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首页> 外文期刊>FEMS Microbiology Letters >Evaluation of two recovery methods for detection of Mycobacterium avium subsp paratuberculosis by PCR: direct-dilution-centrifugation and C-18-carboxypropylbetaine processing
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Evaluation of two recovery methods for detection of Mycobacterium avium subsp paratuberculosis by PCR: direct-dilution-centrifugation and C-18-carboxypropylbetaine processing

机译：通过PCR直接检测离心分离和C-18-羧丙基甜菜碱两种检测方法用于评估鸟分枝杆菌副结核菌副结核病的回收方法

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摘要

A duplex polymerase chain reaction (PCR)-hybridization assay based on Mycobacterium avium subsp. paratuberculosis (MAP)-specific IS900 integration sites was used to evaluate two mycobacterial recovery methods from bovine feces: a direct-dilution-centrifugation method and a C-18-carboxypropylbetaine (CB-18)-based method. All MAP PCR results were confirmed for absence of inhibitors using a novel PCR system based on the rpoB gene of plant chloroplasts as an internal control. The detection limits of both MAP recovery methods when coupled with PCR were determined to be between 100 and 1000 organisms. Using culture as a 'gold standard' PCR following the direct-dilution-centrifugation protocol was 92.6% sensitive and 83.7% specific, whereas PCR following the CB-18 method was 100% sensitive and 53.5% specific. Both methods were 100% specific when 60 'true' negatives from two uninfected herds were tested. Both the CB-18 and direct processing methods coupled with a target-specific amplification technique may provide greater sensitivity to diagnose subclinical animals as they were able to detect more positives, on samples derived from infected herds, than conventional culture methods; however, more extensive investigation and follow-up of suspect animals will be required to fully validate the MAP recovery and molecular detection protocols described. (C) 2003 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved. [References: 34]
机译：基于鸟分枝杆菌亚种的双链聚合酶链反应（PCR）-杂交测定。副结核病（MAP）特异的IS900整合位点用于评估从牛粪中回收两种分枝杆菌的方法：直接稀释离心法和基于C-18-羧丙基甜菜碱（CB-18）的方法。使用基于植物叶绿体rpoB基因的新型PCR系统作为内部对照，确认所有MAP PCR结果均不含抑制剂。两种MAP回收方法与PCR结合时的检出限被确定为100至1000个生物体。使用培养物作为“金标准”，直接稀释离心操作后的PCR灵敏度为92.6％，特异性为83.7％，而采用CB-18方法的PCR灵敏度为100％，特异性为53.5％。当测试来自两个未感染牛群的60个“真”阴性时，这两种方法都是100％特异性的。与传统的培养方法相比，CB-18和直接加工方法结合靶标特异性扩增技术都可以提供更高的诊断亚临床动物的敏感性，因为它们能够从感染牛群的样品中检测出更多阳性。但是，将需要更广泛的调查和追踪可疑动物，以充分验证所描述的MAP回收和分子检测方案。 （C）2003年欧洲微生物学会联合会。由Elsevier B.V.发布。保留所有权利。 [参考：34]

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