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目的初步建立血浆循环DNA的提取及鉴定方法,定量检测煤工尘肺患者血浆循环DNA水平,探讨循环DNA在煤工尘肺发生发展过程中的作用。方法利用病例对照流行病学研究方法选取研究对象,分为煤工尘肺组(31例)、接尘工人组(38例)和正常对照组(40例),采集研究对象外周血标本,采用磁珠法微量DNA快速提取试剂盒提取全部研究对象的血浆循环DNA,应用荧光定量PCR技术对全部研究对象的血浆循环DNA看家基因β-globin的含量进行检测,用临界阈值时的循环数(Ct值)反映循环DNA的相对水平,并根据Ct值绘出相应ROC曲线进行分析。结果煤工尘肺组、接尘工人组和正常对照组血浆循环DNA的Ct值分别为(18.00±1.07)、(19.20±0.58)和(19.80±1.57),煤工尘肺组血浆循环DNA的平均水平高于接尘工人组和正常对照组,差异具有统计学意义(P<0.05),根据煤工尘肺组与对照组数据所得ROC曲线下面积为0.839,煤工尘肺组与接尘工人组所得ROC曲线下面积为0.859。结论该方法能够方便快速的对血浆循环DNA水平进行检测,血浆循环DNA水平的增高在煤工尘肺病的发生发展过程中可能具有一定的意义。
Objective To establish a method for the extraction and identification of DNA from plasma and to quantitatively detect circulating DNA level in coal workers ’pneumoconiosis and to explore the role of circulating DNA in the development and progression of coal workers’ pneumoconiosis. Methods A case-control study of epidemiological methods was used to select the subjects. The patients were divided into coal worker’s pneumoconiosis group (31 cases), dust-exposed workers group (38 cases) and normal control group (40 cases) The beads DNA microarray rapid extraction kit was used to extract plasma circulating DNA from all the subjects. The content of β-globin, a plasma circulating DNA housekeeping gene, was detected by real-time fluorescence quantitative PCR. Value) reflects the relative level of circulating DNA and draws the corresponding ROC curve for analysis based on the Ct value. Results The Ct values of plasma circulating DNA in coal workers pneumoconiosis group, dust exposure workers group and normal control group were (18.00 ± 1.07), (19.20 ± 0.58) and (19.80 ± 1.57) (P <0.05). According to the ROC curve under the data of coal workers ’pneumoconiosis group and control group, the area under the ROC curve was 0.839. The ROC values of coal workers’ pneumoconiosis group and dust-exposed workers group were higher than those of workers exposed to dust and normal control group The area under the curve is 0.859. Conclusion The method can detect circulating DNA in plasma easily and rapidly. Increasing plasma DNA level may play a role in the development of coal worker ’s pneumoconiosis.