基于ddPCR技术分析EB病毒载量特征及与qPCR技术的比较研究

目的解决临床工作中应用实时荧光定量PCR(qPCR)方法检测EB病毒(EBV)载量与临床诊断不符的问题,探讨微滴式数字PCR(ddPCR)和qPCR方法检测EBV载量的能力并为临床提供可能的解决方案。方法收集510例疑似EBV感染相关疾病患者血浆标本,采用ddPCR和qPCR两种方法测定同一血浆标本的EBV-DNA载量。结果 EBV感染人群中,EBV-DNA载量较其他地区低,载量中位数仅360copies/mL,其中初诊未治鼻咽癌患者中位病毒载量为4 590copies/mL,治疗后鼻咽癌患者中位病毒载量下降为430copies/mL,免疫力低下者中位病毒载量为130copies/mL,而淋巴瘤患者中位病毒载量为840copies/mL;qPCR检测EBV感染以400copies/mL为界值,高于400copies/mL时,ddPCR与qPCR的EBV-DNA测定水平值呈中度相关(r=0.533,P<0.05),低于400copies/mL时,ddPCR与qPCR的EBV-DNA测定水平值呈弱相关(r=0.299 5,P<0.05);以ddPCR为标准,qPCR检测EBV-DNA的灵敏度仅为0.317,以ddPCR检测结果为标准,构建qPCR的受试者工作特征曲线下面积为0.871,此时临界值(qPCR)为10copies/mL,灵敏度为0.824,特异度为0.780。结论采用ddPCR方法或优化qPCR的临界值去检测EBV-DNA载量更能为临床诊断EBV感染提供有利支持。

Analysis of Epstein-Barr virus load characteristic based on ddPCR technology and comparison with qPCR technology

Objective To solve the problem that the real-time fluorescent quantitative PCR(qPCR)is not suitable for the clinical diagnosis of EBV in clinical work,and to explore the ability of droplet digital PCR(ddPCR)and qPCR to detect EBV load and provide a possible solution for the clinical work.Methods The plasma samples of 510 patients suspected of Epstein-Barr virus(EBV)infection were collected and the EBVDNA load of the same plasma sample was measured by ddPCR and qPCR.Results Among EBV-infected populations,the EBV-DNA load was lower than in other regions,and the median load was only 360 copies/mL.The median viral load of patients with newly diagnosed nasopharyngeal carcinoma was 4 590 copies/mL.After treatment,the median viral load in patients with pharyngeal cancer was 430 copies/mL.The median viral load in patients with low immunity was 130 copies/mL,and the median viral load in patients with lymphoma was840 copies/mL.The limit value of EBV infection detected by qPCR was 400 copies/mL.Above 400 copies/mL,ddPCR and qPCR EBV-DNA levels were moderately correlated(r=0.533,P<0.05),which was lower than400 copies/mL,the correlation between ddPCR and qPCR EBV-DNA levels were weakly correlated(r=0.299 5,P<0.05);The results were based on ddPCR,and the sensitivity of qPCR to detect EBV-DNA was only 0.317.Based on the results of ddPCR,the receiver operating characteristic curve for qPCR was constructed and the area under the curve was 0.871,the cut-off value(qPCR)was 10 copies/mL,the sensitivity was 0.824,and the specificity was 0.780.Conclusion Detecting EBV-DNA load using ddPCR method or optimizing the cut-off value of qPCR can provide favorable support for clinical diagnosis of EBV infection.