进口生牛皮中细菌的PCR-RFLP图谱建立

旨在建立存在于进口生牛皮中细菌的PCR-RFLP图谱。对从进口生牛皮中分离到的15种细菌,通过VITEK鉴定,分别进行基因组DNA提取,然后以所提取的DNA为模版,用16SrDNA的通用引物27f和1492r进行PCR扩增,扩增产物以17种内切酶,即AvaⅠ、BamHⅠ、BglⅡ、DraⅠ、EcoRⅠ、EcoRⅡ、HindⅢ、Hinf、HpaⅠ、PstⅠ、SmaⅠ、TaqⅠ、XbaⅠ、XmaⅠ、AluⅠ、XhoⅠ、PvuⅠ进行酶切,酶切产物经琼脂糖凝胶电泳分析,建立每种菌16SrDNA RFLP图谱。结果表明,进口生牛皮中分离到的15种细菌分别是侧芽短芽孢杆菌、产色葡萄球菌、黑色消化球菌、环状芽孢杆菌、解鸟氨酸拉乌尔菌、枯草芽孢杆菌、蜡样芽孢杆菌、耐热芽孢杆菌、桥石芽孢杆菌、球型芽孢杆菌、溶血不动杆菌、少动桥氨醇单孢菌、苏云金芽孢杆菌、中间葡萄球菌和分支杆菌,除中间葡萄球菌和产色葡萄球菌外,其他13种菌均扩增出目的条带。说明,PCR-RFLP方法可以快速、可靠地分析、鉴定进口皮毛中的细菌,在进出口动物皮毛中细菌的检测方面具有一定的应用潜能。

Development of PCR-RFLP Fingerprints of Bacteria Isolated from Imported Cattle Rowhide

To develop a PCR-RFLP fingerprint of bacteria isolated from imported cattle rowhide, VITEK was used to identify bacteria isolated from imported cattle rowhide and total genome DNA of each bacteria was extracted. PCR was carried out to amplify the 16S rDNA with universal Primers 27f and 1492r. The amplified products were digested by 17 endonucleases individually and products of digestion were analyzed by electrophoresis. The result showed that the bacteria isolated from cattle rowhide include:Brevibacillus laterosporus,Staphylococcus chromogenes,Peptococcus niger, Bacillus circulans, Raoultella ornithinolytica, Bacillus subtilis, Bacillus cereus, Bacillus thermobacillus, Bacillus choshinensis, Bacillus sphaericus, Acinetobacter haemolyticus, Sphingomonas paucimobilis, Bacillus thuringiensis, Staphylococcus intermedius, Mycobacteria.Targets bands were amplified successfully except Staphylococcus intermedius and Staphylococcus chromogenes. Therefore, the PCR-RFLP fingerprints based on bacterial 16S rDNA is a fast and reliable method to indentify the bacteria existed in imported cattle rowhide and has a potential application in inport and export quarantine.