BACKGROUND & AIMS:
Hepatitis delt virus (HDV) far exceeds our expected level, there remains a lack of reliable quantitative assays for HDV RNA detection. We sought to develop a new method based on digital droplet PCR (ddPCR) for HDV quantitative detection.
With plasmid (pMD19T) containing HDV full-genome, we determined the method for ddPCR-based HDV RNA quantification. To compare various assays for HDV detection, 30 cases diagnosed hepatitis D and 14 controls were examined by ELISA, RT-PCR and ddPCR. 728 HBV-related patients including 182 chronic hepatitis B (CHB), 182 liver cirrhosis (LC), 182 hepatocellular carcinoma (HCC) and 182 liver failure (LF) were screened for HDV infection.
The detection limit of ddPCR for HDV is significantly low, which lower limit of detection (LLoD) and lower limit of quantitation (LLoQ) to be 0.29 IU/ml (95%CI: 1.93*10-3 IU/ml—1.22 IU/ml) and 8.76 IU/ml (95%CI: 1.83 IU/ml -1.03*106 IU/ml), respectively. Among the 44 samples, ELISA detected 30 cases positive, ddPCR reported 24 samples and RT-PCR reported 10 samples positive for HDV RNA. Moreover, the positive rates of anti-HDV were 1.1%, 3.3%, 2.7% and 7.1% in patients with CHB, LC, HCC, and LF; the detection rates of RT-PCR in HDV RNA were 0%, 16.67%, 15.4% and 20%, however, the detection rates of ddPCR were 0%, 33.33%, 30.77% and 60%.
We establish a high sensitivity and specificity quantitative HDV RNA detection method based on ddPCR. HBV-related end-stage liver disease, especially liver failure, are associated with a remarkably high rate of HDV infection.