Steele N M, Williamson J H, Thresher R, Laven R A, Hillerton J E
DairyNZ, Private Bag 3221, Hamilton, 3240, New Zealand; Department of Dairy Sciences, Virginia Polytechnic Institute and State University, Blacksburg 24061.
DairyNZ, Private Bag 3221, Hamilton, 3240, New Zealand.
J Dairy Sci. 2017 May;100(5):3816-3824. doi: 10.3168/jds.2016-11752. Epub 2017 Feb 23.
The performance of a commercial, real-time PCR assay was compared with traditional bacterial culture for the identification of Streptococcus uberis and Staphylococcus aureus in bovine milk collected at different stages of lactation. Initial validation tests using fresh and frozen quarter milk samples identified factors that affected the success of the PCR. Therefore, the standard protocol was adjusted for samples collected at the first milking postpartum (colostrum) and from clinical mastitis cases. The adjustment involved PCR testing both undiluted and diluted (1 in 10 with sterile water) DNA extracts. The performance comparison between culture and the PCR assay used milk samples collected aseptically from individual quarters of mixed-age spring-calving dairy cows, during early, mid, and late lactation. Bacterial culture results were used to select a subset of samples for PCR testing (n = 315) that represented quarters with a current or prior Strep. uberis or Staph. aureus infection. Compared with culture, PCR had a sensitivity of 86.8% and specificity of 87.7% for detecting Strep. uberis (kappa = 0.74) and 96.4% and 99.7%, respectively, for detecting Staph. aureus (kappa = 0.96). The dilution of DNA extracts for colostrum and clinical samples increased the relative sensitivity from 79.2% to 86.8% for Strep. uberis detection and from 92.9% to 96.4% for Staph. aureus, presumably through diluting unidentified PCR inhibitors. The sensitivity for detecting Strep. uberis using PCR, relative to culture, was similar throughout lactation (85-89%), whereas relative specificity was lowest immediately postcalving (64%) but improved in mid and late lactation (98%). Specificity estimates for samples collected in early lactation can be optimized by reducing the cutoff cycle threshold (Ct) value from the recommended value of 37 to 34. Although using this value improved specificity (77%), it reduced test sensitivity (77%). The PCR assay lacked agreement with culture in early lactation, specifically for diagnosing Strep. uberis. Thus, PCR should not be used as the only tool for diagnosing mastitis in early lactation.
将一种商业实时聚合酶链反应（PCR）检测方法与传统细菌培养法进行比较，以鉴定在不同泌乳阶段采集的牛乳中的乳房链球菌和金黄色葡萄球菌。使用新鲜和冷冻的四分乳样品进行的初始验证试验确定了影响PCR成功的因素。因此，针对产后首次挤奶（初乳）时采集的样品以及临床乳腺炎病例的样品，对标准方案进行了调整。调整内容包括对未稀释和稀释（用无菌水按1:10稀释）的DNA提取物进行PCR检测。培养法与PCR检测方法的性能比较使用了从不同年龄的春季产犊奶牛个体乳腺中无菌采集的乳样，这些奶牛处于泌乳早期、中期和晚期。细菌培养结果用于选择一部分样品进行PCR检测（n = 315），这些样品代表当前或先前感染过乳房链球菌或金黄色葡萄球菌的乳腺。与培养法相比，PCR检测乳房链球菌的灵敏度为86.8%，特异性为87.7%（kappa = 0.74），检测金黄色葡萄球菌的灵敏度分别为96.4%和99.7%（kappa = 0.96）。初乳和临床样品DNA提取物的稀释将乳房链球菌检测的相对灵敏度从79.2%提高到86.8%，将金黄色葡萄球菌检测的相对灵敏度从92.9%提高到96.4%，这可能是通过稀释未鉴定的PCR抑制剂实现的。相对于培养法，在整个泌乳期使用PCR检测乳房链球菌的灵敏度相似（85 - 89%），而相对特异性在产后立即最低（64%），但在泌乳中期和晚期有所提高（98%）。通过将截止循环阈值（Ct）值从推荐的37降低到34，可以优化泌乳早期采集样品的特异性估计。尽管使用这个值提高了特异性（77%），但降低了检测灵敏度（77%）。PCR检测方法在泌乳早期与培养法不一致，特别是在诊断乳房链球菌方面。因此，PCR不应作为泌乳早期乳腺炎诊断的唯一工具。
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