数字PCR在EB病毒核酸检测中的临床价值  

The clinical value of digital PCR in Epstein-Barr virus nucleic acid testing

作　　者：黄金印 陈芊如 何晓静[1] 欧子豪 蔡贞[1] 司徒博[1] 郭永[2] 郑磊[1] Huang Jinyin;Tan Chianru;He Xiaojing;Ou Zihao;Cai Zhen;Situ Bo;Guo Yong;Zheng Lei(Department of Laboratory Medicine,Nanfang Hospital,Southern Medical University,Guangzhou 510515,China;School of Biomedical Engineering,Tsinghua University,Beijing 100084,China)

机构地区：[1]南方医科大学南方医院检验医学科,广州510515 [2]清华大学生物医学工程学院生物医学工程,北京100084

出　　处：《中华检验医学杂志》2024年第6期649-657,共9页Chinese Journal of Laboratory Medicine

摘　　要：目的评估EB病毒(EBV)多拷贝和单拷贝基因的数字PCR(dPCR)核酸检测方法在EBV核酸定量检测中的性能,并探讨其在临床应用中的适用性。方法对多拷贝BamHI-W基因及单拷贝EBNA1基因dPCR体系的灵敏度、特异性、精密度、检测下限(LoD)和线性进行性能验证比对,采用最小二乘线性回归分析评估其线性。同时,收集2022年1—7月就诊于南方医科大学南方医院疑似EBV感染相关疾病患者的血浆样本182例,使用dPCR和实时荧光定量PCR(qPCR)同步检测EBV DNA载量,采用最小二乘线性回归分析评估其定量相关性。结果多拷贝和单拷贝基因的dPCR体系均有良好的线性相关(R2分别为0.992、0.997,P均<0.001),BamHI-W基因dPCR体系LoD为188 IU/ml,EBNA1基因dPCR体系LoD为358 IU/ml;BamHI-W基因dPCR体系和EBNA1基因dPCR体系中高浓度样本(1000000 IU/ml)对数变异系数(CV)值分别为0.34%和0.21%,中低浓度样本(5000 IU/ml)对数CV值分别为0.98%和0.64%;7种常见临床感染病原体和EB病毒阳性样本对照检测中,dPCR检测体系中只有EBV阳性样本产生阳性信号,与其他病原体无交叉反应。在182份样本检测中,BamHI-W基因dPCR阳性率为47.80%(87/182),EBNA1基因dPCR阳性率为35.16%(64/182),qPCR阳性率为43.41%(79/182)。定量相关性分析中,BamHI-W基因dPCR体系和EBNA1基因dPCR体系与qPCR检测浓度值线性拟合R2值分别为0.837和0.763(P均<0.001)。临床样本中的BamHI-W基因的拷贝数为3~18拷贝,不同患者感染的EBV的BamHI-W基因拷贝数不同。对于每例患者,多拷贝BamHI-W基因dPCR体系和单拷贝EBNA1基因dPCR体系检测的载量监测变化曲线趋势具有较高的一致性。结论基于dPCR技术的检测多拷贝BamHI-W基因和单拷贝EBNA1基因的EBV检测方法均具有较高的灵敏度和特异性,精密度和定量准确性均适用于临床样本检测。多拷贝BamHI-W基因dPCR方法在提高检测灵敏度方面具有较大的优势,可作为目前EBV DNA载量检测方法Objective This study aims to evaluate the performance of digital PCR(dPCR)detecting multiple and single copies genes of the Epstein-Barr virus(EBV)for nucleic acid quantification and explore their applicability in clinical settings.Methods Compared the sensitivity,specificity,precision,lower limit of detection(LoD),and linearity for multicopy BamHI-W dPCR and single-copy EBNA1 dPCR systems.Linear regression analysis using the least squares method was employed to evaluate the linearity.Additionally,we analyzed plasma samples from 182 patients with suspected EBV-related diseases between January and July 2022 at the Southern Medical University Southern Hospital,using both dPCR and quantitative PCR(qPCR)for EBV DNA quantification.Linear regression analysis using the least squares method was conducted to assess their quantitative correlation.Results The dPCR systems for both multicopy and single-copy genes showed excellent linearity(R2 values of 0.992 and 0.997,respectively,both P<0.001).The LoD were 188 IU/ml for BamHI-W gene and 358 IU/ml for EBNA1 gene dPCR systems.The logarithmic coefficient of variation(CV)values for high-concentration samples(1000000 IU/ml)were 0.34%and 0.21%for the BamHI-W gene and EBNA1 gene dPCR assays,respectively,while for low-concentration samples(5000 IU/ml)were 0.98%and 0.64%,respectively.In the detection of seven common clinical infectious pathogens and EBV positive samples,only EBV-positive samples yielded positive signals in the dPCR detection system,with no cross-reaction with other pathogens.In 182 samples,the positive detection rates were 47.80%(87/182)for BamHI-W gene and 35.16%(64/182)for EBNA1 gene dPCR,compared to 43.41%(79/182)for qPCR.Linear correlation analysis with qPCR showed R²values of 0.837 for BamHI-W gene and 0.763 for EBNA1 gene dPCR(both P<0.001).The BamHI-W gene copy number ranged from 3 to 18 copies per clinical sample,with patient-specific variations.There was a high consistency in viral load trends between the multicopy BamHI-W gene and single-copy EBNA1 gene d

关 键 词：爱泼斯坦巴尔病毒感染 数字聚合酶链反应 多拷贝基因 单拷贝基因 核酸检测 

分 类 号：R440[医药卫生—诊断学]