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首页> 外文期刊>Journal of Veterinary Diagnostic Investigation >Experimental validation of a nested polymerase chain reaction targeting the genetic element ISMAP02 for detection of Mycobacterium avium subspecies paratuberculosis in bovine colostrum.
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Experimental validation of a nested polymerase chain reaction targeting the genetic element ISMAP02 for detection of Mycobacterium avium subspecies paratuberculosis in bovine colostrum.

机译：靶向遗传元件ISMAP02的巢式聚合酶链反应用于检测牛初乳中鸟分枝杆菌亚种>副结核病的实验验证。

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摘要

Colostrum samples experimentally inoculated with Mycobacterium avium subsp. paratuberculosis (MAP; strain K-10) at increasing concentrations between 1x101 and 1x109 cells/ml were tested for recovery of MAP DNA using a nested ISMAP02 target polymerase chain reaction initially developed for detecting MAP DNA in fecal samples. The following detection rates were achieved for sample replicates inoculated with unsonicated MAP pure stock: 100% between 1x107 and 1x109 cells/ml, 75% between 1x103 and 1x106 cells/ml, and 50% between 1x101and 1x102 cells/ml replicates. Detection rates achieved for the colostrum sample replicates inoculated with sonicated MAP cell suspension were 75% for 1x109 cells/ml, 100% between 1x107 and 1x108 cells/ml, 75% for 1x106 cells/ml, 0 for 1x104 cells/ml, and 25% between 1x101 and 1x103 cells/ml. When negative control colostrum samples were tested, 16 of 18 (89%) samples were correctly detected as negative for MAP DNA using the current assay. In conclusion, the MAP DNA detection rates of the present assay improved with increasing concentrations of MAP in the colostrum sample replicates, although MAP DNA was also detected in 2 of 18 (11%) negative control samples, suggesting an undefined technical problem with the assay or, perhaps, sample contamination during preparation. Overall, the present findings suggest a potential role of the proposed polymerase chain reaction assay to detect MAP in colostrum. However, adoption of this test for use in routine screening of field colostrum for MAP awaits findings from an ongoing field validation study.
机译：实验性接种禽分枝杆菌亚种的初乳样品。使用浓度在1x10 1 和1x10 9 细胞/ ml之间递增的浓度的副结核（MAP; K-10菌株）检测MAP DNA的回收率最初开发用于检测粪便样品中MAP DNA的嵌套ISMAP02目标聚合酶链反应。对于未超声处理的MAP纯原种接种的样品重复样品，达到以下检测率：1x10 7 和1x10 9 细胞/ ml之间100％，1x10 3之间75％ 和1x10 6 个细胞/毫升，以及50％的1x10 1 和1x10 2 个细胞/毫升重复。超声处理的MAP细胞悬液接种的初乳样品重复样品的检出率为1x10 9 细胞/ ml为75％，1x10 7 和1x10 8 < / sup> cells / ml，对于1x10 6 细胞/ ml为75％，对于1x10 4 细胞/ ml为0，在1x10 1 和1x10 3 细胞/ ml。当检测阴性对照初乳样品时，使用当前的检测方法，可以正确检测出18个样品中的16个（89％）对MAP DNA阴性。总之，尽管在18个阴性对照样品中的2个（11％）阴性样品中也检出了MAP DNA，但随着初乳样品重复样品中MAP浓度的增加，本测定的MAP DNA检测率也提高了，这表明该测定存在不确定的技术问题或者在准备过程中样品污染。总体而言，目前的发现表明，所提出的聚合酶链反应测定法可检测初乳中的MAP。但是，该测试用于常规筛查初乳的MAP尚待进行中的现场验证研究的结果。

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