Development And Application Of A New Ultra Sensitive Assay Based On Droplet Digital PCR For The Quantification Of HBV-DNA In Peripheral Blood

ObjectiveEstablish a new ultra sensitive assay based on droplet digital PCR(ddPCR) for the quantification of HBV-DNA in peripheral blood, and assess its specificity and sensitivity. And further explore the application value of long-term monitoring and treatment in patients with chronic hepatitis B(CHB).MethodsHBV plasmid DNA serially diluted were detected by ddPCR amplification systems. By this way,the sensitivity and accuracy of ddPCR was evaluated.To compare the sensitivity and accuracy between ddPCR and qRT-PCR amplification system, HBV-DNA in patient samples were detected respectively by ddPCR and qRT-PCR.Multiple concentration gradient of HBV-DNA quality control serum were extacted by using QIAamp UltraSens Virus Kit, and then detected by ddPCR. Through the simulative test of HBV-DNA quality control serum,the sensitivity, accuracy and stability of the method were evaluated.While peripheral blood HBV-DNA below the lower limit of detection,patients with CHB under antiviral therapy were followed up by the above-stated method. By this way, the significance and value of practical application of the method were evaluated.ResultsThe results of ddPCR displayed a high goodness of fit(r=0.997,P=0.000). The ddPCR system detected as little as 1 copy of HBV-DNA plasmid input. The measured results indicated that the ddPCR system is a sensitive and accurate method for the detection of templates ranging from 1to 105 copies of HBV-DNA.A high degree of linearity and quantitative correlation(r=0.931,P=0.000) were showed between ddPCR and qRT-PCR. However, ddPCR assays generated more reproducible results and detected lower copy numbers than qPCR assays.In the test of quality control serum, there was a good correlation(r=0.993, P=0.000) and consistency between ddPCR and the theoretical value of quality control serum. We achieved a detection rate of 50% in the detection of the concentration of 0.93log10 IU/mL of quality control serum.And the detection rate of other concentration gradient of quality control serum were all 100%. These results showed good reproducibility and stability.In follow-up of patients, we found that some patients had been in low viral load status for long or short period of time in both HBeAg positive and negative group, and low viral load status reappeared in a few patients while negative conversion. However, routine virological detection failed to find them.ConclusionIn the detection of HBV-DNA, ddPCR showed good accuracy and sensitivity. A high degree of linearity and quantitative correlation wereshowed between ddPCR and qRT-PCR. However, ddPCR assays generated more reproducible results and detected lower copy numbers than qPCR assays. The method that detecting the concentrated HBV-DNA by dd PCR showed excellent accuracy, sensitivity and reproducibility. And the method could find low viral load status in the serum while routine virological detection failed to observe. Therefore, it can play an important role in the long-term monitoring and treatment of CHB.