细胞游离DNA定量分析用于浆膜腔积液鉴别诊断研究  

Quantitative analysis of cell-free DNA in serous fluid for classification of serous effusions

作　　者：潘永军[1] 周国平[1] 

机构地区：[1]常州市第三人民医院检验科,江苏常州213001

出　　处：《检验医学》2008年第6期638-641,共4页Laboratory Medicine

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。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-glohin DNA sequence, primers and probe were designed and the system of TaqMan with marked bi-fluorescenee 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 transudafivc and exudative fluid was examintcd . 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 copics/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 baeteritic 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.

关 键 词：细胞游离DNA 实时荧光定量PCR 浆膜腔积液 

分 类 号：Q503[生物学—生物化学]