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A novel one-day phage-based test for rapid detection and enumeration of viable Mycobacterium avium subsp. paratuberculosis in cows' milk.
Applied Microbiology and Biotechnology ( IF 5 ) Pub Date : 2020-09-24 , DOI: 10.1007/s00253-020-10909-0
Antonio C G Foddai 1 , Irene R Grant 1
Affiliation  

Abstract

Bacteriophage-based methods for the rapid detection of viable Mycobacterium avium subsp. paratuberculosis (MAP) in veterinary specimens are a recent addition to the Johne’s disease diagnostic toolbox. Here, we report the use of D29 mycobacteriophage-coated tosylactivated paramagnetic beads to capture and concentrate MAP cells from samples (termed phagomagnetic separation, PhMS) and then naturally lyse viable MAP cells (from the inside out) to provide DNA for IS900 qPCR purposes. Transmission electron microscopy confirmed that D29 phages had bound to beads in the correct orientation and that the phage-coated beads captured MAP cells from a suspension. During test optimization, conventional IS900 PCR results were used to subjectively assess the effect of different phage:bead coating ratios, differing amounts of coated beads during PhMS, optimal incubation time post-PhMS to obtain maximal MAP DNA, and the potential benefit of a brief heat shock (55 °C/1 min) prior to IS900 TaqMan qPCR. The limit of detection 50% (LOD50%) of the optimised PhMS-qPCR assay was 10.00 MAP cells/50 ml milk (95% CI 1.20–82.83). Finally, in order to demonstrate the new assay’s ability to detect viable MAP in naturally contaminated milk, bulk tank milk samples from 100 dairy farms were tested. Forty-nine (49%) of these tested PhMS-qPCR-positive, with viable MAP numbers detected ranging from 3–126 MAP/50 ml. The novel PhMS-qPCR assay is a sensitive, specific and easy-to-apply phage-based assay for viable MAP, with potential application for milk surveillance or diagnosis of Johne’s disease.

Key points

• Phage-coated magnetic beads could capture, concentrate and lyse MAP cells from milk.

• PhMS-qPCR assay proved to be a rapid, sensitive and specific test for viable MAP.

• A potential application of PhMS-qPCR assay for milk surveillance was demonstrated.



中文翻译:

一种新型的基于一日噬菌体的测试,用于快速检测和计数活的鸟分枝杆菌亚种。牛奶中的副结核病。

摘要

基于噬菌体的方法快速检测活的鸟分枝杆菌亚种。副结核病兽医标本中的(MAP)是Johne病诊断工具箱中的最新功能。在这里,我们报道了使用D29分枝噬菌体包被的甲苯磺酰活化的顺磁珠捕获和浓缩样品中的MAP细胞(称为吞磁分离,PhMS),然后自然裂解活的MAP细胞(由内而外)以提供用于IS900 qPCR目的的DNA。透射电子显微镜证实D29噬菌体以正确的方向结合到珠子上,并且噬菌体包被的珠子从悬浮液中捕获了MAP细胞。在优化测试过程中,常规的IS900 PCR结果用于主观评估不同噬菌体:珠子的包被率,PhMS期间包被的珠子数量不同,PhMS之后的最佳孵育时间以获得最大的MAP DNA,以及在IS900 TaqMan qPCR之前进行短暂热冲击(55°C / 1分钟)的潜在好处。检出限50%(LOD优化的PhMS-qPCR分析的50%是10.00 MAP细胞/ 50 ml牛奶(95%CI 1.20-82.83)。最后,为了证明新检测方法检测天然污染牛奶中可行的MAP的能力,对来自100个奶牛场的散装罐装牛奶样品进行了测试。这些测试的PhMS-qPCR阳性物中有四十九(49%)位,检测到的可行MAP值为3–126 MAP / 50 ml。新型PhMS-qPCR测定法是一种灵敏,特异且易于应用的基于噬菌体的可行MAP测定法,具有潜在的牛奶监测或诊断约翰尼氏病的潜力。

关键点

•噬菌体包被的磁珠可以捕获,浓缩和溶解牛奶中的MAP细胞。

•PhMS-qPCR分析被证明是可行的MAP的快速,灵敏和特异的检测方法。

•证明了PhMS-qPCR测定法在牛奶监测中的潜在应用。

更新日期:2020-10-17
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