六种常见细菌16S rDNA基因的PCR-RFLP指纹图谱检测方法的建立

Establishment of 16S rDNA-based PCR-RFLP fingerprint for detection of six species of bacteria

为建立针对肺炎克雷伯氏菌、产色葡萄球菌、莫拉菌、睾丸酮丛毛单胞菌、炭疽芽胞杆菌和布氏杆菌的快速检测鉴定方法,提取上述6种菌的基因组DNA,然后以提取的DNA为模板,用16SrDNA的通用引物27f和1492r进行PCR扩增。用17种内切酶,即AvaⅠ、BgⅡ、BamHⅠ、DraⅠ、EcoRⅠ、EcoRⅡ、HindⅢ、Hinf、HpaⅠ、PstⅠ、SmaⅠ、TaqⅠ、XbaⅠ、XmaⅠ、AluⅠ、XhoⅠ和PvuⅠ,分别对6种菌的16S rDNA基因的扩增产物进行酶切。酶切产物经琼脂糖凝胶电泳分析。结果显示,每种菌形成了特异的16S rDNA RFLP指纹图谱。结果表明,建立的方法能够对6种细菌进行准确的鉴定,适用于口岸动物皮毛中6种细菌的快速鉴定。

The study aimed to develop an identification method for rapid detection of Klebsiella pneumoniae,Staphylococcus chromogenes,Moraxella,Comamonas testosteroni,Bacillus anthracis and Brucella.Genomic DNAs of these bacteria extracted by Bacterial Genome DNA Extraction Kit were used as templates for amplification of their 16SrDNA.In these assay,the primers were 27fand 1492r.Thereafter,the PCR products were digested with 17endonucleases,including AvaⅠ,BgⅡ,BamHⅠ,DraⅠ,EcoRⅠ, EcoRⅡ,HindⅢ,Hinf,HpaⅠ,PstⅠ,SmaⅠ,TaqⅠ,XbaⅠ,XmaⅠ,AluⅠ,XhoⅠ and PvuⅠ.Electrophoresis results showed that specific fingerprints of restriction fragment length polymorphism(RFLP) were formed for each bacteria.The results indicated that the developed method could differentiate 6bacteria from each other and could be applied to bacteria detection for imported animal hair and wools.