Objective:To evaluated a new fluorescent quantitative PCR method for HBV cccDNA.Methods:The specimens were obtained by hepatic puncturation.We designed a pair of specific primers to amplify not HBV genome but cccDNA as a method to detect it,using the ATP dependent Dnase to increase the specificity greatly.Results:We have established a new fluorescent quantitative PCR method for HBV cccDNA successfully,the linear range is from 2.5?101to 2.69?109copies per milliliter.The three of five patients were shown HBV positive and the other two were negative using the new method to analyze hepatic puncturation specimens.The HBV positive patients have all received one year long anti-virus therapy.1) the two HBV negative patients were both cccDNA negative and tDNA negative.2) the patient who has received one year long Lamivudine therapy had a DNA concentration of 7.10?103copies tDNA per milliliter serum and 2.64?103copies cccDNA per milliliter serum.The patient who has received one year long entecavir therapy had a DNA concentration of 1.0?105copies tDNA per milliliter serum and 4.04?103copies cccDNA per milliliter serum in his blood.tDNA concentration in the third patient was 6.62?103copies per milliliter serum,3.3?102copies cccDNA per milliliter serum before anti-virus therapy,but after one year long Lamivudine therapy,the tDNA and cccDNA concentration reduced to 5.19?103copies and 1.51?102copies per milliliter serum respectively.Conclusion:This study shown that the fluorescent quantitative PCR had a good linear range,high sensitivity,specificity and accuracy,so it needs less specimen,for example 100 milligram hepatic puncturation specimen is enough.Although further study is needed,the fluorescent quantitative PCR will definitely become an efficient tool for anti-virus therapeutic effect monitoring because of its high sensitivity,specificity,efficiency and accuracy.

OBJECTIVETo establish a sensitive and specific technique for detecting HBV DNA in serum using HBV DNA probe labeled directly by alkaline phosphatase (AlkPhos Direc probe).

METHODSThe probe that purified HBV DNA sequence was labeled directly by alkaline phosphatase and chemiluminescent substrate CDP-star for AP was used in the hybridization assay. HBV DNA was detected by autoradiography on the film. The test compared the chemiluminescen dot blot hybridization assay for 80 samples with digoxigenin-labeled HBV DNA probe detective method. The correlation of 70 samples test results between fluorescent quantitative HBV DNA PCR method and dot blot hybridization assay by AlkPhos Direc probe was analysed.

RESULTSThe sensitivity of the probe labeled directly by alkaline phosphatase was 10pg at least. The coincidence was 100% compared with digoxigenin-labeled HBV DNA probe detection. A correlation coefficient of HBV DNA quantitative results between fluorescent quantitative HBV DNA PCR (QPCR) method and dot blot hybridization assay by AlkPhos Direc probe was 0.98 (P<0.01).

CONCLUSIONSThe method detecting HBV DNA in serum by HBV DNA AlkPhos Direc probe is sensitive and specific. The results between two methods with AlkPhos Direc and digoxigenin-labeled HBV DNA probe are coincident completely. The correlation of HBV DNA quantitative results between fluorescent QPCR method and dot blot hybridization assay by AlkPhos Direc probe is satisfactory.

Alkaline Phosphatase ; chemistry ; metabolism ; Animals ; DNA Probes ; chemistry ; genetics ; DNA, Viral ; blood ; genetics ; Hepatitis B ; blood ; diagnosis ; virology ; Hepatitis B virus ; genetics ; Humans ; Molecular Diagnostic Techniques ; methods ; standards ; Polymerase Chain Reaction ; methods ; Sensitivity and Specificity