A simplified PCR assay for fast and easy mycoplasma mastitis screening in dairy cattle
A simplified PCR assay for fast and easy mycoplasma mastitis screening in dairy cattle
复制标题
DOI：
10.4142/jvs.2011.12.2.191
复制
发表时间：
2011-06-01
影响因子：
1.8
通讯作者：
Nagahata, Hajime
中科院分区：
文献类型：
--
作者：
Higuchi, Hidetoshi;Iwano, Hidetomo;Nagahata, Hajime
建立了一种简便快速筛查奶牛支原体乳房炎的聚合酶链式反应(PCR)方法。主要支原体的种类[支原体(M.)牛、精氨酸分枝杆菌、牛黄分枝杆菌、卡氏分枝杆菌、牛黄分枝杆菌、碱性分枝杆菌和加拿大分枝杆菌]培养的牛乳样品，采用简化的聚合酶链式反应和标准的聚合酶链式反应技术进行检测。简化聚合酶链式反应检测支原体的最低检测浓度约为2.5×10(3)cfu/m L，与标准聚合酶链式反应的检测结果相近。我们比较了简化的聚合酶链式反应和培养方法的特异性和敏感性。在用支原体肉汤培养的1,685个牛奶样本中，简化的PCR检测到152个支原体DNA，根据培养试验也是阳性的。简化的聚合酶链式反应检测支原体的灵敏度为98.7%，特异度为99.7%。简化后的聚合酶链式反应结果与标准聚合酶链式反应的结果一致。该方法不需要DNA纯化，可在3h内对约300个培养样本进行分析，可用于规模化奶牛场支原体乳房炎的筛查。
A simplified polymerase chain reaction (PCR) assay was developed for fast and easy screening of mycoplasma mastitis in dairy cattle. Species of major mycoplasma strains [Mycoplasma (M.) bovis, M. arginini, M. bovigenitalium, M. californicum, M. bovirhinis, M. alkalescens and M. canadense] in cultured milk samples were detected by this simplified PCR-based method as well as a standard PCR technique. The minimum concentration limit for detecting mycoplasma by the simplified PCR was estimated to be about 2.5 x 10(3) cfu/mL and was similar to that of the standard PCR. We compared the specificity and sensitivity of the simplified PCR to those of a culture method. Out of 1,685 milk samples cultured in mycoplasma broth, the simplified PCR detected Mycoplasma DNA in 152 that were also positive according to the culture assay. The sensitivity and specificity of the simplified PCR were 98.7% and 99.7%, respectively, for detecting mycoplasma in those cultures. The results obtained by the simplified PCR were consistent with ones from standard PCR. This newly developed simplified PCR, which does not require DNA purification, can analyze about 300 cultured samples within 3 h. The results from our study suggest that the simplified PCR can be used for mycoplasma mastitis screening in large-scale dairy farms.