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.
METHODS:
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.
RESULTS:
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.
