实时定量PCR检测大肠埃希菌在LB培养基和血中对抗生素的药物敏感性

Antibiotic Susceptibility of Escherichia Coli in LB Broth and Blood Determined by Real-time Quantitative PCR~ △

目的 建立以实时定量PCR快速检测大肠埃希菌在LB液体培养基和健康人全血中对抗生素药物敏感性的方法。方法 临床血培养获得大肠埃希菌株，以常规纸片法检测其对抗生素的药物敏感性。分别在LB液体培养基和健康人新鲜全血中加入该大肠埃希菌至终浓度为0．5麦氏单位，采用不加抗生素的标本作为生长对照组，加入耐受或敏感的抗生素的标本作为耐受药物组或敏感药物组，37℃震荡培养，分别提取0、1、2、3、4小时的基因组DNA，以大肠埃希菌特异β-右旋半乳糖苷酶基因的引物和TaqMan探针，对标本进行实时定量PCR检测。结果 实时定量PCR方法有较好的重复性（CT值变异系数0．26％～1．56％）和精确度（大肠埃希菌DNA提取效率在LB培养基中为43．6％，在血中为26．7％），标准曲线线性关系好（R^2≥0．998）。培养1～3小时后，耐受药物组与生长对照组的大肠埃希菌DNA拷贝数均呈对数增长，在LB液体培养基中增至约19～120倍，在新鲜全血中增至约6～11倍；敏感药物组大肠埃希菌DNA拷贝数呈减少趋势，在LB液体培养基中减至0．22～0．12倍，在新鲜全血中为1．29～0．14倍。上述药敏结果与常规纸片法一致。结论 采用实时定量PCR检测大肠埃希菌在LB液体培养基和健康人全血中对抗生素的药物敏感性，结果与常规纸片法一致，但更快速。

Objective To establish the real-time quantitative PCR （RQ-PCR） assay in fast determining the antibiotics susceptibility of Escherichia coli （ E. coli） in LB broth and whole blood of healthy volunteer. Methods The strain of E. coli was obtained by clinical blood bacterial culture, and antibiotics susceptibility was determined by conventional disk diffusion test. E. coli was spiked into LB broth and whole blood of healthy volunteer respectively to get a final concentration of 0.5 McFarland standard. The samples without antibiotics served as growth control, other samples were treated with resistant or sensitive antibiotics. All the samples were incubated at 37℃ by the shaking method. The genomic DNA were extracted at time points of 0, 1, 2, 3, and 4 hours respectively, and the RQ-PCR assays were performed using primers and TaqMan probe which specifically targets the β-D-galactosidase gene of E. coli. Results The reproducibility （CVs of CTvalues were 0. 26% -1. 56% ） and precision （the recover rates of E. coli DNA were 43.6% in LB broth and 26.7% in blood, respectively） of the RQ-PCR assays were excellent, and the correlation coefficient of the standard curve was 〉10. 998. In the growth control and resistant group, the quantity of E. coli DNA increased by 19-120 times in LB broth and 6-11 times in whole blood after 1-3 hours, and both of them performed as exponential trend. While in the susceptible group, the quantity of E. coli DNA performed as decreased trend, and it decreased by 0.22-0. 12 times in LB broth, and 1.29-0. 14 times in whole blood. The results were in accordance with the conventional disk diffusion test. Conclusion The antibiotic susceptibility of E. coli in LB broth and whole blood of healthy volunteer using RQ-PCR assay parallels the conventional disk diffusion test but with higher speed.