应用 IS6110 实时 PCR 检测新鲜组织中牛分枝杆菌的特异性提高。
Improved specificity for detection of Mycobacterium bovis in fresh tissues using IS6110 real-time PCR.
机构信息
Infectious Bacterial Diseases Research Unit, National Animal Disease Center, Agricultural Research Service, United States Department of Agriculture, Ames, Iowa, USA.
出版信息
BMC Vet Res. 2011 Aug 25;7:50. doi: 10.1186/1746-6148-7-50.
BACKGROUND
Culture of M. bovis from diagnostic specimens is the gold standard for bovine tuberculosis diagnostics in the USA. Detection of M. bovis by PCR in tissue homogenates may provide a simple rapid method to complement bacterial culture. A significant impediment to PCR based assays on tissue homogenates is specificity since mycobacteria other than M. bovis may be associated with the tissues.
RESULTS
Previously published IS6110 based PCR diagnostic assays, along with one developed in house, were tested against environmental mycobacteria commonly isolated from diagnostic tissues submitted to the National Veterinary Services Laboratory. A real-time PCR assay was developed (IS6110_T) that had increased specificity over other IS6110 based assays. Of the 13 non-tuberculous mycobacteria tested with IS6110_T only M. wolinskyi was positive. Thirty M. bovis infected tissue homogenates and 18 control tissues were used to evaluate the potential for the assay as a diagnostic test. In this small sample, IS6110_T detected 20/30 samples from M. bovis infected animals and 0/18 control tissues.
CONCLUSIONS
The IS6110_T assay provides a PCR based assay system that is compatible with current diagnostic protocols for the detection of M. bovis in the USA and compliments current testing strategies.
背景
从诊断标本中分离牛分枝杆菌是美国牛结核病诊断的金标准。组织匀浆中 M. bovis 的 PCR 检测可能提供一种简单快速的方法来补充细菌培养。基于组织匀浆的 PCR 检测的一个主要障碍是特异性，因为除 M. bovis 以外的分枝杆菌可能与组织相关。
结果
先前发表的基于 IS6110 的 PCR 诊断检测方法，以及一个内部开发的方法，针对通常从提交给国家兽医服务实验室的诊断组织中分离的环境分枝杆菌进行了测试。开发了一种实时 PCR 检测方法（IS6110_T），其特异性高于其他基于 IS6110 的检测方法。在使用 IS6110_T 测试的 13 株非结核分枝杆菌中，只有 M. wolinskyi 呈阳性。用 30 份感染 M. bovis 的组织匀浆和 18 份对照组织评估该检测方法作为诊断检测的潜力。在这个小样本中，IS6110_T 检测到 30 份感染 M. bovis 的动物组织样本中的 20 份，而在 18 份对照组织中未检测到阳性结果。
结论
IS6110_T 检测方法提供了一种基于 PCR 的检测系统，与美国目前用于检测 M. bovis 的诊断方案兼容，并补充了当前的检测策略。
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