摘要
目的建立前列腺癌组织中胶原三股螺旋重复蛋白1(collagen triple helix repeat containing 1,CTHRC1)基因转录水平的微滴数字PCR(droplet digital PCR,ddPCR)检测方法,并进行验证。方法根据GenBank中登录的CTHRC1基因序列设计引物,扩增CTHRC1保守区基因片段,并合成含有该基因片段的PUC57质粒。经2%琼脂糖凝胶电泳验证引物的特异性后,采用PUC57质粒验证ddPCR法的正确度、精密性,并确定检测限及线性范围。采用建立的ddPCR法检测复旦大学附属中山医院泌尿外科住院患者73份前列腺穿刺组织样本(前列腺癌组织40份,非前列腺癌组织33份)中的CTHRC1基因转录水平,同时采用试剂盒检测患者血清中的总前列腺特异抗原(total prostate specific antigen,TPSA)水平,将相应结果进行ROC曲线分析,以获得CTHRC1-TPSA联合检测对诊断前列腺癌的预测概率。结果经2%琼脂糖凝胶电泳验证设计的引物具有良好的特异性。检测值和理论值之间的Log值差异均<1,且拟合度高,R2=0.9959,表明该方法具有良好的正确度;重复性验证CV均<10%,中间精密性验证CV均<15%;该方法的检测限为100copies/μL;检测值在100~104copies/μL范围内,与理论值呈良好的线性相关性,R2=0.9972。ddPCR法检测前列腺癌组织中CTHRC1基因转录水平显著高于非前列腺癌组织(Z=-4.339,P<0.001)。CTHRC1与TPSA联合诊断前列腺癌的ROC曲线下面积(area under the curve,AUC)为0.847,灵敏度为83.3%,特异性为67.7%。结论ddPCR法可用于前列腺癌组织中CTHRC1基因转录水平的检测,与TPSA指标联合可提高临床诊断前列腺癌的准确性。
Objective To develop and verify a droplet digital PCR(ddPCR)method for determination of transcription level of collagen triple helix repeat containing 1(CTHRC1)gene in prostate cancer.Methods Primers were designed based on CTHRC1 gene sequence in GenBank,with which the gene fragment in conserved region of CTHRC1 was amplified,and PUC57 plasmid containing the fragment was synthesized.The specificity of primers was verified by 2%agarose gel electrophoresis.The developed ddPCR method was verified for accuracy and precision with PUC57 plasmid,and determined for detection limit and linear range.The transcription levels of CTHRC1 gene in 73 prostate biopsy tissue samples(40 prostate cancer tissues and 33 non-prostate cancer tissues)from inpatients in the Department of Urology,Zhongshan Hospital affiliated to Hehai University were determined by the developed ddPCR method,while the total prostate specific antigen(TPSA)level in sera of patients with the corresponding kits.All the results were analyzed by ROC curve to abtain the predicted probability of diagnosis of prostate cancer by CTHRC1-TPSA.Results Verification with 2%agarose gel electrophoresis showed good specificity of the designed primers.All the differences of Log values between the measured and theoretical values were less than 1,with a high fitting degree,R2=0.9959,indicating high accuracy of the method.All the CVs in verification for reproducibility were less than 10%,while those for intermediate precision were less than 15%.The detection limit of the method was 100 copies/μL,while the measured values within a range of 100~104 copies/μL showed a good linear relationship with the theoretical values,R2=0.9972.The transcription level of CTHRC1 gene in prostate cancer tissue determined by ddPCR was significantly higher than that in non-prostate cancer tissue(Z=-4.339,P<0.001).The area under the curve(AUC)of ROC of prostate cancer diagnosed by CTHRC1 combined with TPSA was 0.847,with a sensitivity of 83.3%and a specificity of 67.7%.Conclusion The developed ddPCR method may be used for determination of transcription level of CTHRC1 gene in prostate cancer tissue,which improved the accuracy of clinical diagnosis of prostate cancer in combination with TPSA index.
作者
杨德平
张艳
刘莹
张培燕
孔娜娜
郑江花
YANG De-ping;ZHANG Yan;LIU Ying;ZHANG Pei-yan;KONG Na-na;ZHENG Jiang-hua(Department of Laboratory Medicine,Shanghai University of Medicine&Health Sciences Affifiliated Zhoupu Hospital,Shanghai 201318,China)
出处
《中国生物制品学杂志》
CAS
CSCD
北大核心
2022年第11期1378-1383,共6页
Chinese Journal of Biologicals
基金
国家自然科学基金面上项目(81372318)
上海市科委生物医药科技支撑专项(21S11902100)
上海市浦东新区卫生和计划生育委员会领先人才培养计划(PWRL2017-07)
上海健康医学院2020年师资人才百人库(B3-0200-20-311008)
2019年周浦医院院级科研项目(ZPXM-2019A-11).
关键词
前列腺癌
胶原三股螺旋重复蛋白1
微滴数字PCR法
总前列腺特异抗原
Prostate cancer
Collagen triple helix repeat containing 1(CTHRC1)
Droplet digital PCR(ddPCR)
Total prostate specific antigen(TPSA)
