ABSTRACT
<p><b>OBJECTIVE</b>To establish a sensitive and specific technique for detecting HBV DNA in serum using HBV DNA probe labeled directly by alkaline phosphatase (AlkPhos Direc probe).</p><p><b>METHODS</b>The 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.</p><p><b>RESULTS</b>The 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).</p><p><b>CONCLUSIONS</b>The 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.</p>
Subject(s)
Animals , Humans , Alkaline Phosphatase , Chemistry , Metabolism , DNA Probes , Chemistry , Genetics , DNA, Viral , Blood , Genetics , Hepatitis B , Blood , Diagnosis , Virology , Hepatitis B virus , Genetics , Molecular Diagnostic Techniques , Methods , Reference Standards , Polymerase Chain Reaction , Methods , Sensitivity and SpecificityABSTRACT
Objective we studied the effect of the Purine mucleoside Valaciclovir on anti-duck hepatitis virus(DHBV) in vivo to provide an experimental basis for clinical treatment of patients with hepatitisB.Methods The Chongqing duck hepatitis B virus model was treated with Valaciclovir once a day for a month at the doses of 50mg.kg－1、100mg.kg-1、200mg.kg－1of body weight per day. Serum DHBV DNA was detected four times in the course of the treatment,ALT and AST in serum and DHBV DNA in liver were detected simultaneously.Results Valaciclovir could signsificantly lower the serum DHBV DNA level. Serum ALT of several ducks in serum rose slightly during the treatment,but became normal after 1 week stopping Valaciclovir. Examination of DHBV DNA in liver with Southern Blot indicated Valaciclovir could inhibit DHBV DNA replication,but could not completely eliminate DHBV SC DNA.Conclusion The study confirms the safety and potent antihepaticviral activity of Valaciclovir in vivo.
ABSTRACT
Objective:To gain insight into the mechanism responsible for clearance of natural hepa DNA virus infections.Methods:A group of seven 2～3 month old ducks were infected intravenously with 10～20 ml DHBV positive serum containing 5?10 7 genomes/ml.Following inoculation,ducks were bled at weekly intervals to obtain serum samples for analysis of DHBV DNA and DHBsAg and anti DHBV antibodies.Peripheral blood mononuclear cells were collected at 10,35 days postinoculation(p.i) and used to conduct antigen specific blastogenesis assay.Liver samples were obtained at 5,30,60 days p.i for analysis of DHBV DNA and surface antigens and liver histology.Results:Infection of all 7 animals with approximately 5?10 8～1?10 9 DHBV genomes led to a transient viremia after an incubation period of 1 to 2 weeks.Liver samples contained multiple copies of all of the expected species of DHBV DNA replicative intermediates,including DHBV cccDNA during the peak of viremic phase.Further analysis showed that the absence of a prolonged viremia could be explained by immediate antigen specific blastogenesis and high titer of antibody response.Meanwhile,there was no obvious evidence of liver cell injury during transient DHBV infection.Conclusion:These results demonstrate that noncytopathic antiviral mechanisms make a role in hepa DNA virus clearance.