实时荧光定量PCR检测结直肠癌患者粪便肿瘤型M2-PK DNA及临床应用研究  

Clinical application of real-time fluorescence quantitative PCR for the detection of fecal tumor M2-pyruvate kinase in colorectal cancer patients

作　　者：刘玉兰[1] 何凤屏[1] 徐新[1] 吴青松[1] 李定云[1] 马占忠[1] 郭艳乐[1] 唐盛[2] 尹卫东 龚海涛 刘艺 林恒先 

机构地区：[1]汕头大学医学院附属粤北人民医院,广东韶关512026 [2]深圳市迈科龙生物技术有限公司,广东518057

出　　处：《国际检验医学杂志》2017年第11期1444-1446,共3页International Journal of Laboratory Medicine

基　　金：广东省重大科技专项资金项目(2013A022100011);国家临床医药研究专项基金项目(L2014074)

摘　　要：目的建立适用于结直肠癌患者粪便标本肿瘤型M2丙酮酸激酶(M2-PK)DNA检测的实时荧光定量聚合酶链反应(PCR)方法,分析自建方法的临床应用价值。方法采用PCR方法扩增肿瘤型M2-PK DNA片段(162bp),纯化后采用TA克隆法转入PGM-T载体,构建重组质粒。以重组质粒为模板,建立实时荧光定量PCR检测方法,评价自建方法的敏感性、特异性和重复性。选择2014年1月至2016年6月确诊的结直肠癌患者200例,同期体检健康者100例,采用自建方法对受试者粪便和血清标本进行肿瘤型M2-PK DNA检测,并与酶联免疫吸附法肿瘤型M2-PK检测结果进行比较。结果测序结果证实重组质粒构建成功。自建方法检测肿瘤型M2-PK DNA的灵敏度为10copy/mL,对肌肉型M2-PK DNA和M1型丙酮酸激酶(M1-PK)DNA检测结果为阴性,批内及批间变异系数为0.3%~2.9%。实时荧光定量PCR检测患者粪便标本肿瘤型M2-PK DNA阳性率为92.50%,ELISA检测肿瘤型M2-PK阳性率为80.00%。粪便和血清标本肿瘤型M2-PK DNA实时荧光定量法检测结果与肿瘤病理分期密切相关。结论本研究建立的实时荧光定量PCR法适用于结直肠癌患者粪便标本肿瘤型M2-PK DNA检测,具有特异性强、灵敏度高的特点,可用于结直肠癌早期诊断。Objective To investigate the application valve of real-time fluorescence quantitative polymerase chain reaction(RTPCR)for the detection of tumor M2-pyruvate kinase(tM2-PK)DNA in patients with colorectal cancer(CRC).Methods Fragment of tM2-PK DNA(162bp)was amplified and inserted into PGM-T vector to construct recombinant plasmid,which was used to develop RT-PCR method.Sensitivity,specificity and repeatability of RT-PCR for the detection of tM2-PK were analyzed.From Jan.2014 to Jun.2016,200 CRC patients and 100 healthy subjects were enrolled and detected for fecal and serum tM2-PK DNA by using RT-PCR,and the detected results were compared with those detected by using enzyme linked immunosorbent assay(ELISA).Results Recombinant plasmid was successfully constructed,which was certified by sequencing.The sensitivity of RT-PCR for the detection of tM2-PK DNA was 10copy/mL,with high specificity and 0.3%-2.9% of coefficient of variation.In patients,the positive rate of fecal tM2-PK DNA,detected by RT-PCR,was 92.50%,and that of ELISA to detect tM2-PK was 80.00%.Fecal and serum levels of tM2-PK were correlated with the pathologic stages of tumour.Conclusion Self-established RT-PCR could be specificity and sensitivity for the detection of fecal tM2-PK,which could be used for the early diagnosis of CRC.

关 键 词：结直肠癌 粪便 肿瘤型M2丙酮酸激酶 实时荧光定量聚合酶链反应 

分 类 号：R730.43[医药卫生—肿瘤] R735.34[医药卫生—临床医学]