• 主题
高级搜索  >
历史搜索 清空历史
首页> 外文期刊>BMC Veterinary Research >Evaluation of combined high-efficiency DNA extraction and real-time PCR for detection of Mycobacterium avium subsp. paratuberculosis in subclinically infected dairy cattle: comparison with faecal culture, milk real-time PCR and milk ELISA
【24h】

Evaluation of combined high-efficiency DNA extraction and real-time PCR for detection of Mycobacterium avium subsp. paratuberculosis in subclinically infected dairy cattle: comparison with faecal culture, milk real-time PCR and milk ELISA

机译：评估高效DNA提取和实时PCR结合检测鸟分枝杆菌亚种。亚临床感染奶牛的副结核病：与粪便培养，牛奶实时PCR和牛奶ELISA的比较

获取原文
           
页面导航
  • 摘要
  • 著录项
  • 相似文献
  • 相关主题

摘要

Background Johne’s disease is caused by Mycobacterium avium subsp. paratuberculosis (Map) and it is one of the most important diseases in cattle worldwide. Several laboratory tests for Map detection are available; however, these are limited by inadequate sensitivity and specificity when used in subclinically infected populations. To identify Map shedders in subclinically infected cattle, we used a new, high-yield method for DNA-extraction from Map in faeces combined with quantitative real-time PCR (qPCR) for amplification of the insertion sequence IS 900 of Map (HYDEqPCR). Evaluation of HYDEqPCR was carried out in comparison with faecal culture, milk qPCR, and milk enzyme-linked immunosorbent assay (ELISA), on 141 faecal and 91 milk samples, from 141 subclinically infected dairy cattle. Results The qPCR proved to be highly sensitive, with a detection limit of 2 IS 900 DNA copies/μl in 67?% of the reactions. It also showed 100?% specificity, as determined from 50 Map and non-Map strains, and by the sequencing of qPCR amplicons. The detection limit of HYDEqPCR was 90 Map/g Map-spiked faeces, which corresponds to 2.4 colony forming units/g Map-spiked faeces, with an estimated efficiency of 85?% (±21?%). When tested on the field samples, HYDEqPCR showed 89?% of the samples as positive for Map, whereas faecal culture, milk qPCR, and milk ELISA detected 19?%, 36?% and 1?%, respectively. Fisher’s exact tests only show statistical significance ( p ≤0.05) for the correlation between HYDEqPCR and faecal culture. The agreement between HYDEqPCR and milk qPCR and milk ELISA was poor, slight, and non-significant. Conclusions This study highlights the advantages of HYDEqPCR for detection of Map in subclinically infected populations, in comparison with faecal culture, milk qPCR and milk ELISA. HYDEqPCR can detect low-level Map shedders that go undetected using these other methods, which will thus underestimate the proportions of Map-shedders in herds. Identification of these shedding animals is extremely important for prevention of the spread of Map infection in an animal population. Due to the relatively high sensitivity and specificity of HYDEqPCR, it can be applied to test for Map at the herd or individual level, regardless of animal age or production stage. HYDEqPCR will allow early detection and control of Map in any population at risk.
机译：背景Johne病是由鸟分枝杆菌亚种引起的。副结核病（地图），它是全世界牛中最重要的疾病之一。有几种用于地图检测的实验室测试;但是，当在亚临床感染人群中使用时，由于灵敏度和特异性不足而受到限制。为了鉴定亚临床感染牛中的Map脱落者，我们使用了一种新的高产率方法，从粪便中的Map中提取DNA，并与定量实时PCR（qPCR）结合以扩增Map的插入序列IS 900（HYDEqPCR）。 HYDEqPCR的评估与粪便培养，牛奶qPCR和牛奶酶联免疫吸附测定（ELISA）相比，对来自141例亚临床感染奶牛的141份粪便和91份牛奶样品进行了评估。结果qPCR被证明是高度敏感的，在67％的反应中检测限为2 IS 900 DNA拷贝/μl。根据50个Map和非Map菌株，以及通过qPCR扩增子的测序确定，它还显示出100％的特异性。 HYDEqPCR的检出限为90 Map / g Map掺入粪便，相当于2.4个菌落形成单位/ g Maps掺入粪便，估计效率为85％（±21％）。在现场样品上进行测试时，HYDEqPCR显示89％的样品对Map呈阳性，而粪便培养，牛奶qPCR和牛奶ELISA分别检测到19％，36％和1％。 Fisher的精确测试仅显示HYDEqPCR与粪便培养之间的相关性具有统计学意义（p≤0.05）。 HYDEqPCR与牛奶qPCR和牛奶ELISA之间的一致性差，轻微且不显着。结论本研究强调了与粪便培养，牛奶qPCR和牛奶ELISA相比，HYDEqPCR在亚临床感染人群中检测Map的优势。 HYDEqPCR可以检测使用其他方法无法检测到的低水平Map脱落菌，因此会低估畜群中Map脱落菌的比例。识别这些脱落的动物对于预防Map感染在动物种群中的传播极为重要。由于HYDEqPCR的相对较高的敏感性和特异性，因此无论动物年龄或生产阶段如何，都可用于畜群或个体水平的Map检测。 HYDEqPCR将允许在任何有风险的人群中早期检测和控制Map。

著录项

相似文献

  • 外文文献
  • 专利
获取原文

客服邮箱：kefu@zhangqiaokeyan.com

京公网安备：11010802029741号 ICP备案号：京ICP备15016152号-6 六维联合信息科技 (北京) 有限公司©版权所有

  • 客服微信

  • 服务号