الفهرس 
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Abstract
Summary
Hepatitis B infection is considered one of the most important global viral infections. It affects about 240 million people worldwide, especially the few and middle-income countries. And Egypt is at the forefront of countries that suffer from the spread of infection with this virus.
1. Sample collection:
Ninety-five positive serum samples for the hepatitis B virus were collected from different Egyptian governorates (Cairo, Qalyubia and Giza). All patients were diagnosed HBV infection with no co-infection with other hepatitis viruses such as HCV, HDV, or HIV. All samples had been aliquot and stored at -20°C until use
2. DNA Extraction:
DNA of HBV was extracted and Purified according to the instruction of viral DNA extraction kit protocol.
3. Amplification of target genes of HBV (surface antigen):
The Pre-S region amplified using polymerase chain reaction (PCR) test by primer for gene.
4. Detection of PCR Product:
PCR products were detected by electrophoresis in 1% agarose gel stained with Ethidium bromide. The agarose gel was visualized using UV Transilluminator to detect PCR product comparing with positive and negative controls.
5. Recovery of PCR product from agarose gel:
After PCR products were separated by 1% agarose gel electrophoresis, PCR products were recovered from gel according to gel extraction kit protocol.
6. Hepatitis B surface antigen region Sequencing:
Amplified HBV DNA fragments were sequenced directly using the ABI Prism Big Dye Terminator V.3.1 Cycle sequencing Kit on an ABI 310 DNA automated sequencer (Applied Biosystems).
7. Removal of unincorporated:
DyeEx purification Kits had been used to remove all unincorporated dye terminator directly from sequencing reaction. The steps of removing the residual of dye were carried out.
8. Sequencing results and Bioinformatics analysis:
8.1. Preparing and Loading Samples for sequencing:
Sequencing had been carried out at Genetic Engineering Research Department (VACSERA). Electrophoresis process was performed on ABI Prism 310 Genetic Analyzer, by usingABI Prism 310 data base collection.
8.2. Sequencing assembly and Phylogenetic tree:
The sequence results generated by the forward and reverse sequencing primers were analyzed with the software program sequencing analysis 5.3.1. The six sequence of HBV isolates we are regestrited in Gen Bank under accession numbers (MT004774, MT004773, MT004772, MT004771, MT004770 and MT004769). For sequence comparisons of Large s of the sixHBV isolates retrieved from Gen Bank, sequence alignment was performed using the multiple-alignment algorithm in Misalign (DNASTAR, Window version 3.12e).
Results can be summarized in the following
We can finally summarize the results as follows, from 135 samples only Ninety-five samples had a positive to HBV, we examined six of them. The structure of HBsAg genome were determined by PCR product that amplified with specific primer for HBV.samples which diagnosed were three mutated viruses were diagnosed in the surface gene of the hepatitis B virus, where the mutated viruses were isolated from patients when analyzing blood samples taken from them and the results gave their positives to the surface antigen gene.
Then the mutated viruses were deposited into the hepatitis B virus, genetically type D, to confirm that the genetic type of the hepatitis B virus, the most common in Egypt, is the genotype D. A sequence of gene essences was also made, which was also attributed to the genetic type D.
This finding will allow the introduction of a new vaccine for HBV or help the emergence of a new diagnostic kit for HBV and to Control of these HBV mutants, who will require new drugs, vaccines, and treatment strategies, will become the next major challenge on the path to eventual elimination of HBV infection.