空肠弯曲菌分离株ERIC-PCR分型和生化分型的比较研究

建立空肠弯曲菌(Campylobacter jejuni)的ERIC-PCR分子生物学分型技术,比较ERIC-PCR分型和生化分型的分型效果。从菌株TY1273出发,运用L16(54)正交试验,对Mg2+、dNTPs、引物和TaqDNA聚合酶浓度等因素在较大范围水平内进行反应体系条件摸索,得到一个初步的优化体系;在此基础之上进行单因素的进一步小范围水平内的微调优化,得到最终的优化体系;最后以优化的ERIC-PCR方法对24株C.jejuni分离株分型;同时根据API Campy生化反应结果进行生化分型,比较ERIC-PCR分子分型方法和生化分型方法。结果显示ERIC-PCR方法将24株菌扩增均得到大小在100 bp-3000 bp之间的条带,并可将其分为22个基因型,分辨系数为0.92,具有较高的分辨力。生化分型将24株菌分为19个生化型,显示了菌株基因的多样性。表明ERIC-PCR技术比生化分型能更好的体现菌株的遗传多样性,且具有简便和分辨力搞等优点,可用于C.jejuni的多样性研究。

Comparison of the Typing Methods of ERIC?PCR and Biochemical for Campylobacter jejuni Isolates

To establish an ERIC?PCR molecular typing method for Campylobacter jejuni genotyping, the subtyping effect of ERIC-PCR and biochemical typing methods were compared. L16 (54) orthogonal text was designed with four factors of Mg2+, dNTPs, primers, TaqDNA polymerase concentration to explore the suitable reaction conditions in a wide range. Then three factors (except the primers), template concentration and annealing temperature were further optimized in a small range for final optimization. The optimized ERIC?PCR method was used to type the 24 C. jejuni isolates. According to the results of biochemical reactions to biochemical type the strains, the ERIC-PCR molecular typing method was compared with biochemical typing method. C. jejuni strains were grouped into 22 types. The ERIC?PCR method exhibited better disciminative results in molecular typing with discrimination index of 0.92. The C. jejuni isolates were classified to 19 biochemical types by biochemical typing method, which showed the diversity of genes in the strains. The ERIC?PCR subtyping method showed the genetic diversity of the strains better than biochemical typing method, which was an efficient method for typing and tracking analyses. It was suitable for genetic diversity analysis of of C. jejuni strains.