变性梯度凝胶电泳技术在食源性沙门氏菌分子分型中的应用研究

Application of polymerase chain reaction-denaturing gradient gel electrophoresis technique in the molecular typing of foodborne Salmonella

目的建立基于变性梯度凝胶电泳(polymerase chain reaction-denaturing gradient gel electrophoresis,PCR-DGGE)技术的沙门氏菌分型方法。方法以5株代表性沙门氏菌为供试菌,用引物对GC-rpoB1/ropB3R扩增获得目的片段后,经变性凝胶电泳分离获得谱图,量化分析谱带多样性及均一性,依据菌株谱图量化相似性构建聚类分析图,分析该技术用于沙门氏菌分型的可行性。结果 5个供试菌的PCR-DGGE谱图差异大,其中以标准菌株肠炎沙门氏菌(ATCC9184)的多样性及丰富度最高,标准菌株鼠伤寒沙门氏菌的最低;就谱带相似性而言,分离自鸭腿样品的2株沙门氏菌的相似性最高,戴斯系数高达85.7%,而分属于不同血清型的两个标准菌株则相似度指数为0%;基于谱带相似性建立的聚类树可将供试菌株很好地分为不同簇。结论本研究所建立的基于PCR-DGGE技术的沙门氏菌分型方法准确度和分辨力均较理想,具有一定的理论及实际意义。

Objective To establish a typing method for Salmonella based on polymerase chain reaction-denaturing gradient gel electrophoresis（PCR-DGGE）. Methods With 5 representative Salmonella isolates as sample, the target fragment was amplified by using primer pair GC-ropB1/ropB3 R. The spectrum was obtained by denatured gel electrophoresis, and the diversity and evenness of the spectrum were quantitatively analyzed. The cluster analysis chart was constructed according to the similarity of the bacterial strain, and the feasibility of using this technology for the classification of Salmonella was analyzed. Results The PCR-DGGE spectra of the 5 tested strains were significantly different, with the highest diversity and richness of the standard strain Salmonella Enteritidis（ATCC 9184） and the lowest of the standard strain Salmonella Typhimurium. In terms of spectral band similarity, the 2 Salmonella strains isolated from duck leg samples had the highest similarity, with the Deiss coefficient as high as 85.7%. The similarity index of 2 standard strains belonging to different serotypes was 0%. The cluster tree based on spectral band similarity could divide the tested strains well into different clusters. Conclusion The PCR-DGGE based Salmonella typing method established in this study is ideal in accuracy and resolving power, which is of certain theoretical and practical significance.