Establishment And Application Of A Fluorescent Quantitative PCR Method For CccDNA Of HBV

Hepatitis B virus infected 350 million people worldwide, most havechronic intection, and around 5 percent will develop to hepatic cirrhosis orcancer as a final result, which leads to 1.5 million deaths per year.Moreover the rate of infected people is higher in China, around 10 to 20percent. Most anti-virus therapy can decrease the virus concentrationunder the lower detective limit successfully, but it can not eliminate thecccDNA which is the duplicate template of HBV offspring. The detectionof the anti-virus drug's effect to cccDNA has not been widely used.Objective:To evaluated a new fluorescent quantitative PCR method forHBV cccDNA. Method:The specimens were obtained by hepaticpuncturation. We designed a pair of specific primers to amplify not HBVgenome but cccDNA as a method to detect it, using the ATP dependentDnase to increase the specificity greatly. Results: We have establisheda new fluorescent quantitative PCR method for HBV cccDNA successfully,the linear range is from 2.5×101 to 2.69×109 copies per milliliter. Thethree of five patients were shown HBV positive and the other two werenegative using the new method to analyze hepatic puncturation specimens.All of the HBV positive patients have received one year long anti-virustherapy. 1) The two HBV negative patients were both cccDNA negativeand tDNA negative. 2) the patient who has received one year longLamivudine therapy had a DNA concentration of 7.10×103 copies tDNAper milliliter serum and 2.64×103 copies cccDNA per milliliter serum.The patient who has received one year long entecavir therapy had a DNAconcentration of 1.0×105 copies tDNA per milliliter serum and 4.04×103copies cccDNA per milliliter serum in his blood. tDNA concentration inthe third patient was 6.62×103 copies per milliliter serum, 3.3×102copies cccDNA per milliliter serum before anti-virus therapy, but after oneyear long Lamivudine therapy, the tDNA and cccDNA concentrationreduced to 5.19×103 copies and 1.51×102 copies per milliliter serumrespectively.Conclusion: This study shown that the fluorescent quantitative PCR had agood linear range, high sensitivity, specificity and accuracy, so it needsfewer specimens, for example 100 milligram hepatic puncturationspecimen is enough. Although further study is needed, the fluorescentquantitative PCR will definitely become an efficient tool for anti-virustherapeutic effect monitoring because of its high sensitivity, specificity,efficiency and accuracy.