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
投稿时间:2017-10-06  修订日期:2017-10-24  点此下载全文
引用本文:刘平,余玲玲,王金丹,蔡锚,吴兆明,郑晓群.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[J].医学研究杂志,2018,47(6):38-44
DOI: 10.11969/j.issn.1673-548X.2018.06.011
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作者单位E-mail
刘平 325027 温州医科大学附属第二医院
325035 温州医科大学检验医学院、检验医学教育部重点实验室 
 
余玲玲 325027 温州医科大学附属第二医院  
王金丹 325035 温州医科大学检验医学院、检验医学教育部重点实验室  
蔡锚 325027 温州医科大学附属第二医院  
吴兆明 325035 温州医科大学检验医学院、检验医学教育部重点实验室  
郑晓群 325035 温州医科大学检验医学院、检验医学教育部重点实验室 jszhengxq@163.com 
基金项目:国家级大学生创新创业训练计划项目(201610343003);浙江省科技厅分析测试科技计划项目(2016C37073);浙江省温州市科技局公益性科技计划项目(Y201407320)
中文摘要:目的 建立基于微滴式数字PCR(ddPCR)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ddPCR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ddPCR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+33.17XR2=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cf-DNA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ddPCR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。
中文关键词:ddPCR  cf-DNA  结直肠癌  CEA  CA19-9
 
Establishment of Digital PCR for Detecting Plasma Cell-free DNA and Its Application Value in Colorectal Cancer
Abstract:Objective To establish a method for detecting concentrations of plasma cell-free DNA (cf-DNA) based on the droplet digital PCR (ddPCR), and to explore its diagnosis and treatment value in colorectal cancer. Methods ddPCR 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 ddPCR method established in this experiment can detect as low as 10pg/μl DNA template,which is approximate to 0.29copies/μl, and ddPCR was evaluated as linear in the range of 10pg/μl-10ng/μl (Y=-2.34+33.17X, 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 ddPCR 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 5300copies/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 ddPCR for detecting plasma cf-DNA has been established,which is helpful for the diagnosis and prognosis of colorectal cancer.
keywords:ddPCR  cf-DNA  Colorectal cancer  CEA  CA19-9
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