Kim C H, Khan M, Morin D E, Hurley W L, Tripathy D N, Kehrli M, Oluoch A O, Kakoma I
Department of Veterinary Pathobiology, University of Illinois, Urbana 61802, USA.
J Dairy Sci. 2001 Jan;84(1):74-83. doi: 10.3168/jds.S0022-0302(01)74454-2.
Staphylococcus aureus is an economically important and a major mastitis-causing pathogen that also poses food safety and antimicrobial resistance threats. Substances in mastitic milk inhibit the Taq DNA polymerase reaction (Taq PCR) making it of limited use for detecting S. aureus mastitis. In the study reported here, a set of oligonucleotide primers of 21 and 24 bases was used in Taq-PCR to amplify DNA from S. aureus (isolates from bovine mastitis). A specific amplicon of 270 bp was generated as predicted. Replacing Taq DNA polymerase with Thermus thermophilus (Tth) DNA polymerase alone (Tth-PCR) raised the sensitivity of S. aureus detection in milk from experimentally infected cows from 65 to 80%. Combining the use of Tth DNA polymerase and the purification of crude DNA extract using Chelex-100 before PCR raised the sensitivity to 100%. In a random survey involving 100 milk samples from cattle not infected with S. aureus, the test was 100% specific. With milk samples from clinical cases of bovine mastitis, 100% sensitivity and specificity were also observed. It is concluded that Tth-PCR on milk samples with the purification of crude DNA extracts using Chelex-100 is as sensitive as but faster than conventional milk bacteriological culture techniques and is highly specific. The modified PCR correlates with elevated somatic cell counts, detects evidence of chronic and resolving infection based on S. aureus-specific DNA and circumvents the endogenous inhibitory effects of milk.
金黄色葡萄球菌是一种具有经济重要性的主要致乳腺炎病原体，还构成食品安全和抗菌药物耐药性威胁。患乳腺炎奶牛的乳汁中的物质会抑制Taq DNA聚合酶反应（Taq PCR），使得其在检测金黄色葡萄球菌引起的乳腺炎方面用途有限。在本文报道的研究中，使用一组21个碱基和24个碱基的寡核苷酸引物进行Taq-PCR，以扩增来自金黄色葡萄球菌（从牛乳腺炎分离株）的DNA。如预期的那样，产生了一个270 bp的特异性扩增子。仅用嗜热栖热菌（Tth）DNA聚合酶替代Taq DNA聚合酶（Tth-PCR），将实验感染奶牛乳汁中金黄色葡萄球菌检测的灵敏度从65%提高到了80%。在PCR之前，将Tth DNA聚合酶的使用与使用Chelex-100纯化粗DNA提取物相结合，使灵敏度提高到了100%。在一项对100份未感染金黄色葡萄球菌的奶牛乳汁样本的随机调查中，该检测的特异性为100%。对于牛乳腺炎临床病例的乳汁样本，也观察到了100%的灵敏度和特异性。得出的结论是，对乳汁样本进行Tth-PCR并使用Chelex-100纯化粗DNA提取物，与传统的乳汁细菌学培养技术一样灵敏，但速度更快，且具有高度特异性。改良后的PCR与体细胞计数升高相关，基于金黄色葡萄球菌特异性DNA检测慢性和正在消退感染的证据，并规避了乳汁的内源性抑制作用。
