الفهرس 
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Abstract
Hepatitis B virus (HBV) is responsible for a large number of chronic infections worldwide and HBV is a major cause of hepatocellular carcinoma (HCC). Its chronic infection can lead to chronic liver inflammation and the accumulation of genetic alterations to result in the oncogenic transformation of hepatocytes. HBV control and eradication are considered one of the major public health challenges of the 21st century.
In general mutations incident to hepatitis B virus caused many symptoms of unusual and unexpected in regular. It is an important issue to follow HBV surface antigen because of false-negative results in some serology test was reported. So updating of serology Kit such as rapid test (surface antigen or core antigen cassette (depending on following up mutation of HBV must be continuing. Understanding the gap between serology screening (surface antigen) and nucleic acid test (NAT) may clarify the reason of different between results of immunoassays and (NAT).
Materials and Methods:]
1. Materials:
1.1 Samples collection:
from ninety thousand and five hundred blood samples were screened by campaigns for the company blood transfusion services (EgyBlood-VACSERA) during 2014, only two samples which diagnosed as positive NAT for HBV and negative ChLIA of HBsAg with no co-infection with other hepatitis viruses such as HCV or HIV had been aliquot and stored at -20°C until use.
Blood samples are usually collected in EDTA tubes. Then, samples were centrifuged and the plasma was transferred to a screw-cap cryo tube within 1 h of collection.
1.2 Primers:
A simple and efficient way for virus primer design was used based on the alignment of HBV genotype sequences published at gene banks database. One set of primers were selected for surface antigen. The selected primers had been tested to confirm that it will be suitable for all HBV genotypes with all genotype retraved from GenBank database.
2. Methods:
2.1 DNA Extraction:
DNA of HBV was extracted and Purified according to the instruction of viral DNA extraction kit protocol.
2.2. PCR amplification:
. PCR amplification was done using Taq PCR master Mix Kit. PCR reactions were done in 100μl mixture reaction, according to the instruction of the manufacturer.
2.3 Detection of PCR Product:
PCR products were detected by horizontal electrophoresis in 2% agarose gel stained with Ethidiume bromide. The agarose gel was visualized using ultra violate (UV) to detect PCR product comparing with positive and negative controls.
2.4 Recovery of PCR product from agarose gel:
After PCR products were separated by horizontal electrophoresis 2% agarose gel, PCR products were recovered from gel according to gel extraction kit protocol.
2.5 Cloning
When direct sequencing for amplified fragments were failed according to the protocol provided by the manufacturer of DNA sequence kit. The PCR products ≈1200 bp were cloned into pPCR -ScriptTM Amp SK+ vector (Stratagene, La Jolla, sCA, USA) according to manufacture protocol.