture method with a previously published F57 based quantitative real-time PCR (F57qPCR) method, to determine their relative abilities to count the number of three different MAP isolates in suspensions with the same optical densities (OD). McFarland turbidity standards were also compared with F57qPCR and culture, due to its frequent inclusion and use in MAP studies.FINDINGS: The numbers of MAP in two-fold serial dilutions of isolates with respective OD measurements were determined by F57qPCR and culture. It was found that culture provided lower MAP CFU counts by approximately two log10, compared to F57qPCR. The McFarland standards (as defined for E. coli) showed an almost perfect fit with the enumeration of MAP performed by F57qPCR.CONCLUSIONS: It is recommended to use culture and/or qPCR estimations of MAP numbers in experiments where all subsequent counts are performed using the same method. It is certainly not recommended the use of culture as the standard for qPCR experiments and vice versa.