数字PCR检测血浆游离DNA方法的建立及在结直肠癌中的初步应用

Establishment of Digital PCR for Detecting Plasma Cell-free DNA and Its Application Value in Colorectal Cancer

目的建立基于微滴式数字PCR(dd PCR)技术定量检测血浆游离DNA(cf-DNA)含量的方法,并探讨其在结直肠癌诊疗中的应用价值。方法设计β-actin特异性引物和探针,建立dd PCR检测cf-DNA绝对定量方法,根据不同浓度标准品验证该检测方法的敏感度、线性以及重复性。该法检测68例结直肠癌患者、33例结直肠良性肿瘤患者及60例健康对照者血浆cf-DNA水平,化学发光法检测其血清CEA、CA19-9水平,分析血浆cf-DNA水平与临床病理参数及血清CEA、CA19-9水平的关系,并用ROC曲线评估其诊断效能。结果本实验所建立的dd PCR方法能够检测低至10pg/μl的微量DNA模板,检测敏感度为0.29copies/μl;且在模板浓度为10pg/μl^10ng/μl的范围内线性良好(Y=-2.34+33.17X,R2=0.999);批内CV为9.85%,批间CV为11.04%。该法检测结直肠癌组、结直肠良性肿瘤组和健康对照组血浆cf-DNA水平分别为6700(3800~9925)、3100(2300~4000)、2500(1775~4000)copies/ml,结直肠癌组血浆cf-DNA水平显著高于结直肠良性肿瘤组(P=0.000)和健康对照组(P=0.000),而结直肠良性肿瘤组与健康对照组比较,差异无统计学意义(P=0.167)。结直肠癌患者血浆cfDNA水平在不同年龄、性别、肿瘤部位、分化程度、淋巴结转移以及肿瘤浸润程度间比较,差异均无统计学意义(P均>0.05),在TNM分期间差异有统计学意义(P<0.01)。结直肠癌患者血浆cf-DNA水平与CEA(r=0.210,P=0.099)和CA19-9(r=0.125,P=0.233)无相关性。ROC曲线结果显示,以5300copies/ml为cut off值,cf-DNA诊断结直肠癌ROC曲线下面积(AUC)、敏感度、特异性分别为0.931、73.3%和98.3%,高于CEA(0.762、51.7%、95.6%)和CA19-9(0.548、45.0%、82.0%)。结论建立了一种敏感、特异的dd PCR定量检测血浆cf-DNA的方法,有助于结直肠癌的辅助诊断及预后判断。

Objective To establish a method for detecting concentrations of plasma cell-free DNA（ cf-DNA） based on the droplet digital PCR（ dd PCR）,and to explore its diagnosis and treatment value in colorectal cancer. Methods dd PCR specific primers and probes for β-actin were designed to establish an absolute quantitative method for the detection of cf-DNA. The sensitivity,linearity and reproducibility of the method were validated by the detection of standards with different concentrations. The plasma cf-DNA levels were detected by this method in 68 patients with colorectal cancer,33 patients with colorectal benign tumor and 60 healthy controls,and the concetrations of carcinoembryonic antigen（ CEA）,carcinoembrvonic antigen 19-9（ CA19-9） were assayed by chemiluminescence,the correlation between plasma cf-DNA levels and clinicopatholoical parameters and serum CEA,CA19-9 was analyzed,and Receiver Operqting Characreristic（ ROC） curves were established to illustrate the diagnostic performance. Results The dd PCR method established in this experiment can detect as low as 10 pg/μl DNA template,which is approximate to 0. 29 copies/μl,and dd PCR was evaluated as linear in the range of 10 pg/μl-10 ng/μl（ Y =-2. 34 ＋ 33. 17 X,R2= 0. 999）; Coefficient of Variance（ CV） of intra-ssay was 9. 85%,CV of inter-assay was 11. 04%. The levels of cf-DNA detected by dd PCR among the plasma samples of patients with colorectal cancer,colorectal benign tumor and healthy controls were 6700（ 3800-9925）,3100（ 2300-4000）,2500（ 1775-4000） copies/ml,respectively. There were significant differences between colorectal cancer and colorectal benign tumor patients（ P = 0. 000）,colorectal cancer patients and healthy individuals（ P = 0. 000）,and there was no significant difference between colorectal benign tumor patients and healthy individuals（ P = 0. 167）. No statistically significant difference of cf-DNA in age,gender,tumor sites,tumor differentiation,lymph node metastasis and the degree of tumor invasion was found.. But there was significant difference in the stage TNM（ P〈0.01）. There was no correlation between cf-DNA and CEA（ r = 0. 210,P = 0. 099） and CA19-9（ r = 0. 125,P = 0. 233）. The ROC curve showed that when cut off value was 5300 copies/ml,the area under the ROC curve（ AUC）,sensitivity and specificity of plasma cf-DNA in colorectal cancer patients were 0. 931,73. 3% and 98. 3%,respectively,which were significantly higher than those of CEA（ 0. 762,51. 7%,95. 6%） and CA19-9（ 0. 548,45. 0%,82. 0%）. Conclusion A sensitive,specific dd PCR for detecting plasma cf-DNA has been established,which is helpful for the diagnosis and prognosis of colorectal cancer.