Farmers prefer fast, sensitive, and on-site tests for treatment decisions on mastitis. Due to the time to results of the currently available diagnostic tools, these are rarely used for that purpose. Genotypic tests that do not require a growth step may be suitable for on-site testing, for example loop-mediated isothermal amplification (LAMP), which has been described as a sensitive test that can be used on-site. Therefore, this study aimed to develop and evaluate LAMP assays for the detection of a subset of mastitis-causing pathogens, Escherichia coli, Klebsiella pneumoniae, Staphylococcus aureus, and Streptococcus spp., in milk from cows with clinical mastitis. Furthermore, a generic nucleic acid lateral flow immunoassay (NALFIA) was evaluated as a potential on-site readout of the LAMP assays. For each assay of LAMP and NALFIA, the limit of detection and analytical specificity were determined using isolates, and the diagnostic specificity was determined using selected samples with known etiology. In addition, the diagnostic specificity of LAMP was determined using field samples with unknown etiology at testing. Bacteriological culture with identification by mass spectrometry was used as a reference method. The 4 assays had a kappa ≥0.73 with the reference method when testing the selected samples, but ≥0.47 when testing field samples. After correcting for prevalence, kappa was ≥0.80 for the E. coli, K. pneumoniae, and Staph. aureus assays. The Streptococcus spp. assay had a kappa of 0.47 (0.48 after correction) with the reference method, probably caused by the assay broadly targeting a genus instead of a particular species. The NALFIA readout was found to have kappa ≥0.81 for the E. coli, Staph. aureus, and Streptococcus spp. assays at a generic runtime, but for the K. pneumoniae assay a shorter runtime could be used. In conclusion, LAMP is a promising method for fast on-site tests for mastitis-causing pathogens if the current elaborate method for sample preparation is replaced by a simplified protocol. The NALFIA is an easy and reliable readout for on-site use, with the observation that for the current assay designs a generic runtime is not yet possible for the chosen set of pathogens. If associated with a simple and fast sample preparation protocol, the combination of LAMP and NALFIA has the potential to enable fast and reliable on-site testing of clinical mastitis milk samples.

农民更喜欢快速，敏感和现场测试来确定乳腺炎的治疗方案。由于无法获得当前可用的诊断工具的结果，因此很少将其用于此目的。不需要生长步骤的基因型测试可能适用于现场测试，例如环介导的等温扩增（LAMP），已被描述为可以现场使用的灵敏测试。因此，本研究旨在开发和评估LAMP检测方法，以检测引起乳腺炎的病原体，大肠杆菌，肺炎克雷伯菌，金黄色葡萄球菌和链球菌spp。，来自患有临床乳腺炎的奶牛。此外，评估了通用核酸侧向免疫测定（NALFIA）作为LAMP测定的潜在现场读数。对于LAMP和NALFIA的每种测定，使用分离株确定检测限和分析特异性，并使用病因已知的选定样品确定诊断特异性。此外，使用病原学未知的田间样品在测试中确定了LAMP的诊断特异性。通过质谱鉴定的细菌培养用作参考方法。在测试所选样本时，这四种测定的kappa值≥0.73（参考方法），而在测试现场样本时的kappa值≥0.47。校正患病率后，大肠杆菌的kappa≥0.80 ，肺炎克雷伯菌和葡萄球菌。金黄色葡萄球菌测定。该链球菌属。该测定法的kappa值为0.47（校正后为0.48），可能是由于该测定法广泛针对某个属而不是特定物种。该NALFIA读数被发现有卡帕≥0.81对大肠杆菌，金黄色葡萄球菌。金黄色葡萄球菌和链球菌属。常规运行时进行测定，但用于肺炎克雷伯菌可以使用更短的运行时间。总而言之，如果将当前详细的样品制备方法替换为简化的方案，则LAMP是一种快速现场检测引起乳腺炎的病原体的有前途的方法。NALFIA是一种简便可靠的读数，可用于现场使用，因为对于当前的分析设计而言，对于所选病原体尚无法实现通用运行时间。如果与简单，快速的样品制备方案相关联，则LAMP和NALFIA的组合具有实现对临床乳腺炎牛奶样品进行快速可靠的现场测试的潜力。