Developing a Real-Time PCR assay to determine the Hepatitis B virus DNA in serum

Hepatitis B virus is an important infectious agent, infected two billion people worldwide and caused chronic infection in 400 millions. Turkey is one of the modereately endemic countries with an average HBsAg positivity rate of 5% (differs between 1 and 14,3 % according to the region).Virologic diagnosis and follow-up of HBV infection is made by the detection of specific antigens, antibodies and HBV DNA. Presence of HBV DNA in blood is a reliable indicator for active HBV infection and is an important marker for staging and managing the infection and following the sucess of the treatment. HBV DNA can be determined 3-5 weeks earlier than HBsAg in blood, therefore is useful for the early diagnosis and currently in use for screening of the donors in blood banksNucleic acid amplification assays using PCR method are frequently used for HBV DNA determination. There are some limitations of conventional qualitative PCR assays, such as high sensitivity, low reproducibility and high carryover contamination rates. Real-time PCR method has a lower contamination risk compared to classical PCR since the detection is performed during amplification. In addition, quantitative results of real time PCR is more accurate, because the quantification is made according to the number of the cycle in which the signal crosses the threshold (cycle threshold, ct) by measuring the probe signal in every cyclus during amplification. Therefore, real-time PCR has a wider dynamic range of quantification. These advantages make the real-time PCR method ideal tool for HBV DNA monitoring which fluctates over a wide dynamic range (> 9 logarithma).The aim of this study was to develop a quantitative real-time PCR assay for HBV DNA that can be used for routine patient management, with a performance similar to commercial assay but with a lower cost. The new assay is expected to have an internal control and to detect all six genotypes of HBV.An enveloped, cell cultured DNA virus, bovine herpes virus type 1 (BHV-1), which does not infect human, used as an internal control. Internal control added to the patient sample before the extraction in order to monitor the problems of both extraction and amplification steps.Plasmid standart carrying the amplicon was generated and transfected into Escherichiae coli. Absolute quantitation method was used by drawing a standart curve with five plasmid diluents in each run.Dynamic range of the developed HBV DNA assay was between 5x101 and 5x1010 IU/ml and the limit of detection was 48 IU/ml (between 32 and 129 IU/ml). These parameters were suitable for diagnosis and monitoring of the HBV infection.Reproducibility of the assay is important in order to determine the reliable differences in HBV DNA levels during the follow-up of the patients. Intra- and interassay CV% values of the developed assay were 0.7- 4.1% and 0.3 - 1.9%, respectively. These values were comparable to commercial assays.The developed assay was able to detect all the samples of a HBV genotyping panel, containing genotype A, B, C, D and G. However, quantitative results of the samples containing genotype B and C were approximately 2 log10 below the expected value. The cause of this variation could not be determined since primers and probes were designed to detect all the genotypes. It is unlikely that this quantitation problem of the developed assay will cause a major problem in the routine patient service, since D is the only genotype detected in Turkey to date.The accuracy of the new assay was tested with the QCMD - 2007 HBV DNA proficiency panel. Results were correlated with expected results (r2=0.92).The correlation of the developed assay and the commercial test used in the routine laboratory was evaluated by 180 HBV DNA positive and 80 HBV DNA negative plasma samples. The sensitivity and specificity were 99% and 94%, respectively, when the commercial assay was accepted as the gold standart. Quantitative results of the samples detected by two assays (178 samples) showed a strong and significant correlation (r=0.86, p=0.000, R2=0.74).No false positivity was detected by the new assay, when 26 HCV-RNA positive, HBsAg-negative samples were tested.Cost analysis showed that the new assay was 45 TL/test cheaper than the commercial assay. If the annual HBV DNA test number (approximately 1500) taken into account, the benefit would be 67500 TL/year, if the developed assay is used for the routine diagnostics.As a result, we developed a quantitative real-time PCR assay for HBV DNA. Sensitivity, specificity, reproducibility and accuracy of test results and cost benefit showed that it is convenient for routine use.