荧光定量PCR检测血浆循环DNA及其在肝细胞癌诊断中的意义

Detection of plasma circulating DNA by fluorescence quantitative PCR and its clinical significance in diagnosis of hepatocellular carcinoma

目的建立血浆中循环DNA的定量检测方法并探讨其在肝细胞癌诊断中的应用价值。方法先提取正常健康人全血DNA,采用PCR方法扩增人球蛋白基因,然后把该内参基因的扩增产物进行梯度稀释来制备标准品,再用荧光定量PCR方法扩增不同浓度的标准品,从而建立检测血浆循环DNA的标准曲线。然后分别收集肝细胞癌患者术前血浆标本132例、肝硬化患者血浆标本55例,用已建立的方法定量检测这些患者血浆标本中的循环DNA。结果建立了检测血液循环DNA的荧光定量PCR方法,该方法扩增效率较高;其标准曲线的Slope值为-0.308,Intercept值为28.283,R2值为0.997。肝细胞癌患者血浆中DNA浓度为(332±38)ng/m L,显著高于肝硬化患者的(57±6)ng/m L和正常健康人的(15±4)ng/m L,差异有统计学意义(P<0.05)。结论荧光定量PCR技术可用于检测血液循环DNA浓度。血循环DNA检测对肝细胞癌的诊断具有重要的应用价值。

Objective To establish fluorescence quantitative PCR （FQ -PCR） for detecting plasma circulating DNA and explore its role in the diagnosis of hepatocellular carcinoma （HCC）. Methods Total DNA was extracted from whole blood of normal healthy persons. Human globin gene as a reference gene was amplified by using ordinary PCR. Af- ter gradient dilution, the PCR products were used as standard substance and amplified by FQ - PCR at different concentra- tions. Thereby the standard curve to detect circulating DNA in plasma was established. Then the circulating DNA in plas- ma samples from 132 patients with HCC, 55 patients with liver cirrhosis and 30 healthy volunteers were detected by the abovementioned FQ - PCR method. Results High amplification efficiency was found in the FQ - PCR method established for detecting plasma circulating DNA. The standard curve of the above FQ - PCR had slope of O. 308, intercept of 28. 283 and RE of 0. 997. Plasma DNA level in patients with HCC was significantly higher than in patients with liver cirrhosis （57 ± 6 ng/mL） in healthy controls （ 15 ± 4 ng/mL） （P 〈 0. 05）. Conclusion Circulating DNA in blood sample, which could be quantitatively measured by FQ - PCR, is valuable for the diagnosis of HCC.