Cook Kimberly L, Britt Jenks S
AWMRU, USDA-ARS, 230 Bennett Lane, Bowling Green, KY 42104, USA.
J Microbiol Methods. 2007 Apr;69(1):154-60. doi: 10.1016/j.mimet.2006.12.017. Epub 2006 Dec 30.
Detection of Johne's disease, an enteric infection of cattle caused by Mycobacterium avium subsp. paratuberculosis (M. paratuberculosis), has been impeded by the lack of rapid, reliable detection methods. The goal of this study was to optimize methodologies for detecting M. paratuberculosis in manure from an infected dairy cow or in contaminated soil samples using a quantitative, real-time PCR (QRT-PCR) based analysis. Three different nucleic acid extraction techniques, the efficiency of direct versus indirect sample extraction, and sample pooling were assessed. The limit of detection was investigated by adding dilutions of M. paratuberculosis to soil. Results show that the highest yield (19.4+/-2.3 microg(-1) DNA extract) and the highest copy number of the targeted M. paratuberculosis IS900 sequence (1.3+/-0.2x10(8) copies g(-1) manure) were obtained with DNA extracted from manure using Qbiogene's Fast DNA Spin kit for soil. Pooling ten samples of M. paratuberculosis-contaminated soil improved the limit of detection ten fold (between 20 and 115 M. paratuberculosis cells g(-1) soil). Detection was between 65% and 95% higher when samples were extracted directly using bead-beating than when using pre-treatment with cell extraction buffers. The final soil-sampling and extraction regime was applied for detection of M. paratuberculosis in pasture soil after the removal of a M. paratuberculosis culture positive dairy cow. M. paratuberculosis remained in the pasture soil for more than 200 days. Results from these studies suggest that DNA extraction method, sampling protocol and PCR conditions each critically influence the outcome and validity of the QRT-PCR analysis of M. paratuberculosis concentrations in environmental samples.
副结核分枝杆菌（M. paratuberculosis）引起的牛肠道感染——副结核病的检测，一直因缺乏快速、可靠的检测方法而受到阻碍。本研究的目的是优化检测方法，利用基于定量实时PCR（QRT-PCR）的分析，检测受感染奶牛粪便或受污染土壤样本中的副结核分枝杆菌。评估了三种不同的核酸提取技术、直接与间接样本提取的效率以及样本合并情况。通过向土壤中添加副结核分枝杆菌稀释液来研究检测限。结果表明，使用Qbiogene的土壤快速DNA提取试剂盒从粪便中提取DNA时，获得了最高产量（19.4±2.3微克-1 DNA提取物）和最高拷贝数的靶向副结核分枝杆菌IS900序列（1.3±0.2×108拷贝克-1粪便）。合并十个受副结核分枝杆菌污染的土壤样本，检测限提高了十倍（每克土壤中副结核分枝杆菌细胞数在20至115个之间）。与使用细胞提取缓冲液预处理相比，使用珠磨法直接提取样本时的检测率高出65%至95%。在移除一头副结核分枝杆菌培养阳性的奶牛后，将最终的土壤采样和提取方案应用于牧场土壤中副结核分枝杆菌的检测。副结核分枝杆菌在牧场土壤中留存了200多天。这些研究结果表明，DNA提取方法、采样方案和PCR条件均对环境样本中副结核分枝杆菌浓度的QRT-PCR分析结果和有效性产生关键影响。
