Detection of Mycobacterium avium subsp paratuberculosis in ovine faeces by direct quantitative PCR has similar or greater sensitivity compared to radiometric culture
Detection of Mycobacterium avium subsp paratuberculosis in ovine faeces by direct quantitative PCR has similar or greater sensitivity compared to radiometric culture
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DOI：
10.1016/j.vetmic.2007.05.002
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发表时间：
2007-11-15
影响因子：
3.3
通讯作者：
Whittington, Richard J.
中科院分区：
文献类型：
--
作者：
Kawaji, Satoko;Taylor, Deborah L.;Whittington, Richard J.
本研究的目的是建立一种新的基于IS900的实时荧光定量聚合酶链式反应(QPCR)方法，用于禽分枝杆菌亚种的检测和定量。粪便中的副结核(MAP)DNA，并用它来检测受感染的绵羊。MAP的C株和S株均可用QPCR法检测，与其他51种分枝杆菌无交叉反应，其中10株分枝杆菌含有IS900样序列。克隆到pCR2.1中的IS900片段和MAP基因组DNA分别为1个拷贝和1个FG，而在添加的粪便样品中，检测下限为每克绵羊粪便10个活MAP。总共检测了506个单独的绵羊粪便样本和27个培养结果已知的集合绵羊粪便样本。在69个BACTEC培养阳性个体粪便中，QPCR方法检出68个，且在培养上清液中检测的时间与QPCR检测的DNA含量之间有很强的相关性(r=-0.70)。在混合粪便样本中，定量聚合酶链式反应也与培养结果一致(kappa=0.59)。从接触MAP的绵羊粪便标本中检测到MAP DNA，表明该方法对粪便中MAP具有很高的分析灵敏度，并能检测到绵羊粪便中的无效MAP。未暴露于MAP的176只绵羊的粪便样本中，QPCR检测结果均为阳性。这是首次报道一种直接粪便定量聚合酶链式反应分析方法，其灵敏度与金标准放射培养方法类似。(C)2007 Elsevier B.V.保留所有权利。
The aims of this study were to develop a new real-time quantitative PCR (QPCR) assay based on IS900 for detection and quantification of Mycobacterium avium subsp. paratuberculosis (MAP) DNA in faeces, and to use this to detect infected sheep. Both the C and S strains of MAP were detected by the QPCR assay, and no cross reactions were detected with 51 other species of mycobacteria including 10 which contained IS900-like sequences. One copy of IS900 fragment cloned into plasmid pCR2.1 and 1 fg of MAP genomic DNA were consistently detected, while in spiked faecal samples the detection limit was 10 viable MAP per gram of ovine faeces. A total of 506 individual ovine faecal samples and 27 pooled ovine faecal samples with known culture results were tested. The QPCR assay detected 68 of 69 BACTEC culture positive individual faeces and there was a strong relation between time to detection in culture and DNA quantity measured by QPCR (r = -0.70). In pooled faecal samples, QPCR also agreed with culture (kappa = 0.59). MAP DNA was detected from some culture negative faecal samples from sheep exposed to MAP, suggesting that the QPCR has very high analytical sensitivity for MAP in faecal samples and detects non-viable MAP in ovine faeces. None of the faecal samples from 176 sheep that were not exposed to MAP were positive in QPCR. This is the first report of a direct faecal QPCR assay that has similar sensitivity to a gold standard radiometric culture assay. (C) 2007 Elsevier B.V. All rights reserved.