Gao Anli, Mutharia Lucy, Raymond Melinda, Odumeru Joseph
Laboratory Services Division (Gao, Odumeru), University of Guelph, 95 Stone Rd W, Guelph, ON Canada N1H 8J7.
J Microbiol Methods. 2007 May;69(2):417-20. doi: 10.1016/j.mimet.2006.10.019. Epub 2007 Jan 9.
Factors affecting the detection of Mycobacterium avium subsp. paratuberculosis (MAP) by PCR in raw milk and their interactions were investigated. Three day old bulk tank raw milk (50 ml) samples were seeded with MAP at a level of an estimated 30 CFU/ml. Heat-treatment of raw milk before centrifugation significantly affected the partitioning of MAP in the cream, whey and pellet fractions. Based on the IS900 PCR results, MAP preferentially partitioned into the cream fraction in unheated raw milk, and into the pellet fraction in the heat-treated milk. Treatment with 0.75% hexadecylpyridinium chloride (HPC) helped collect MAP in cream fraction. Heat treatment, use of pooled cream and pellet fractions and treatment with HPC improved the detection by PCR significantly, while washing of pellets prior to DNA extraction did not. The limit of detection using our optimized procedure was an estimated 15-50 CFU in 50 ml, or <or=1 CFU/ml.
研究了影响通过聚合酶链反应（PCR）检测生牛奶中副结核分枝杆菌（MAP）的因素及其相互作用。将估计每毫升含30个菌落形成单位（CFU）的MAP接种到3日龄的大容量罐生牛奶（50毫升）样本中。离心前对生牛奶进行热处理显著影响了MAP在乳脂、乳清和沉淀部分的分配。基于IS900 PCR结果，MAP在未加热的生牛奶中优先分配到乳脂部分，而在热处理的牛奶中则分配到沉淀部分。用0.75%十六烷基吡啶氯化物（HPC）处理有助于将MAP收集到乳脂部分。热处理、使用合并的乳脂和沉淀部分以及用HPC处理显著提高了PCR检测效果，而在DNA提取前洗涤沉淀则没有这种效果。使用我们优化的程序，检测限估计为50毫升中15 - 50个CFU，或≤1 CFU/毫升。
