Veterinary Research Institute, Hudcova 70, 621 00 Brno, Czech Republic.
Vet Microbiol. 2011 Apr 21;149(1-2):133-8. doi: 10.1016/j.vetmic.2010.10.009. Epub 2010 Oct 21.
This study focused on the development of a reliable and cost-efficient DNA isolation procedure for the detection of Mycobacterium avium subsp. paratuberculosis (MAP) in faeces by previously developed IS900 and F57 quantitative real time PCR (qPCR) and their comparison with culture. The recovery of MAP DNA from the spiking experiments ranged from 29.1 to 102.4% of the input amount of MAP with median 37.9%. The limit of detection was determined to be 1.03 × 10(4) for F57 qPCR and 6.87 × 10(2)MAP cells per gram of faeces for IS900 qPCR, respectively. The developed technique for DNA isolation was coupled with IS900 qPCR and compared to traditional MAP culture using a cohort of 1906 faecal samples examined from 12 dairy cattle farms in our laboratory. From those 1906 original faecal samples, 875 were positive by IS900 qPCR and 169 by culture. None of the culture positive samples was negative by IS900 qPCR. This data facilitated development of a predictive model capable of estimating the probability of being culture positive by estimating the absolute number of MAP per gram of faeces as determined IS900 qPCR without performing the culture.
本研究旨在开发一种可靠且经济高效的 DNA 分离程序，用于通过先前开发的 IS900 和 F57 定量实时 PCR(qPCR)检测粪便中的禽分枝杆菌副结核亚种(MAP)，并将其与培养法进行比较。从添加实验中回收的 MAP DNA 量为 MAP 输入量的 29.1%至 102.4%，中位数为 37.9%。F57 qPCR 的检测限为 1.03×10(4)，IS900 qPCR 的检测限为每克粪便 6.87×10(2)个 MAP 细胞。开发的 DNA 分离技术与 IS900 qPCR 相结合，并与传统的 MAP 培养法在我们实验室的 12 个奶牛场的 1906 个粪便样本中进行了比较。在这 1906 个原始粪便样本中，875 个样本通过 IS900 qPCR 呈阳性，169 个样本通过培养呈阳性。没有一个培养阳性样本在 IS900 qPCR 中呈阴性。该数据有助于开发一种预测模型，通过估计 IS900 qPCR 确定的每克粪便中 MAP 的绝对数量，而无需进行培养，从而估计培养阳性的概率。
