数字PCR在人巨细胞病毒核酸定量检测中的应用

Quantitative detection of human cytomegalovirus nucleic acid by droplet digital PCR

目的分析微滴式数字PCR(ddPCR)和实时荧光定量PCR(qPCR)检测人巨细胞病毒(HCMV)核酸的结果,比较两种方法的差异性,并评估ddPCR技术的临床有效性和实用性。方法收集306例疑似HCMV感染相关疾病患者血浆标本,采用ddPCR和qPCR两种方法测定同一份标本的HCMVDNA载量。结果疑似HCMV感染人群中,基于ddPCR的检测结果,男性和女性的阳性率差异无统计学意义(χ^(2)=1.07,P>0.05);不同年龄段的病例阳性率差异无统计学意义(χ^(2)=0.03,P>0.05);不同疾病类型的样本阳性率差异有统计学意义(χ^(2)=10.38,P<0.05)。采用ddPCR方法检测的阳性率为46.41%(120/306),采用qPCR方法检测的阳性率为22.86%(70/306),阳性率差异有统计学意义(χ^(2)=103.80,P<0.05)。受试者工作特征曲线(ROC)分析显示,ddPCR检测的灵敏度为0.96,特异性为0.97,ROC曲线下面积(Area Under Curve,AUC)为0.98。结论采用ddPCR检测HCMVDNA比qPCR具有更好的可定量性,尤其是在低浓度样本检测中更具优势,可以进一步作为临床诊断的直接方法,对现有qPCR检测体系做出有益补充和升级。

Objective To analyze the results of droplet digital PCR(ddPCR)and quantitative real-time(qPCR)for detection of human cytomegalovirus(HCMV)nucleic acid,to compare the differences be-tween the two methods,and to evaluate the clinical effectiveness and utility of ddPCR.Methods Plasma samples were collected from 306 patients with suspected HCMV infection-related diseases.The HCMV DNA load of each sample was determined using both ddPCR and qPCR technology.Results There was no signifi-cant difference in positive rates between males and females based on ddPCR assays in the population with sus-pected HCMV infection(χ^(2)=1.07,P>0.05),nor among cases of different ages(χ^(2)=0.03,P>0.05).However,the difference in positive rates between samples with different disease types was statistically significant(χ^(2)=10.38,P<0.05).In the HCMV infected population,46.41%of the samples tested by the ddPCR method and 22.86%of the samples tested by the qPCR method were positive(χ^(2)=103.80,P<0.05).Receiver operating characteristic curve(ROC)analysis showed that the ddPCR assay had a sensitivity of 0.96,a specificity of 0.97,and an area under the curve(AUC)of 0.98.Conclusion The use of ddPCR to detect HCMV DNA has better quantifiability than qPCR,especially for the detection of low concentration samples.It can also beused as a direct method for clinical diagnosis,which will be beneficial in supplementing and upgrading the existing qPCR assay system.