Griffioen Karien, Cornelissen Jan, Heuvelink Annet, Adusei Daniela, Mevius Dik, Jan van der Wal Fimme
Faculty of Veterinary Medicine, Department of Farm Animal Health, Utrecht University, PO Box 80151, 3508 TD Utrecht, the Netherlands.
Wageningen Bioveterinary Research, Department of Infection Biology, Wageningen UR, PO Box 65, 8200 AB Lelystad, the Netherlands.
J Dairy Sci. 2020 Sep;103(9):8407-8420. doi: 10.3168/jds.2019-18035. Epub 2020 Jun 18.
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.
农民们在做出乳腺炎治疗决策时，更喜欢快速、灵敏且能现场检测的方法。由于现有诊断工具得出结果所需时间较长，这些工具很少用于此目的。无需培养步骤的基因检测可能适用于现场检测，例如环介导等温扩增技术（LAMP），该技术已被描述为一种可用于现场的灵敏检测方法。因此，本研究旨在开发并评估用于检测临床型乳腺炎奶牛乳汁中部分致乳腺炎病原体（大肠杆菌、肺炎克雷伯菌、金黄色葡萄球菌和链球菌属）的LAMP检测方法。此外，还评估了一种通用核酸侧向流动免疫分析（NALFIA）作为LAMP检测潜在的现场读数方法。对于LAMP和NALFIA的每种检测方法，使用分离株确定检测限和分析特异性，并使用已知病因的选定样本确定诊断特异性。此外，使用检测时病因不明的现场样本确定LAMP的诊断特异性。采用质谱鉴定的细菌培养作为参考方法。在检测选定样本时，这4种检测方法与参考方法的kappa值≥0.73，但在检测现场样本时≥0.47。校正患病率后，大肠杆菌、肺炎克雷伯菌和金黄色葡萄球菌检测方法的kappa值≥0.80。链球菌属检测方法与参考方法的kappa值为0.47（校正后为0.48），可能是因为该检测方法广泛针对一个属而非特定物种。发现对于大肠杆菌、金黄色葡萄球菌和链球菌属检测方法，NALFIA读数在通用运行时间下的kappa值≥0.81，但对于肺炎克雷伯菌检测方法，可以使用更短的运行时间。总之，如果将当前复杂的样本制备方法替换为简化方案，LAMP是一种用于快速现场检测致乳腺炎病原体的有前景的方法。NALFIA是一种易于使用且可靠的现场读数方法，但观察到对于当前的检测设计，对于所选病原体集尚未能实现通用运行时间。如果与简单快速的样本制备方案相结合，LAMP和NALFIA的组合有可能实现对临床型乳腺炎乳汁样本的快速可靠现场检测。
