Tasara T, Stephan R
Institute for Food Safety and Hygiene, Vetsuisse Faculty, University of Zurich, Winterthurerstr. 272, CH-8057 Zurich, Switzerland.
Appl Environ Microbiol. 2005 Oct;71(10):5957-68. doi: 10.1128/AEM.71.10.5957-5968.2005.
A light cycler-based real-time PCR (LC-PCR) assay that amplifies the F57 sequence of Mycobacterium avium subsp. paratuberculosis was developed. This assay also includes an internal amplification control template to monitor the amplification conditions in each reaction. The targeted F57 sequence element is unique for M.avium subsp. paratuberculosis and is not known to exist in any other bacterial species. The assay specificity was demonstrated by evaluation of 10 known M. avium subsp. paratuberculosis isolates and 33 other bacterial strains. The LC-PCR assay has a broad linear range (2 x 10(1) to 2 x10(6) copies) for quantitative estimation of the number of M. avium subsp. paratuberculosis F57 target copies in positive samples. To maximize the assay's detection sensitivity, an efficient strategy for isolation of M. avium subsp. paratuberculosis DNA from spiked milk samples was also developed. The integrated procedure combining optimal M. avium subsp. paratuberculosis DNA isolation and real-time PCR detection had a reproducible detection limit of about 10 M. avium subsp. paratuberculosis cells per ml when a starting sample volume of 10 ml of M. avium subsp. paratuberculosis-spiked milk was analyzed. The entire process can be completed within a single working day and is suitable for routine monitoring of milk samples for M. avium subsp. paratuberculosis contamination. The applicability of this protocol for naturally contaminated milk was also demonstrated using milk samples from symptomatic M. avium subsp. paratuberculosis-infected cows, as well as pooled samples from a dairy herd with a confirmed history of paratuberculosis.
开发了一种基于荧光定量PCR（LC-PCR）的检测方法，用于扩增副结核分枝杆菌的F57序列。该检测方法还包括一个内部扩增对照模板，以监测每个反应中的扩增条件。靶向的F57序列元件是副结核分枝杆菌所特有的，在其他细菌物种中不存在。通过对10株已知的副结核分枝杆菌分离株和33株其他细菌菌株的评估，证明了该检测方法的特异性。LC-PCR检测方法具有较宽的线性范围（2×10¹至2×10⁶拷贝），用于定量估计阳性样品中副结核分枝杆菌F57靶标拷贝数。为了最大限度地提高检测方法的灵敏度，还开发了一种从添加了副结核分枝杆菌的牛奶样品中高效分离副结核分枝杆菌DNA的策略。将最佳的副结核分枝杆菌DNA分离方法与实时PCR检测相结合的综合程序，当分析10ml添加了副结核分枝杆菌的牛奶起始样品时，其可重复检测限约为每毫升10个副结核分枝杆菌细胞。整个过程可以在一个工作日内完成，适用于对牛奶样品中副结核分枝杆菌污染的常规监测。使用来自有副结核分枝杆菌感染症状奶牛的牛奶样品以及来自有确诊副结核病史的奶牛群的混合样品，也证明了该方案对自然污染牛奶的适用性。
