摘要
目的 采用液滴式数字PCR(ddPCR)法检测晚期非小细胞肺癌(NSCLC)患者外周血循环肿瘤DNA(ctDNA) EGFR 基因突变的情况,探讨试剂盒的性能、检测结果的可靠性和准确性以及ddPCR法在外周血ctDNA中的应用价值。方法 使用标准突变比例的模拟cfDNA样本ddPCR法对试剂检测限、准确性、特异性、精密度、抗干扰能力等指标进行分析及评价;使用ddPCR法检测262例NSCLC患者外周血ctDNA,随机选取30例样本同时进行Super-ARMS法检测,并从方法学标准化和检测结果准确性两方面进行统计学分析。结果 检测试剂在检测限以上的准确性、特异性均为100%,定量精密度(突变比例对数的绝对值变异系数 CV )<5%,关键环节质量控制各主要参数的相关性和线性关系均符合要求;Super-ARMS和ddPCR法同步检测30例NSCLC患者外周血ctDNA的结果具有较好的一致性( Kappa =0.783, P =0.000)。结论 ddPCR法灵敏度高,可绝对定量,是血液检测的有效的方法,但其在检测过程中存在较多对检测结果产生影响的环节,故而检测方法标准化至关重要。
Objective To detect the mutation status of EGFR gene in peripheral blood cell-free tumor DNA (ctDNA) from the patients with advanced non-small cell lung cancer (NSCLC) by the droplet digital PCR (ddPCR), and evaluate the performance and accuracy of the ddPCR method and its application value in the detection of peripheral blood ctDNA. Methods The simulated cfDNA samples with standard mutation ratios were detected with the ddPCR method repeatedly, and the detection limit, accuracy, specificity, precision, and anti-interference ability of the method were analyzed. The peripheral blood ctDNA samples from 262 patients with NSCLC were detected by the ddPCR method, and 30 of these samples were selected randomly and detected by the Super-ARMS method. The methodology and accuracy of the two methods were compared. Results Both of accuracy and specificity of the ddPCR method above the detection limit were 100%. The quantitative precision ( CV ) of the logarithm of mutation ratios was less than 5%. The correlation and linear relationship of the main parameters for quality control were all in accordance with the requirements. The detection results of the peripheral blood ctDNA from 30 NSCLC between the Super-ARMS and the ddPCR had good consistency ( Kappa =0.783, P = 0.000 ). Conclusion The ddPCR method has high sensitivity and may be quantified absolutely, which is an effective method for blood detection. However, there are many factors which may affect the detection results in the process of detection, so the standardization of the detection method is very important.
作者
李霄
丁颖
季盼
张智弘
LI Xiao;DING Ying;JI Pan;ZHANG Zhihong(Department of Pathology,Jiangsu Provincial People′s Hospital&The First Affiliated Hospital of Nanjing Medical University,Nanjing 210029,Jiangsu,China)
出处
《临床检验杂志》
CAS
2019年第8期579-582,共4页
Chinese Journal of Clinical Laboratory Science
