Optimization of the PCR for detection of Staphylococcus aureus nuc gene in bovine milk
Optimization of the PCR for detection of Staphylococcus aureus nuc gene in bovine milk
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DOI：
10.3168/jds.s0022-0302(01)74454-2
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发表时间：
2001-01-01
影响因子：
3.5
通讯作者：
Kakoma, I
中科院分区：
文献类型：
--
作者：
Kim, CH;Khan, M;Kakoma, I
金黄色葡萄球菌（Staphylococcus aureus）是一种经济上重要的乳腺炎致病菌，也对食品安全和抗菌素耐药性构成威胁。乳汁中的物质抑制Tag DNA聚合酶反应（Taq PCR），使其在检测金黄色葡萄球菌乳腺炎中的应用受到限制。在本文报道的研究中，一组21和24个碱基的寡核苷酸引物用于Taq-PCR扩增金黄色葡萄球菌（牛乳腺炎分离株）的DNA。结果与预测一致，产生了一个270 bp的特异性扩增子。用单纯嗜热热菌（th） DNA聚合酶（th- pcr）替代Tag DNA聚合酶，使金黄色葡萄球菌检测的敏感性从65%提高到80%。结合Tth DNA聚合酶的使用和PCR前Chelex-100粗DNA提取物的纯化，将灵敏度提高到100%。在对未感染金黄色葡萄球菌的牛的100份牛奶样本进行的随机调查中，该测试具有100%的特异性。对临床牛乳腺炎病例的乳样，也观察到100%的敏感性和特异性。综上所述，Chelex-100对牛奶样品粗DNA萃取物进行纯化后的Tth-PCR与传统的牛奶细菌培养技术一样灵敏，但速度更快，特异性强。改进的PCR与体细胞计数升高相关，基于金黄色葡萄球菌特异性DNA检测慢性和解决感染的证据，并绕过牛奶的内源性抑制作用。
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 Tag 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 Tag 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.