荧光定量聚合酶链反应检测血液制品中细菌污染方法的建立  

Establishment of fluorescence quantitative PCR for detecting bacterial contamination of blood products

作　　者：应燕玲[1] 陶苏丹[1] 和艳敏[1] 许先国[1] 朱发明[1] 吕杭军[1] 严力行[1] 

机构地区：[1]浙江省血液中心输血研究所卫生部血液安全研究重点实验室,浙江杭州310006

出　　处：《中华医院感染学杂志》2011年第1期205-208,共4页Chinese Journal of Nosocomiology

摘　　要：目的建立快速检测血液制品中细菌污染的荧光定量聚合酶链反应(PCR)方法。方法以大肠埃希菌和金黄色葡萄球菌分别作为革兰阴性菌和革兰阳性菌代表菌,模拟不同程度细菌污染的血液标本,利用3种不同抽提方法提取细菌基因组DNA进行荧光定量PCR(FQ-PCR);以大肠埃希菌系列浓度梯度基因组DNA为模板建立标准曲线,将14株不同菌株模拟细菌污染的血液标本进行FQ-PCR检测。结果根据Ct值和扩增效果,3种基因组抽提方法中以DNA过柱纯化法效果最佳;以大肠埃希菌梯度稀释(1.2×108)～(1.2×102)CFU/ml进行FQ-PCR反应,得到标准曲线为Y=-0.2211X+8.80(R2=0.998);14株不同细菌均能在103CFU/ml检测出阳性,但Ct值差异有统计学意义。结论建立了检测血液制品中细菌污染的荧光定量诊断技术,该方法快速可靠,在预防和诊断输血相关性细菌污染上具有一定的应用价值。OBJECTIVE To establish a fluorescence quantitative PCR method for rapid detecting contamination bacterium of blood products. METHODS The different concentration of contamination bacterium in blood samples were simulated with a series of graded dilution Escherichia coli and Staphylococus aureus. The genomic DNAs were extracted by three methods and detected by fluorescence quantitative PCR（FQ-PCR）. The standard curve of FQ-PCR was established using a series of graded concentrations of E. coli genomic DNA as template. 14 different strains were detected by FQ-PCR method after simulating contamination bacterium in blood samples. RESULTS According to the Ct values and amplification efficiency, DNA purification column method was the best one among the three extraction methods. The standard curve of the FQ-PCR was Y=-0. 2211Xq-8.80 （R2 =0. 998） ,which obtained with gradient dilution of E. coli （1. 2×10^8- 1. 2 × 10^2CFU/ml）. Fourteen different strains were detected as positive in 103CFU/ml concentration, with different Ct values. CONCLUSION A rapid and accurate FQ-PCR method for detecting contamination bacterium in blood samples has been established, which can also benefit for the prevention and diagnosis of bacterial contamination in transfusion.

关 键 词：血液细菌污染 荧光定量聚合酶链反应 分子诊断 

分 类 号：R446.1[医药卫生—诊断学]