应用实时荧光定量PCR方法检测血小板制品细菌污染

The real-time fluorescence quantitative PCR assay for detection of bacterial contamination in platelet concentrates

目的应用实时荧光定量PCR方法检测血小板制品中细菌的探讨。方法选取金黄色葡萄球菌及大肠埃希氏杆菌,用改良的Chelex-100法抽提细菌基因组DNA,进行荧光定量PCR检测。并应用过滤法去除反应体系中潜在的细菌及其基因组DNA污染。结果针对16srRNA基因保守序列进行扩增的荧光定量PCR方法具有较高的特异性和灵敏度,与人类淋巴细胞及病毒等的基因组无交叉反应。在金黄色葡萄球菌检测中,应用过滤法后最低含菌量组与阴性对照组Ct值有极显著差异(P<0.001)。该方法对金黄色葡萄球菌的最低检出量为0.3CFUs/PCR,与阴性对照Ct值有显著差异(P<0.01),在大肠埃希氏杆菌中该法可检测出0.1CFUs/PCR,与阴性对照Ct值有显著性差异(P<0.01)。结论改良Chelex-100法抽提细菌基因组及后续的荧光定量PCR分析用于检测血小板制品中的细菌污染,检测实验缩短到3-4h,操作简单,灵敏性高,特异性好,为在临床标本大规模检测中的应用提供理论基础和实验数据。

Objective To establish a real-time PCR assay to detect bacterial contamination in plateler concentrates （PCs）. Methods PCs were spiked with serial dilutions of Staphylococcus aureaus and Eschetichia coil, and the bacterial genomic DNA was isolated by modified Chelex-100 method and then quantitatively detected by Tagman realtime PCR. Filtration was performed to avoid contamination from other bacterial DNA in the reagents of extraction kit as well as the PCR mixture. Results The real-time PCR targeting conservative 16S rRNA gene showed high sensitivity and specificity in detecting bacterial contamination in PCs, and no cross-reaction with human genomic DNA and viruses was found. The Ct value of the lowest bacterial dose groups after filtration in both S. aureaus and E. coli were statistically different from the negative controls, with a minimal detection quality of 0.3CFUs/PCR for S. aureaus, and 0.1CFUs/PCR for E. coll. , respectively. Conclusion The modified Chelex-100 method to extract bacterial genomic DNA and the subsequent real-time PCR are convenient, sensitive and specific, and may be applied to the large scale clinical detection of bacterial contamination in PCs.