The Establishment And Application Of A Nanotechnology Method For Detection Of Hepatitis B Virus Gene Mutation

Objective: 1.To establish a QDs-mediated fluorescent method for the detection of hepatitis B virus(HBV)gene mutation(M204I mutation)based on nanotechnology research.2.To evaluate the significance of this method in detecting M204 I mutation in chronic hepatitis B patients with poor response to nucleos(t)ide analogues(NAs).Materials and methods: The serum samples with the viral load of 107 IU/m L were diluted to different concentrations of HBV DNA as the serum standard(theoretical value).The experiment was divided into three groups,and the serum samples were detected by three different methods: magnetic nanobeads method(reagent 1),magnetic bead method(reagent 2)and boiling method(reagent 3)respectively.The results were compared with the standard above.To investigate the consistency of magnetic nanobeads method for quantitative detection of HBV DNA,quantitative linear relationship and reproducibility.Primers were designed for the mutant M204 I in the HBV polymerase gene.And then detection of HBV M204 I mutation by quantum dot labeling fluorescent probe hybridization.To evaluate the efficiency of the method by compared with the direct sequencing.And the application of this method in the detection of HBV M204 I mutation in CHB patients.Results: Correlation of results showed high consistent between test and theoretical values with r value of 0.986(reagent 1,P<0.001),0.950(reagent 2,p=0.001)and 0.979(reagent 3,P<0.001)respectively,and the difference was statistically significant.Consistency--the theoretical values for the lower level detection of HBV DNA is 101 IU/ml,and the extraction results are 3.520×101 IU/ml by reagent 1,9.123×103 IU/ml by reagent 2,6.195×101 IU/ml by reagent 3.Reproducibility--the average relative deviation of the three methods are 0.243±0.405(reagent 1),1.189±0.855(reagent 2),-0.439±0.618(reagent 3)respectively,the average relative deviation of reagent 1 at different times is 0.505±0.659.The limit of the method for detection of hepatitis B virus genetic variation by fluorescence quantum dots is 103 IU/ml,and the higher the viral load,the more intensive the light spots.The HBV M204 I mutation was detected by the new method in 28 patients with chronic hepatitis B with poor response to the nucleoside(s)treatment.The results of the serum samples between the new sequencing method and the direct sequencing method were consistent.Conclusion: The serum HBV DNA extraction combined with quantum dot system are convenient and reproducible,providing a new technology for the quantitative detection of HBV DNA.Quantum dot can be used as fluorescent probes to detect viral HBV DNA polymerase gene variants.This QDs-mediated fluorescent method provides an attractive platform for detection of Hepatitis B Virus M204 I mutation.And HBV DNA polymerase mutations may be one of the causes of poor response in CHB patients with the nucleoside(s)treatment.