摘要
      快速准确地检测农牧业中的抗生素耐药性病原体很重要，因为这些病原体对公众健康构成了重大威胁。虽然人们一直致力于检测现在常见的耐药细菌，如耐甲氧西林金黄色葡萄球菌，但很少有方法评估金黄色葡萄菌（SA）对大环内酯类药物的耐药性。在此，我们报道了乳制品中大环内酯类耐药SA的可视化现场检测系统。首先，对天津周边奶牛场采集的生乳、牛粪、水和气溶胶沉积物进行宏基因组测序，以确定最丰富的大环内酯类抗性基因，该基因被发现是macB基因。同时，筛选SA持家基因以进行SA的选择性鉴定，从而选择SAOUHSC_01275基因。接下来，开发了靶向上述基因的LAMP测定法，并通过琼脂糖凝胶电泳进行解释。对于现场应用，比较了不同的pH敏感比色LAMP指示剂，从而选择聚二乙炔（PDA）作为最敏感的候选者。此外，通过智能手机分析RGB信息，可以实现半定量检测，牛奶样本的基因组DNA检测限为1.344×10−7 ng/μL。最后，通过使用冷冻干燥试剂和便携式设备，在从样品到结果的1小时内，在真实的农场成功地实施了所提出的方法。这是PDA首次用于检测LAMP产品，这种通用的读出系统可以扩展到其他抗生素抗性基因和细菌。
Abstract
Rapidly and accurately detecting antibiotic-resistant pathogens in agriculture and husbandry is important since these represent a major threat to public health. While much attention has been dedicated to detecting now-common resistant bacteria, such as methicillin-resistant Staphylococcus aureus, fewer methods have been developed to assess resistance against macrolides in Staphylococcus aureus (SA). Here, we report a visual on-site detection system for macrolide resistant SA in dairy products. First, metagenomic sequencing in raw milk, cow manure, water and aerosol deposit collected from dairy farms around Tianjin was used to identify the most abundant macrolide resistance gene, which was found to be the macB gene. In parallel, SA housekeeping genes were screened to allow selective identification of SA, which resulted in the selection of the SAOUHSC_01275 gene. Next, LAMP assays targeting the above-mentioned genes were developed and interpreted by agarose gel electrophoresis. For on-site application, different pH-sensitive colorimetric LAMP indicators were compared, which resulted in selection of polydiacetylene (PDA) as the most sensitive candidate. Additionally, a semi-quantitative detection could be realized by analyzing the RGB information via smartphone with a LOD of 1.344 × 10−7 ng/μL of genomic DNA from a milk sample. Finally, the proposed method was successfully carried out at a real farm within 1 h from sample to result by using freeze-dried reagents and portable devices. This is the first instance in which PDA is used to detect LAMP products, and this generic read-out system can be expanded to other antibiotic resistant genes and bacteria.

https://www.sciencedirect.com/science/article/pii/S0956566322008648