布鲁氏菌种及种内部分生物型快速PCR鉴定方法的建立及应用研究

Development and application of one-step polymerase chain reaction（PCR） for rapid identification of Brucella species and some biovars

目的建立一步法PCR快速鉴定布鲁氏菌种及种内部分生物型。方法设计6对引物，建立一步法PCR反应，将布鲁氏菌6个种和种内某些生物型进行鉴定，应用该方法检测了27株布鲁氏菌参考菌株和239株临床分离的布鲁氏菌，并与生物学分型方法进行比较。结果通过一步法PCR能够将待检菌株准确的鉴定到布鲁氏菌种，对于猪种可以定位到生物型和疫苗株；对于牛种可以鉴定到牛种生物型1、3、4与2、5、6、7、9两组生物型；对于羊种可以鉴定到1与2、3两组生物型和羊种疫苗株。生物学分型方法和PCR方法准确鉴定到种水平的符合率分别为98．33％和100％。结论一步法PCR是一种快速、特异、简便而低费用的鉴定布鲁氏菌种的分子分型方法。

Objective To develop a one-step PCR assay for rapid discrimination of six BruceUa species and some intraspecific biovars. Methods Using 6 pairs of primers in one-step PCR to differentiate six classical Brucella species and some biovar in ordinary PCR instrument. The tested strains including 27 reference strains of six Brucella species and 239 Brucella strains were estimated by the PCR assay and biolog- ical identification methods. Results The six Brucella species could be precisely differentiated by the one- step PCR assay from the tested strains. Five biovars and vaccine strain of B. su/s species could be deter- mined, and biovars 1, 3, 4 and biovars 2, 5, 6, 7, 9 of B. abortus species could be identified at the level of their biovar, moreover, biovars 1, 2 and 3, and vaccine strain Rev 1 of B. melitensis species were also discriminated at the biovar and strain level. The accurate rates of the biological identification method and the PCR assay were 98.33% and 100% respectively. Conclusion One-step PCR assay was a rapid, specific, and low cost method for identification of Brucella species and discriminating hiovars in ordinary PCR instru- ment.