Abstract：
Mycobacterium avium ssp. paratuberculosis (MAP) is the bacterium responsible for causing Johne\'s disease (JD), which is endemic to dairy cattle and also implicated in the etiology of Crohn\'s disease. The difficulty in diagnosing asymptomatic cows for JD makes this disease hard to control. Johne\'s disease is considered a priority under the One Health approach to prevent the spread of the causative agent to humans. Environmental screening is a strategic approach aimed at identifying dairy herds with animals infected with MAP. It serves as the initial step toward implementing more intensive actions to control the disease. Quantitative PCR (qPCR) technology is widely used for diagnosis. Given that genome sequencing is now much more accessible than ever before, it is possible to target regions of the MAP genome that allow for the greatest diagnostic sensitivity and specificity. The aim of this study was to identify among the published qPCR assays targeting IS900 the more cost-effective options to detect MAP and to validate them in the diagnostic context of JD. Mycobacterium avium ssp. paratuberculosis IS900 is a prime target because it is a multicopy genetic element. A total of 136 publications have reported on the use of IS900 qPCR assays over the past 3 decades. Among these records, 29 used the SYBR Green chemistry, and 107 used TaqMan technology. Aside from the 9 reports using commercial assays, 72 TaqMan reports cited previously published work, leaving us with 27 TaqMan qPCR designs. Upon closer examination, 5 TaqMan designs contained mismatches in primer or probe sequences. Additionally, others exhibited high similarity to environmental microorganisms or non-MAP mycobacteria. We assessed the performance of 6 IS900 qPCR designs and their sensitivity when applied to clinical or environmental samples, which varied from 4 to 56 fold overall. Additionally, we provide recommendations for testing clinical and environmental samples, as certain strategies used previously should be avoided due to poor qPCR design (e.g., the presence of mismatches) or a lack of specificity.
摘要：
鸟分枝杆菌ssp。副结核病（MAP）是导致约翰氏病（JD）的细菌，这是奶牛特有的，也与克罗恩病的病因有关。诊断无症状奶牛的JD的困难使得这种疾病难以控制。在“一个健康”方法下，JD被认为是防止病原体传播给人类的优先事项。环境筛查是一种战略方法，旨在识别感染MAP的动物的奶牛群。它是实施更密集的行动来控制疾病的第一步。定量聚合酶链反应(qPCR)技术广泛用于诊断。鉴于基因组测序现在比以往任何时候都更容易获得，可以靶向MAP基因组的区域,这些区域具有最大的诊断灵敏度和特异性.这项研究的目的是在针对IS900的已发表的qPCR分析中确定更经济有效的选择来检测MAP，并在JD疾病的诊断背景下验证它们。MAPIS900是主要目标，因为它是多拷贝遗传元件。在过去的30年中，共有136篇出版物报道了IS900qPCR测定法的使用。在这些记录中,29例使用SYBRGreen化学，107例使用TaqMan技术。除了使用商业检测的9份报告外，72TaqMan报告引用了以前发表的工作，给我们留下了27个TaqManqPCR设计。经过仔细检查，TaqMan设计在引物或探针序列中含有错配。此外,其他与环境微生物或非MAP分枝杆菌表现出高度相似性。我们评估了6种IS900qPCR设计的性能及其应用于临床或环境样品时的灵敏度，总体上从4到56倍不等。此外,我们为测试临床和环境样本提供建议，由于qPCR设计不佳，应避免以前使用的某些策略(例如，错配的存在)或缺乏特异性。
