应用多重PCR鉴定微生物肥料常用芽孢杆菌  

Multiplex-PCR approach to identify Bacillus species applied in microbial fertilizers

作　　者：曹凤明 中国农业科学院农业资源与农业区划研究所 沈德龙[1] 李俊[2] 关大伟[2] 姜昕[2] 李力[1] 冯瑞华[2] 杨小红[2] 陈慧君[2] 葛一凡[2] 

机构地区：[1]中国农业科学院农业资源与农业区划研究所,北京100081 [2]农业部微生物肥料和食用菌菌种质量监督检验测试中心,北京100081

出　　处：《微生物学报》2008年第5期651-656,共6页Acta Microbiologica Sinica

基　　金：国家科技支撑项目(2006BAD25B04)~~

摘　　要：【目的】枯草群芽孢杆菌中枯草芽孢杆菌(Bacillus subtilis)、解淀粉芽孢杆菌(B.amyloliquefaciens)、地衣芽孢杆菌(B.licheniformis)和短小芽孢杆菌(B.pumilus)是微生物肥料中常用菌种,用传统方法鉴定费时费力,有必要建立检测和鉴定这些芽孢杆菌的种特异性PCR方法。【方法】利用已登录的gyrA、rpoA和16SrRNA基因序列分别设计和筛选上述菌种的特异引物并建立多重PCR反应体系。【结果】以基因组DNA为模板,扩增芽孢杆菌、类芽孢杆菌和短芽孢杆菌3属15种的标准菌株(共33株),4个目标种分别产生了大小不同的唯一的产物,除个别种与短小芽孢杆菌引物有交叉反应外,其余参考菌株均为阴性。从23株枯草群菌株的基因组DNA扩增发现,PCR鉴定与常规鉴定结果一致。【结论】本文建立的多重PCR方法具有较好的特异性,可快速准确鉴定枯草群的4个种,在微生物肥料检测方面有良好的实用前景。[Objective] To discriminate the strains of Bacillus subtilis group including B. subtilis, B. amyloliquefaciens, B. licheniformis, and B. pumilus, a rapid and accurate distinguishing method is essential for the identification of the target strains to ensure the quality and safety of microbial fertilizers. [Methods] By analyzing unique nucleotide sequences of the rpoA, gyrA and 16S rDNA genes, 4 pairs of species-specific primers were optimized and the multiplex PCR was developed to discriminate and identify B. subtilis, B. amyloliquefaciens, B. licheniformis and B. pumilus. [Results] Thirty-three reference strains belonging to three genera of Bacillus, Paenibacillus and Brevibacillus were tested and the anticipated results appeared except for four species with cross amplification results with the primers of B. pumilus. However, the four species can be easily discriminated by morphology characters. In addition, the multiplex-PCR results of 23 strains of B. subtilis group isolated from MF products were identical with the biochemical assay. [Conclusion] The newly constructed multiplex-PCR assay is species-specific and effective. This method can be used to detect and identify the strains of B.subtilis group from microbial fertilizers products.

关 键 词：多重PCR 种特异引物 鉴定 枯草群芽孢杆菌 微生物肥料 

分 类 号：S144[农业科学—肥料学]