Herthnek D, Englund S, Willemsen P T J, Bölske G
National Veterinary Institute (SVA), Uppsala, Sweden.
J Appl Microbiol. 2006 May;100(5):1095-102. doi: 10.1111/j.1365-2672.2006.02924.x.
To develop a fast and sensitive protocol for detection of Mycobacterium avium subsp. paratuberculosis (MAP) in bovine semen and to make a critical evaluation of the analytical sensitivity.
Processed semen was spiked with known amounts of MAP. Semen from different bulls as well as semen of different dilutions was tested. The samples were treated with lysing agents and beadbeating and the DNA was extracted with phenol and chloroform. Real-time PCR with a fluorescent probe targeting the insertion element IS900 detected as few as 10 organisms per sample of 100 mul semen. PCR-inhibition was monitored by inclusion of an internal control. Pre-treatment with immunomagnetic separation was also evaluated, but was not shown to improve the overall sensitivity.
Real-time PCR is a sensitive method for detection of MAP in bovine semen. Lysis by mechanical disruption followed by phenol and chloroform extraction efficiently isolated DNA and removed PCR-inhibitors.
The high sensitivity of the applied method allows reliable testing of bovine semen used for artificial insemination to prevent the spread of Johne's disease, caused by MAP.
开发一种快速且灵敏的检测牛精液中副结核分枝杆菌（MAP）的方法，并对其分析灵敏度进行严格评估。
向处理后的精液中添加已知量的MAP。对来自不同公牛的精液以及不同稀释度的精液进行检测。样品用裂解剂处理并进行珠磨，然后用苯酚和氯仿提取DNA。使用靶向插入元件IS900的荧光探针进行实时PCR检测，每100微升精液样品中低至10个菌体即可被检测到。通过加入内对照监测PCR抑制情况。还评估了免疫磁珠分离预处理，但未显示其能提高总体灵敏度。
实时PCR是检测牛精液中MAP的灵敏方法。通过机械破碎进行裂解，随后用苯酚和氯仿提取，可有效分离DNA并去除PCR抑制剂。
所应用方法的高灵敏度使得对用于人工授精的牛精液进行可靠检测成为可能，以防止由MAP引起的副结核病传播。
