多重PCR检测奶牛乳腺炎金黄色葡萄球菌、无乳链球菌、停乳链球菌和酵母菌方法的建立与应用  

Development of Mutiplex PCR for Simultaneous Detection of Staphycococcus aureus,Streptococcus agalactiae,Streptococcus dysgalactiae and Yeasts in Bovine Mastitis Milk Samples

作　　者：马保臣[1] 秦卓明[2] 蔡玉梅[1] 董玉兰[3] 柴同杰[1] 

机构地区：[1]山东农业大学动物科技学院,泰安271018 [2]山东省农业科学院,济南250000 [3]中国农业大学动物医学院,北京100094

出　　处：《畜牧兽医学报》2006年第11期1202-1208,共7页ACTA VETERINARIA ET ZOOTECHNICA SINICA

基　　金：德国政府基金会(DAAD)国际优秀学者资助项目[423-china;Mongolei](2004.06-2006.05)

摘　　要：参照GenBank发表的序列,在金黄色葡萄球菌、无乳链球菌和停乳链球菌16S rRNA与23S rRNA之间的区域设计了3对引物,参照念珠菌和隐球菌的18S rRNA的序列设计1对引物,建立了检测金黄色葡萄球菌、无乳链球菌、停乳链球菌和酵母真菌4种乳腺炎主要致病菌的多重PCR方法。参照Skladny的方法制备模拟了细菌感染临床标本。结果表明:本试验建立的多重PCR方法具有较好的特异性,多重PCR方法检测乳样中的金黄色葡萄球菌的细菌最小浓度为104CFU/mL,检测无乳链球菌、停乳链球菌和酵母真菌的细菌最小浓度分别为102CFU/mL、103CFU/mL和103CFU/mL。通过对采自临床型乳腺炎(46个)和隐性乳腺炎(167个)动物共计213个乳样分别用传统细菌学培养法和多重PCR方法进行检测,多重PCR对金黄色葡萄球菌和酵母真菌的检测具有更高的检出率(P<0.01),但该方法对无乳链球菌和停乳链球菌的检出率与培养法差异不显著(P>0.05)。A mutiplex polymerase chain reaction（PCR） assay was developed for the simultaneous detection of the four major agents of bovine mastitis, Staphycococcus aureus, Streptococcus agalactiae, Streptococcus dysgalactiae and yeast. The target sequences of Staphycococcus aureus, Streptococcus agalactiae,and Streptococcus dysgalactiae were the 16S to 23S rRNA spacer regions. The target sequence of yeast was 18S rRNA region. The results showed that the assay was specific. Simulation bacteria infection samples were made according to Skladny＇s method. The sen- sitivity of the mutiplex PCR was lower in detecting Staphycococcus aureus, Streptococcus dysgalactiae and yeast of the milk. The sensitivity of mutiplex PCR in detecting Staphycococcus aureus, Streptococcus agalactiae, Streptococcus dysgalactiae and yeasts was 10^4CFU/mL, 10^2CFU/mL, 10^3 CFU/mL and 10^3CFU/mL respectively. The performance of the mutiplex PCR and traditional culture method were evaluated by examining 46 clinical mastitis milk samples and 167 subclinical mastitis milk samples. The results showed that mutiplex PCR was more sensitive than culture in detecting Staphycococcus aureus and yeasts（P〈0. 01）, but was not different from the culture method in detecting Streptococcus agalactiae and Streptococcus d ysgalactiae （ P〉0. 05）.

关 键 词：多重PCR 乳腺炎 金黄色葡萄球菌 无乳链球菌 停乳链球菌 酵母真菌 

分 类 号：S855[农业科学—临床兽医学] S854.43[农业科学—兽医学]