快速鉴别奶牛乳腺炎细菌或真菌的双重PCR方法  

Development and Application of Double-PCR Method for Quick Detection of Bacteria and Yeast from Bovine Mastitis

作　　者：戈胜强[1] 柴同杰[1] 马保臣[1] 李晓霞[1] 蔡玉梅[1] 吕静[1] 秦梅[1] 王炳晓[1] 

机构地区：[1]山东农业大学动物医学院,泰安271018

出　　处：《畜牧兽医学报》2008年第11期1554-1561,共8页ACTA VETERINARIA ET ZOOTECHNICA SINICA

摘　　要：综合多种细菌、真菌DNA模板的提取方法,采用β-巯基乙醇裂解细胞壁,利用蜗牛酶和溶菌酶消化细胞壁,再利用石英砂机械破壁,能彻底破坏细菌、真菌细胞壁,提取高质量的DNA,摸索出一套从奶样中同时提取细菌、真菌核酸的新方法。建立双重PCR方法,同时完成对细菌16S rRNA保守区特异性基因片段和真菌18S rRNA保守区特异性基因片段的扩增,快速诊断奶牛乳房炎是细菌感染还是真菌感染或是混合感染。对采自临床型乳腺炎和隐性乳腺炎病例的共计84个乳样分别用传统细菌学培养法和双重PCR方法对比鉴定。结果表明,双重PCR检测乳样细菌的最小浓度为102CFU/mL,检测乳样真菌的最小浓度为103CFU/mL。双重PCR方法对细菌的检测与平板培养检测方法比较差异不显著(P>0.05),对真菌的检测与平板培养检测方法比较具有更高的检出率(P<0.01)。A double polymerase chain reaction （PCR） was developed for simultaneous detection and germ group classification of bacteria and yeasts of bovine mastitis. We developed a new method that extracts both bacteria and yeast DNA from milk using β-mercaptoethanol, which can decompose cell walls. At the same process we used lysozyme and snailase, which can assimilate cell walls, and quartz sand, which can further abrade the outside of cell. The conserved regions from 16S rRNA and 18S rRNA were selected as target sequences of bacteria and yeast respectively. We evaluated the technique for the detection and identification of which species it was in artificially infected milk and milk from cows with moderate or severe clinical mastitis. The performances of the double-PCR and traditional culture method were evaluated on 84 mastitis milk samples. The results indicated that the sensitivity of double-PCR in detecting bacteria and yeast was 102 CFU/mL and 103 CFU/mL, respectively. There was no significant difference as compared to the culture method in detecting bacteria （P〉0.05）, but double-PCR was more sensitive than the culture method in detecting yeasts （P〈0.01）.

关 键 词：奶牛乳腺炎 细菌 真菌 双重PCR 

分 类 号：S858.23[农业科学—临床兽医学] S857.26[农业科学—兽医学]