Department of Veterinary Sciences and Public Health, University of Milan, Via Celoria 10, 20133 Milano, Italy.
J Dairy Res. 2013 May;80(2):223-6. doi: 10.1017/S0022029913000022. Epub 2013 Mar 12.
Staphylococcus aureus isolates from dairy cow mastitis are not always consistent with the characteristic morphology described, and molecular investigation is often needed. The aim of the study was to develop a duplex real-time PCR assay for rapid identification of Staph. aureus isolates, targeting both nuc and Sa442. Overall, 140 isolates collected from dairy cow mastitis in 90 different herds, were tested. All strains had been identified using morphological and biochemical characteristics. DNA from each strain was amplified in real-time PCR assay, to detect nuc or Sa442. Thereafter, a duplex real-time PCR assay was performed, and specificity of the amplified products was assessed by high resolution melting curve analysis. Out of 124 Staph. aureus isolates, 33 did not show the typical morphology or enzymic activity; in 118 strains, the two melt-curve peaks consistent with nuc and Sa442 were revealed, while 2 isolates showed only the peak consistent with Sa442. Four isolates bacteriologically identified as Staph. aureus, were PCR-negative and were further identified as Staph. pseudintermedius by sequencing. Staph. pseudintermedius and coagulase-negative staphylococci did not carry nuc or Sa442. The results showed the correct identification of all isolates, comprehending also coagulase-or nuc-negative Staph. aureus, while other coagulase-positive Staphylococci were correctly identified as non-Staph. aureus. Both sensitivity and specificity were 100%. High resolution melting analysis allowed easy detection of unspecific products. Finally, the duplex real-time PCR was applied directly to 40 milk samples, to detect infected mammary quarters. The assay confirmed the results of bacteriological analysis, on Staph. aureus-positive or-negative samples. Therefore, the proposed duplex real-time PCR could be used in laboratory routine as a cost-effective and powerful tool for high-throughput identification of atypical Staph. aureus isolates causing dairy cow mastitis. Also, it could be applied directly to milk samples, to detect Staph. aureus mammary infections avoiding bacteriological analysis.
从奶牛乳腺炎中分离出的金黄色葡萄球菌并不总是符合描述的特征形态，因此经常需要进行分子调查。本研究旨在开发一种用于快速鉴定金黄色葡萄球菌分离株的双重实时 PCR 检测方法，该方法针对 nuc 和 Sa442 进行检测。总共对 90 个不同牛群的乳腺炎奶牛中收集的 140 株分离株进行了测试。所有菌株均使用形态和生化特征进行鉴定。使用实时 PCR 检测方法从每种菌株中扩增 DNA，以检测 nuc 或 Sa442。然后，进行双重实时 PCR 检测，并通过高分辨率熔解曲线分析评估扩增产物的特异性。在 124 株金黄色葡萄球菌分离株中，有 33 株未显示典型形态或酶活性；在 118 株菌株中，揭示了与 nuc 和 Sa442 一致的两个熔解曲线峰，而 2 株仅显示与 Sa442 一致的峰。4 株经细菌学鉴定为金黄色葡萄球菌的分离株为 PCR 阴性，进一步通过测序鉴定为中间葡萄球菌。中间葡萄球菌和凝固酶阴性葡萄球菌不携带 nuc 或 Sa442。结果表明，该检测方法正确鉴定了所有分离株，包括凝固酶或 nuc 阴性的金黄色葡萄球菌，而其他凝固酶阳性葡萄球菌则正确鉴定为非金黄色葡萄球菌。该检测方法的灵敏度和特异性均为 100%。高分辨率熔解分析允许轻松检测非特异性产物。最后，将双重实时 PCR 直接应用于 40 个牛奶样本，以检测感染的乳腺象限。该检测方法证实了对金黄色葡萄球菌阳性或阴性样本的细菌分析结果。因此，该双重实时 PCR 可在实验室常规中用作经济高效且强大的工具，用于高通量鉴定引起奶牛乳腺炎的非典型金黄色葡萄球菌分离株。此外，它还可以直接应用于牛奶样本，以检测金黄色葡萄球菌的乳腺感染，避免细菌分析。
