实时定量PCR检测人全血中金黄色葡萄球菌DNA方法的建立

摘要

目的 建立实时定量PCR (RQ-PCR)快速检测人全血中金黄色葡萄球菌DNA的方法,以便早期定量评估肠屏障损伤所致肠道内细菌移位引起或加重的全身感染.方法 对15份模拟全血标本及26份外科发热患者全血标本进行RQ-PCR检测.选择金黄色葡萄球菌高度保守的看家基因fem A基因作为靶基因设计引物和Taqman探针,建立20μl的RQ-PCR反应体系,采用含靶基因扩增片段的重组质粒建立标准曲线,提取血标本中的细菌基因组DNA.结果 引物和探针特异性好,检测限为100拷贝/μ1 (103 CFU/ml),灵敏度为99.7％,特异度为94.6％.标准曲线线性关系好,R2在0.9918 ～0.9997.不同浓度的金黄色葡萄球菌样本检测的平均准确性为(96.25±2.26)％,批内及批间重复性的平均变异系数分别为(8.06±0.07)％和(10.01±4.40)％.全血标本中金黄色葡萄球菌DNA平均回收率为(111.72±20.72)％.临床血标本RQ-PCR检测阳性率为15.4％ (4/26),血培养结果皆为金黄色葡萄球菌阴性.结论 RQ-PCR可用于定量检测全血标本中的金黄色葡萄球菌DNA含量,具有快速、灵敏、特异性强、重复性好的优点.%Objective To establish a rapid real-time quantitative polymerase chain reaction (RQ-PCR)assay in quantifying and detecting Staphylococcus aureus DNA from human venous blood samples,so as to quantificationally evaluate the systemic infection caused or deteriorated by intestinal bacteria translocation.Methods Totally 26 clinical blood samples and 15 simulation blood samples were detected.The primers and TaqMan probe were designed targeting the highly conserved house-keeping femA gene of Staphylococcus aureus,and a 20 μl RQ-PCR amplification reaction system was established.The standard curve was built based on the recombinant plasmid DNA containing the amplicon of the target gene,and genomic DNA was extracted using QIAamp DNA Blood Mini Kit.Results The specificity of primers and probe was excellent,the detecting limit was 100 copies/μl (103 CFU/ml),the sensitivity was 99.7％,and the specificity was 94.6％.The correlation coefficient of the standard curve was between 0.9918 and 0.9997.For samples with different Staphylococcus anreus concentrations,the average accuracy of the RQ-PCR assay was (96.25 ± 2.26) ％ ; the intra-and interassay coefficients of variation were (8.06 ±0.07)％ and (10.01 ±4.40)％,respectively.The average recovery rate of Staphylococcus aureus DNA in blood samples was (111.72 ± 20.72) ％.In clinical blood samples,the positive rate of Staphylococcus aureus DNA was 15.4％ (4/26),while the blood culture of these samples all produced negative result for Staphylococcus aureus.Conclusion RQ-PCR assays is a rapid,sensitive,and specific method with good repetitiveness and can be used in the quantitative detection of Staphylococcus aureus in whole blood samples.