【摘要】 目的应用实时荧光定量PCR检测浆膜腔积液中细胞游离DNA含量,探讨浆膜腔漏出液和渗出液鉴别诊断指标。方法选择β-珠蛋白基因作为细胞游离DNA的代表标志物,设计引物和探针建立双标记荧光素TaqMan系统;以人类全基因组DNA为模板PCR扩增,扩增产物经克隆建立标准物,从而对浆膜腔积液中细胞游离DNA进行定量分析。结果对172份明确诊断的浆膜腔积液标本进行分析,浆膜腔积液中细胞游离DNA浓度的受试者工作特征曲线(ROC)曲线下面积为0.958,95%可信区间(CI)为0.931~0.986(P=0.00);积液中细胞游离DNA的cutoff浓度为306拷贝/mL,对浆膜腔积液漏出液和渗出液的鉴别诊断灵敏度为91.8%,特异性为92.3%;渗出液(细菌性、结核性和恶性)中细胞游离DNA浓度明显高于漏出液(P≤0.001),细菌性积液和结核性积液之间细胞游离DNA浓度差异无统计学意义(P=0.996),但明显高于恶性积液中细胞游离DNA浓度(P≤0.018)。结论浆膜腔积液中细胞游离DNA可作为浆膜腔漏出液和渗出液鉴别诊断指标。更多还原

【Abstract】 Objective Cell-free DNA concentration in serous effusions were measured by real-time fluorescent quantitative polymerase chain reaction(PCR),for a target to classifying the transudative and exudative fluid in serous effusions.Methods β-globin DNA was chosed as the representative marker for cell-free DNA in serous effusions.On the basis of β-globin DNA sequence,primers and probe were designed and the system of TaqMan with marked bi-fluorescence was established;PCR enlarged with the formwork of mankind full gene series DNA,and the enlarging products were cloned and the same matters as the standardized matter were produced.Thereby using this standardized matters,cell-free DNA in serous effusions can be quantitatively analysed.Results Cell-free DNA in 172 samples of the definite transudative and exudative fluid was examinted.The area under the ROC curve was 0.958[95% confidence interval(CI),0.931～0.986(P=0.000)].The ROC curve was plotted to determine the cutoff DNA concentration for classifying serous fluid as transudates and exudates.At 306 copies/mL,cell-free DNA alone correctly classified serous effusions with 91.8% sensitivity and 92.3% specificity respectively;cell-free DNA concentration in the exudative fluid(bacteric,tuberculous and malignant)is obviously higher than the transudative fluid(P≤0.001),there are not markedly differentiation between the bacteric and tuberculous fluid(P=0.996),but the cell-free DNA concentration in the bacteritic and tuberculous fluid is obviously higher than the malignant fluid(P≤0.018).Conclusions Quantitative analysis of cell-free DNA in serous fluid is an useful tool for classification of serous effusions.更多还原