Department of Biotechnology, Acharya Nagarjuna University, Guntur, Andhra Pradesh, 522 510, India.
Department of Veterinary Microbiology, Genomix Molecular Diagnostics Pvt. Ltd, 5-36/207, Prashanthnagar, Kukatpally, Hyderabad, Telangana, 500 072, India.
Braz J Microbiol. 2019 Oct;50(4):1105-1114. doi: 10.1007/s42770-019-00116-z. Epub 2019 Sep 20.
The Mycobacterium avium subspecies paratuberculosis (MAP) causes paratuberculosis (Johne's disease), a systemic and chronic inflammation of intestine that affects bovine, small ruminants like goat and sheep. The disease has a greater economic importance in cattle and in small ruminants. But its effective control is impeded due to lack of rapid and accurate diagnostics. The present study is aimed at developing a LAMP-coupled lateral flow device (LFD) for rapid detection of paratuberculosis in livestock animal species such as cattle and in small ruminants at resource-limited areas. LAMP primers with biotin and FITC end tags were designed for IS900 gene specific for MAP. To determine sensitivity of LAMP assay, 10-fold serial dilutions were made from 10 ng/μl MAP stock DNA and were compared with PCR. The detection limits of LAMP-coupled LFD were defined and reactions were repeated for reproducibility. The specificity was evaluated using other infectious bacteria such as M. bovis, M. tuberculosis, Brucella abortus, Leptospira interrogan, Yersinia enterocolitica, Salmonella typhimurium, Listeria monocytogens, and Staphylococcus aureus. A total of 95 samples turned positive for LAMP-coupled LFD out of 389 fecal samples. All the cultural-positive and PCR-positive samples showed positive in LAMP-coupled LFD. Nine samples with negative cultures turned positive in LAMP assay. The overall sensitivity and specificity of the LAMP-coupled LFD assays were 100% and 97.02% respectively in comparison with the culture as the gold standard method. The sensitivity detection limit of developed assay was 10 fg/μl and specificity was 100%. This assay successfully detected MAP not only by using bacterial DNA but also in clinical fecal samples. The clear band formation at control and test positions was observed on LAMP-coupled LFD. The developed assay is a simple, rapid, easy to perform, and is very useful in early diagnosis of Mycobacterium avium subsp. paratuberculosis at point of care resource-limited areas.
分支杆菌副结核亚种（MAP）引起副结核病（约翰氏病），这是一种影响牛、山羊和绵羊等小反刍动物的肠道的全身性和慢性炎症。这种疾病在牛和小反刍动物中具有更大的经济重要性。但由于缺乏快速准确的诊断方法，其有效控制受到阻碍。本研究旨在开发一种基于环介导等温扩增（LAMP）的侧流装置（LFD），用于在资源有限的地区快速检测牛和小反刍动物等家畜动物中的副结核病。设计了针对 MAP 的 IS900 基因的带有生物素和 FITC 末端标记的 LAMP 引物。为了确定 LAMP 检测的灵敏度，从 10ng/μl MAP 库存 DNA 中进行 10 倍系列稀释，并与 PCR 进行比较。定义了 LAMP 结合 LFD 的检测限，并进行了重复性反应。使用其他传染性细菌，如 M. bovis、M. tuberculosis、Brucella abortus、Leptospira interrogan、Yersinia enterocolitica、Salmonella typhimurium、Listeria monocytogens 和 Staphylococcus aureus 评估了特异性。在 389 份粪便样本中，共有 95 份 LAMP 结合 LFD 呈阳性。所有培养阳性和 PCR 阳性样本在 LAMP 结合 LFD 中均呈阳性。9 份培养阴性样本在 LAMP 检测中呈阳性。与培养作为金标准方法相比，LAMP 结合 LFD 检测的总灵敏度和特异性分别为 100%和 97.02%。开发的检测方法的灵敏度检测限为 10 fg/μl，特异性为 100%。该检测方法不仅可以使用细菌 DNA，还可以使用临床粪便样本成功检测到 MAP。在 LAMP 结合 LFD 上可以观察到对照和测试位置的清晰带形成。该检测方法简单、快速、易于操作，非常有助于在资源有限的基层医疗点进行早期诊断分枝杆菌副结核亚种。
