College of Veterinary Medicine, China Agricultural University, Beijing, China.
Lett Appl Microbiol. 2011 Sep;53(3):278-82. doi: 10.1111/j.1472-765X.2011.03102.x. Epub 2011 Jul 4.
To develop a PCR-based assay to detect Prototheca zopfii (P. zopfii) and its mastitis-related subtype (genotype 2) directly from milk samples.
The DNA extraction method herein is based on the lysing properties of chemical agents, mechanical grinding and DNA-binding properties of silica particles; this method was developed to rapidly extract DNA directly from P. zopfii in bovine milk. Two pairs of primers specific for P. zopfii and genotype 2 were used in the duplex PCR, and a sensitivity test showed that the detection level was 5 × 10(2) colony-forming units (CFU) ml(-1) for P. zopfii and 5 × 10(3) CFU ml(-1) for genotype 2. Furthermore, a practical survey of 23 milk samples showed that the assay produced results that were in accordance with those obtained by the conventional microbiology method.
The DNA extraction method is effective in isolating sufficient quantities of DNA from P. zopfii in milk for PCR analysis. The PCR assay is economical, sensitive and more rapid than the conventional culture method.
The assay could be used as an alternative method for the rapid the detection of bovine mastitis resulting from P. zopfii genotype 2.
开发一种基于 PCR 的检测方法，直接从奶样中检测 Prototheca zopfii（P. zopfii）及其乳腺炎相关亚型（基因型 2）。
本文中的 DNA 提取方法基于化学试剂的裂解特性、机械研磨和硅颗粒的 DNA 结合特性；该方法旨在快速从牛乳中的 P. zopfii 中直接提取 DNA。使用针对 P. zopfii 和基因型 2 的两对引物进行双重 PCR，灵敏度测试表明 P. zopfii 的检测水平为 5×10(2)菌落形成单位 (CFU) ml(-1)，基因型 2 的检测水平为 5×10(3) CFU ml(-1)。此外，对 23 个奶样的实际调查表明，该检测方法的结果与传统微生物学方法的结果一致。
该 DNA 提取方法可有效分离奶中足够数量的 P. zopfii DNA 用于 PCR 分析。与传统培养方法相比，PCR 检测法更经济、更灵敏、更快速。
该检测方法可作为快速检测由 P. zopfii 基因型 2 引起的牛乳腺炎的替代方法。
