Higuchi Hidetoshi, Iwano Hidetomo, Kawai Kazuhiro, Ohta Takehiro, Obayashi Tetsu, Hirose Kazuhiko, Ito Nobuhiko, Yokota Hiroshi, Tamura Yutaka, Nagahata Hajime
Animal Health Laboratory, Faculty of Veterinary Medicine, Rakuno Gakuen University, Hokkaido 069-8501, Japan.
J Vet Sci. 2011 Jun;12(2):191-3. doi: 10.4142/jvs.2011.12.2.191.
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 × 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.
我们开发了一种简化的聚合酶链反应（PCR）检测方法，用于快速、简便地筛查奶牛的支原体乳腺炎。通过这种基于简化PCR的方法以及标准PCR技术，对培养的牛奶样本中的主要支原体菌株[牛支原体（M. bovis）、精氨酸支原体（M. arginini）、牛生殖支原体（M. bovigenitalium）、加利福尼亚支原体（M. californicum）、牛鼻支原体（M. bovirhinis）、碱性支原体（M. alkalescens）和加拿大支原体（M. canadense）]进行了检测。简化PCR检测支原体的最低浓度限值估计约为2.5×10(3) cfu/mL，与标准PCR的限值相似。我们将简化PCR的特异性和灵敏度与培养法的进行了比较。在支原体肉汤中培养的1685份牛奶样本中，简化PCR检测出152份样本中的支原体DNA，这些样本根据培养检测也是阳性的。简化PCR检测这些培养物中支原体的灵敏度和特异性分别为98.7%和99.7%。简化PCR获得的结果与标准PCR的结果一致。这种新开发的简化PCR不需要DNA纯化，可在3小时内分析约300个培养样本。我们的研究结果表明，简化PCR可用于大型奶牛场的支原体乳腺炎筛查。
