基于微滴式数字PCR技术对乙型肝炎低病毒血症患者血浆HBV DNA精准检测

Accurate detection of plasma HBV DNA in patients with low hepatitis B viral load based on droplet digital PCR

目的建立基于微滴式数字PCR(droplet digital PCR,ddPCR)技术检测HBV DNA的方法,并实现对乙型肝炎低病毒血症患者HBV DNA的精准检测。方法构建pUC57-HBV质粒作为阳性对照质粒,设计合成HBV DNA ddPCR引物及探针,以梯度稀释的阳性对照质粒为模板进行ddPCR检测,完成标准曲线构建和检出限分析,并进行检测的重复性和特异性分析。收集首都医科大学附属北京佑安医院19例乙型肝炎低病毒血症患者的血浆样本(HBV DNA<10 IU/mL),提取HBV DNA后进行ddPCR检测。结果建立的基于ddPCR技术检测HBV DNA的方法检测下限低至1 copy/μL(阳性质粒稀释法);对3种不同拷贝的阳性对照质粒(1×10^(2)、1×10^(1)、1×10^(0) copies/μL)重复检测的变异系数分别为18.8%、9.9%、9.7%,且具有100%特异性;应用该方法在低于临床检测下限的乙型肝炎低病毒血症患者中,以1 copy/μL为临界值,HBV DNA的阳性检出率为68.42%。结论建立的ddPCR检测低病毒载量HBV DNA检测方法能够高灵敏和高特异地对乙型肝炎低病毒血症患者HBV DNA实现绝对定量。

Objective To establish a method for detecting hepatitis B virus(HBV)DNA based on droplet digital PCR(ddPCR),and achieve the accurate detection of HBV DNA in patients with low hepatitis B viral load.Methods The pUC57-HBV plasmid was constructed as the positive control plasmid,and the primers and probes of HBV DNA ddPCR were designed and synthesized.The positive control plasmid diluted gradiently was used as the template for the detection of ddPCR to construct the standard curve and determine the detection limit.The repeatability and specificity of the established method were analyzed.Last,plasma samples were collected from 19 patients with low hepatitis B viral load(HBV DNA<10 IU/mL),and the extracted HBV DNA was detected by the established ddPCR.Results The detection limit of the established ddPCR for detecting HBV DNA was as low as 1 copy/μL.The coefficients of variation of repeated detection for three different copies of positive control plasmids,including 1×10^(2),1×10^(1) and 1×10^(0) copies/μL,were 18.8%,9.9%and 9.7%,respectively,with 100%of specificity.When 1 copy/μL was taken as the cut-off value,the positive detection rate of HBV DNA by the established method in the patients with hepatitis B viral load below the clinical detection limit was 68.42%.Conclusion The established ddPCR method for accurately detecting HBV DNA with low viral load can achieve the absolute quantification of HBV DNA in patients with low hepatitis B viral load with high sensitivity and specificity.