Comparison of real-time polymerase chain reaction with the COBAS Amplicor test for quantitation of hepatitis B virus DNA in serum samples

Abstract

INTRODUCTIONIt is estimated that 350 million individuals are chronically infected with hepatitis B virus (HBV) worldwide, and about one-third of these live in China [1] . HBV infection can cause chronic hepatitis, cirrhosis and hepatocellular carcinoma [2] . Among patients with active viral replication, cirrhosis will develop in 15%-20% within 5 year [3] , and 70-90 percent of cases of hepatocellular carcinoma occur against a background of cirrhosis [4] . Quantitation of HBV DNA in serum or plasma has been an important tool in identifying individuals with active hepatitis B, to monitor the efficiency of antiviral treatment, and to predict whether the treatment will be successful [5][6][7][8][9] .Several commercial molecular assays have been developed for quantitation of HBV DNA in serum or plasma samples. One of these assays is the COBAS Amplicor HBV Monitor, which is based on the amplification of DNA targets by the use of HBV-specific primers. Other methods, such as the VERSANT HBV DNA 3.0 assay, which is based on branched-DNA signal amplification, and Hybrid Capture Ⅱ, which is based on hybridization of a chemiluminescent probe, are also routinely used in diagnostic laboratories [10,11] . However, the costs of these assays are too high to be used in developing countries such as China, and in these countries the HBV burden is much heavier than in developed countries.Recently, quantitative real-time PCR has been used to detect and quantify HBV levels in serum or plasma samples. This assay is based on the relationship between the initial DNA target levels and the threshold cycle (Ct) of the amplification products. The Ct value is smaller when the initial DNA target level is higher. Several evaluation studies have shown that real-time PCR has a higher sensitivity, a broader dynamic range, and an accurate quantitation of HBV DNA compared to those of the existing commercial methods [12][13][14][15] .The Fosun real-time PCR HBV kit is a commercial METHODS:The reference sera of the Chinese National Institute for the Control of Pharmaceutical and Biological Products and the National Center for Clinical Laboratories of China, and 158 clinical serum samples were used in this study. The linearity, accuracy, reproducibility, assay time, and costs of the real-time PCR were evaluated and compared with those of the Cobas Amplicor test. RESULTS:The intra-assay and inter-assay variations of the real-time PCR ranged from 0.3% to 3.8% and 1.4% to 8.1%, respectively. The HBV DNA levels measured by the real-time PCR correlated very well with those obtained with the COBAS Amplicor test (r = 0.948). The real-time PCR HBV DNA kit was much cheaper and had a wider dynamic range. CONCLUSION:The real-time PCR assay is an excellent tool for monitoring of HBV DNA levels in patients with chronic hepatitis B.

Usefulness of in-house PCR methods for hepatitis B virus DNA detection

“…A Cobas Amplicor instrument was used for PCR amplification, hybridization, and detection. According to the manufacturer, the Cobas Amplicor HBV Monitor test shows linearity from 2.0 × 10 2 (lower detection limit) to 2.0 × 10 5 HBV DNA copies/mL (Konnick et al, 2005;Shi et al, 2008). Samples with >200,000 HBV DNA copies/mL in the Amplicor assay were diluted 1:900, 1:27,000, or 1:810,000, as necessary, in HBV DNA-negative normal human serum (NHS), and the diluted material was re-extracted, amplified, and analyzed to obtain a result within the analytical measurement range of the Amplicor assay.…”

“…These assays are based on the amplification of DNA targets using HBV-specific primers (COBAS Amplicor HBV Monitor); branched-DNA signal amplification, such as the VERSANT HBV DNA 3.0 assay; the hybridization of a chemiluminescent probe as hybrid capture; or a specific fluorescent probe in real-time PCR (Abbott Real-time HBV or Cobas TaqMan HBV test) (Ciotti et al, 2008;Morris et al, 2013;Shi et al, 2008;Yao et al, 2004;Yuan et al, 2004). In Brazil, HBV DNA testing is commonly conducted to monitor HBV treatment, but commercial assays are generally used.…”

Variations in the S and P Regions of the Hepatitis B Virus Genome Under ImmunosuppressionIn VitroandIn Vivo

“…The automatic fluorescence quantitative flow cytometry (PerkinElmer), and RT-6000 automatic microplate reader (Bio-Tek) were used for analysis. This kit was approved by the State Food and Drug Administration of China for in vitro diagnosis with a low detection limit (21).…”

Simultaneous detection of hepatitis B virus genotypes and mutations associated with resistance to lamivudine, adefovir, and telbivudine by the polymerase chain reaction-ligase detection reaction

“…The kit has been approved by the Chinese Food and Drug Administration for in vitro diagnosis and has a lower limit of detection of 84 IU/mL. 25 …”