ddPCR Facilitates Highly Sensitive, Accurate Detection of Hepatitis B
More than 20 billion people worldwide have been infected with hepatitis B virus (HBV), and HBV is a common cause of liver diseases and liver cancers. The fatality of these diseases as well as their relation to hepatitis infection is well-established; 600,000 HBV-related deaths are estimated to occur every year and 73% of liver cancer deaths worldwide are due to hepatitis viruses, with a higher prevalence in developing countries.
Currently, clinical diagnosis of HBV infection is typically performed using serological tests and quantitative real-time PCR. However, false-negative results are common with these methods because of HBV surface antigen (HBsAg) variation and low HBV copy number.
In particular, the precision limitations of qPCR have prevented distinguishing between small differences in copy number among samples, especially in cases of low copy number. It is difficult to accurately justify the results of qPCR when the quantification cycle (Cq) value of an unknown sample is near the cutoff.
“The development of HCC is strongly associated with HBV. Recently, several new antiviral strategies targeting cccDNA have been established to improve HBV clearance. It is of great clinical significance to provide an accurate and sensitive approach for closed circular DNA (cccDNA) detection. With this method, more and more patients with chronic HBV will have precision treatment available to prevent or delay HCC occurrence, and HCC in patients could be diagnosed at an earlier stage,” explained lead investigator Song-Mei Liu, MD, PhD, of the Center for Gene Diagnosis, Zhongnan Hospital of Wuhan University, Wuhan, China.
For this assay, the team has combined droplet digital PCR (ddPCR) with PCR enabling them to detect cccDNA in several sample types with higher sensitivity than the traditional Southern blotting method, which has more difficulty when detecting low copy numbers of cccDNA.
ddPCR was useful to identify which patients might be harboring hepatocellular carcinoma. Researchers found that almost 90 percent of 68 HCC patients were cccDNA-positive compared to 53 percent of 79 non-HCC patients.
Serum cccDNA copy number was found to be higher in HCC patients compared to non-HCC patients. Combined analysis of serum cccDNA and HBV-DNA distinguished HCC patients from non-HCC patients. “This implicates cccDNA as a risk factor for HCC,” said Dr. Liu.
“Serum cccDNA is indeed a much better and useful diagnostic marker than intrahepatic cccDNA,” said Dr. Liu.
Recently, several new antiviral strategies targeting cccDNA have been found to improve HBV clearance. Therefore, providing an accurate and sensitive approach for cccDNA detection is of great clinical significance for earlier diagnosis and HCC prediction.