蜡样芽孢杆菌ERIC-PCR分子分型方法的建立

Establishment of ERIC-PCR Molecular Typing Method for Bacillus cereus

摘要:

为建立简单快速的蜡状芽孢杆菌(Bacillus cereus)基因间重复共有序列PCR(ERIC-PCR)的分子分型方法，该研究以B. cereus CMCC 63303的基因组DNA为模板，并采用正交设计L16(45)对影响ERIC-PCR反应体系的五个因素（模板DNA、Mg2+、dNTP、Taq DNA聚合酶、引物）在四个浓度水平上进行优化，并通过单因素试验确定最佳退火温度，最后以优化后的反应体系对50株B. cereus 分离株进行ERIC-PCR分子分型和聚类分析验证其稳定性和分型效果。结果显示应用建立的ERIC-PCR反应体系对50株分离株扩增均得到了9~17条、大小在200~4000 bp之间的条带，并可将其分为47个型，且分辨力达到0.996，具有较高稳定性和分辨能力。表明ERIC-PCR技术对B. cereus分型，具有简便和分辨力高等优点，可用于食源性B. cereus分离株的多样性研究。

Abstract:

An efficient enterobacterial repetitive intergenic consensus sequence (ERIC-PCR) molecular typing method was established for Bacillus cereus. Based on the template of B. cereus CMCC 63303 genomic DNA, the ERIC-PCR amplification system in four levels of five factors (DNA template, Mg2+, dNTP, Taq DNA polymerase and primer) was optimized by orthogonal design L16(45) .Then the effects of annealing temperature on the amplification system was discussed through single factor experiment and the optimal reaction condition were determined. Fifty B. cereus strains were typed using the optimal reaction system and cluster analysis. The ERIC-PCR results exhibited better discriminative results in molecular typing with discrimination index of 0.996. B. cereus strains were grouped into 47 types. The ERIC-PCR system was an efficient method for typing and tracking analyses. It was suitable for genetic diversity analysis of foodborne B. cereus strains.