强毒小肠结肠耶氏菌生物素标记基因探针的研究

在构建的小肠结肠耶氏菌毒性质粒DNA基因文库pYB1～8与pYP1～6的基础上,筛选出了pYB7和pYP6克隆株.用限制性内切酶Bam HI消化pYB7,Pst消化pYP6,可分离出3.8kb和6.4kb的插入性DNA片段.以这两个基因片段为目的基因,用生物素化dUTP和光敏生物素标记,获得了生物素标记的基因探针.该探针能检出10pg以上的强毒小肠结肠耶氏菌DNA,不与无毒小肠结肠耶氏菌及大肠杆菌、鼠伤寒沙门氏菌、金黄色葡萄球菌等18种对照菌反应,具有高度的特异性和敏感性.pYB7与pYP6探针对不同血清型及来源的小肠结肠耶氏菌检测,其结果与自凝性试验、依钙试验等结果相符;对小肠结肠耶氏菌强毒株与无毒株检定的准确率为100%.

Study on Biotin - labelled Gene Probes of Virulent Strains of Yersinia enterocolitica

On the basis of pYB1-8 and pYP1-6 gene libraries of virulence plasmids of Yersinia enterocolitica constructed previously, pYB7 and pYP6 recombinant clones were screened and insertion DNA fragments of 3. 8 kb and 6. 4 kb were cut from those recombinant clones with Bam HI and Pst I respectively. The recovered DNA fragments were labelled with biotin-11-dUTP or photobiotin to prepare biotinylated gene probes. These two prepared probes have high sensitivity and specificity, they were able to detect 10pg or more DNAs of virulent strains of Y. enterocal-itica but did not react with DNAs of avirulent strains of Y. enterocolitica and the other 18 species of bacteria, such as E. coli, Salmonella typhimurium, Staphylococcus aureus, etc, used as control. Using the probes to detect and identify the different serotypes and different isolates from various sources, the accuracy rate for differentiating the virulent strains from avirulent strains was 100%. There was complete agreement between the result of probe assay and those of Ca2 dependency test and autoagglutination.