Comparison of branched DNA and real-time polymerase chain reaction methods in quantitation of hepatitis B virus DNA.

H B virus (HBV) is a major causative agent of chronic hepatitis, and can cause liver cirrhosis and hepatocellular carcinoma. Serological assays are not sufficient for the diagnosis of HBV infection. Measurement of HBV DNA levels are routinely used to identify infectious chronic carriers, and to predict and monitor the efficacies of antiviral treatment regiments.1 A number of commercial assays are currently available for the quantification of HBV DNA in patient serum or ethylenediamine tetraacetic acid-plasma, including hybridization, signal, and target amplification based technologies.2,3 The QuantiplexTM HBV DNA branched DNA assay (Versant 3.0, Bayer, Germany) is a signal amplification assay based on branched DNA (bDNA) technology.3 Real-time polymerase chain reaction (PCR) quantitation is based on the evaluation of the threshold cycle (CT) when amplification of a PCR product is first detected. The higher the starting copy number, the sooner amplified product is detected.4 Our aim in this study was to compare bDNA (Versant 3.0, Bayer, Germany) and real-time PCR (Fluorion Iontek, Turkey) assays for quantitation of HBV DNA in sera of patients. The study was carried out in Erciyes University Medical Faculty, Department of Microbiology, Laboratory of Virology, Erciyes, Turkey in May 2005. Blood samples collected from 220 chronic hepatitis B patients were included in the study. The sera of the patients were kept at -20oC until the study. All of the sera were from Turkish patients. The HBV DNA in sera was investigated by the bDNA assay according to the manufacturer’s instructions. The quantification range of the bDNA assay was 2 x 103-108 copy/mL. The results greater than 2 x 103 copy/mL were accepted as positive. If there was no virus detection, it was reported as <2 x 103 copy/mL. The results greater than 108 copy/mL were reported as >108 copy/mL. The HBV DNA in sera was investigated by the real-time PCR assay according to the manufacturer’s instructions. Viral DNA was extracted from 200 μL of serum using the QIAmp MinElute kit (Qiagen, Germany). Reaction mixture was prepared by Fluorion HBV QNP (Quantification probe) Version 2.0 (Iontek, Turkey). Five microliters of extracted DNA were added to the plate containing 20 μL of the reaction mixture. The amplification profile was performed as follows: 30 minutes at 95oC, 30 seconds at 95oC, 90 minutes at 54oC with 50 cycles, and 10 seconds at 22oC. Amplification and detection were performed by ICycler detection system (Biorad, USA). The quantification range of real time PCR was 103-107 copy/mL. The analytical detection limit for real-time PCR was 200 copy/mL. No amplification results were reported as <200 copy/mL. The results between 200-1000 copy/mL were reported as <1000 copy/mL. The results greater than 107 copy/mL were reported as >107 copy/mL. Pearson’s correlation coefficient was used to assess the strength of the linear association between the log-transformed values of real-time PCR and bDNA assay. A 2-tailed p value of less than 0.05 was considered to indicate statistical significances. Furthermore, consistency of the differences between the log10 quantitative values obtained from Versant 3.0, and real-time PCR was assessed according to the analysis as proposed by Bland and Altman.5 The HBV DNA was investigated by real-time PCR and bDNA assay in 220 sera. The results below or above the detection limit of each assay were excluded from calculation. Seventy-one samples (32.3%) were detectable by both real-time PCR and bDNA tests within their quantitation ranges. When quantitative results were compared, significant correlation was Brief Communication