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首页> 外文会议>International Colloquium on Paratuberculosis >Evaluation of three methods of DNA extraction for the detection of Mycobacterium paratuberculosis by polymerase chain reaction in milk
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Evaluation of three methods of DNA extraction for the detection of Mycobacterium paratuberculosis by polymerase chain reaction in milk

机译：用牛奶中聚合酶链反应检测三氧萃取方法三种方法的评价

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Objectives: To evaluate three methods of DNA extraction from milk for the IS900 polymerase chain reaction (PCR) and compare analytical sensitivities of the PCR and modified double incubation radiometric mycobacterial culture (RMC) method. Experimental design: The comparative evaluation of the three DNA extraction methods (Beadbeater, InstaGene and Qiagene) and determination of the detection limits of the RMC and PCR were carried out on triplicate samples of milk inoculated with serial ten-fold dilutions of Mycobactehum paratuberculosis. Results: Among the three protocols of DNA extraction from milk, the Beadbeater method was the most efficient procedure for the preparation of M. paratuberculosis DNA template for the IS900 PCR. The average detection limit of the Beadbeater PCR system was about 70 viable M. paratuberculosis cells/50 ml sample. The InstaGene and Qiagene (QIAamp DNA Stool Kit) methods produced average detection limits by PCR of 600 and 700 cells/50 ml sample, respectively. The analytical sensitivity of the RMC was about 700 viable cells/50 ml sample. Conclusions: The analytical sensitivity of the Beadbeater PCR system is sufficient for this test to be used for the detection of low levels of M. paratuberculosis contamination in milk. Further evaluation of this test on diagnostic samples is warranted.
机译：目的：评估IS900聚合酶链反应（PCR）牛奶中的三种DNA提取方法，并比较PCR的分析敏感性和改良的双重孵化辐射分枝杆菌（RMC）方法。实验设计：三次DNA提取方法（胎儿，液体和沸石）的比较评价和RMC和PCR的检测限的测定是在接种的乳头溶液的连续十倍稀释液的三份牛奶样品上进行的。结果：在牛奶中的三种DNA方案中，珠子制造方法是为IS900 PCR制备M.Alatbulisis DNA模板的最有效的方法。珠子撞击器PCR系统的平均检测限为约70个可行的副伞菌癌细胞/ 50mL样品。 Instagene和Qiapl Qiagene（QIAMAM DNA凳子试剂盒）方法分别产生600和700个细胞/ 50ml样品的PCR平均检测限。 RMC的分析敏感性约为700个活细胞/ 50mL样品。结论：珠映泊PCR系统的分析敏感性足以用于该试验，用于检测牛奶中低水平的副伞菌污染。有必要进一步评估该测试的诊断样本。

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