Institute for Terrestrial and Aquatic Wildlife Research, University of Veterinary Medicine Hannover, Foundation, Hannover, Germany.
Research Center for Emerging Infections and Zoonoses, University of Veterinary Medicine Hannover, Foundation, Hannover, Germany.
J Appl Microbiol. 2019 Jul;127(1):47-58. doi: 10.1111/jam.14284.
The aim of this study was to design an assay for the identification of Mycobacterium avium subsp. paratuberculosis (MAP) to be used in faeces and milk samples of small ruminants with a loop-mediated isothermal amplification (LAMP) system, as a time-saving and user-friendly method in contrast to real-time PCR.
For the detection of MAP in milk and faeces of small ruminants, we developed a set of primers, specific for the target gene ISMap02. The analytical sensitivity of LAMP, when targeting ISMap02, showed a DNA detection limit of 10 fg μl . After performing spiking experiments with two MAP reference strains, DSM 44133 and ATCC 19698 , the limit of detection, using the LAMP protocol described herein were 3·8 MAP CFU per ml milk and 12·5 MAP CFU per gram faeces. All LAMP results during the establishment of the assay were compared to those of the real-time PCR results. An internal amplification control was incorporated into the assay to exclude false-negative results produced and had no significant negative impact on the analytical sensitivity. Validation of the assay was confirmed by testing field samples of faeces and revising the results with real-time PCR.
Our study conducted the first MAP detection system with a LAMP targeting ISMap02. Due to the positive results we encourage the use of LAMP in combination with ISMap02, when detecting MAP in faeces samples, as an alternative to targeting other genes as f57 or IS900. Further research on MAP detection in different matrices like raw milk, tissue or sperm with this system is recommended.
This study provides new achievements in MAP diagnostic. Especially small ruminants do not show signs of diarrhoea until the terminal stage of the illness. The greatest task in fighting MAP is to rule out animals, which shed MAP with faeces and milk before showing symptoms of Johne's disease. Worldwide there is a need to eradicate animals, which are low MAP shedders to stop the illness spreading in animal holdings. MAP detection with LAMP is time saving, easy to use, does not need expensive equipment, as, for example, PCR kits and can be used without access to laboratories. The target gene ISMap02 was shown to be a specific insertion element for MAP and is a reliable aim in future MAP detection studies.
本研究旨在设计一种用于小反刍动物粪便和牛奶样本中鉴定鸟分枝杆菌副结核亚种（MAP）的检测方法，该方法采用环介导等温扩增（LAMP）系统，与实时 PCR 相比，是一种节省时间且用户友好的方法。
为了检测小反刍动物的牛奶和粪便中的 MAP，我们开发了一套针对靶基因 ISMap02 的引物。当靶向 ISMap02 时，LAMP 的分析灵敏度显示出 DNA 检测限为 10 fg μl。用本文所述的 LAMP 方案进行了用两个 MAP 参考株 DSM 44133 和 ATCC 19698 进行的污染实验后，使用该方案的检测限为每毫升牛奶 3.8 MAP CFU 和每克粪便 12.5 MAP CFU。在建立检测方法的过程中，所有的 LAMP 结果都与实时 PCR 结果进行了比较。在检测中加入了内部扩增对照，以排除产生的假阴性结果，且对分析灵敏度没有显著负面影响。通过测试粪便的现场样本并结合实时 PCR 对结果进行修正，验证了该方法的验证。
我们的研究首次使用针对 ISMap02 的 LAMP 建立了 MAP 检测系统。由于结果为阳性，我们鼓励在检测粪便样本中的 MAP 时，将 LAMP 与 ISMap02 结合使用，作为针对其他基因（如 f57 或 IS900）的替代方法。建议使用该系统在不同基质（如原奶、组织或精子）中进一步研究 MAP 检测。
本研究在 MAP 诊断方面取得了新的成果。特别是在小反刍动物中，直到疾病的终末期才会出现腹泻症状。与 MAP 作斗争的最大任务是排除在没有出现约翰氏病症状之前就通过粪便和牛奶排泄 MAP 的动物。在全球范围内，需要消除低 MAP 排泄量的动物，以阻止疾病在动物养殖场传播。LAMP 检测节省时间，易于使用，不需要昂贵的设备，例如 PCR 试剂盒，并且可以在没有实验室的情况下使用。已经证明靶基因 ISMap02 是 MAP 的特异性插入元件，是未来 MAP 检测研究的可靠目标。
