| Hepatitis B is caused by the pathogens of Hepatitis B virus (HBV) which belongs to hepadnaviridae. The HBV genome is a double-strand with a closed circular DNA and its molecular weight is 3.2kb. The RNA is transcripted from the negative strand DNA nucleotide containing four ORF called, S, C, P, X. Hepatitis B is an infectious disease that spreads worldwide. China is a high-prevalence area having the control situation serious. Hepatitis B virus is transmissed by the blood, sex and mother-to-infant breeding. Early diagnosis can lessen victim with hepatitis B virus number and enhance the cured probability, reducing cross-infection and dissemination. It is of great significance in prevention and treatment. At present, the detection of HBV virus can be divided into antigen detection and DNA tests. Antigen test is a main detection method. DNA detection is to detect the existence or the number of pathogens in vivo for diagnosis. The clinical applications include viral load detection. Detection of HBV DNA is important in hepatitis B prevention and treatment.Real-time fluorescence quantitative PCR technology is an approach with fluorescence added in the reaction and the accumulative signal monitoring whole process, which can analyze the unknown template from a standard curve. It has accurate characteristics, high sensitivity, fast speed and good reproducibility. There is no need to have electrophoresis after PCR. Since the inventions of this technology, it is widely used in DNA or RNA absolute quantitative analysis, analysis of gene expression and tumor gene detection etc. This technology also can be used as assessment before and after treatment by monitoring, clinical examination and drug treatment effect. The primes were designed at conservative areas of HBV pre-C, C and X region. These target sequences were amplified from pBR322 plasmid containing three HBV genomes and.then cloned into pBS-T vector which had the similar size with HBV through AT clone strategy. The fragment sizes of pre-C, C and X respectively were 113bp,101bp and 57bp. The primary identification process was blue and white maculation screening and colony PCR. And the sequencer confirmed the final identification. BLAST analysis indicated that we had obtained the desired subclones successfully. The copy numbers of new clones are higher than those of the original plasmid. So it solved the problem about the source of standard plasmid. The pre-C region had the highest sensitivity. This conclusion obtained from the optimization on annealing temperature and sensitivity by convention PCR with pre-C, C and X region. To establish the FQ-PCR reaction system with TaqMan probe and to make the standard curve, it took the pBS-T plasmid of different concentration containing pre-C conventional sequence as a template. The standard curve were generated after the reaction. The correlation coefficient of standard curve was 0.995 which revealed the high reliability. The linear ranged from 5×10~3 copies/ul to 5×10~9 copies/ul. The coefficient of variation were less than 5% in the same sample and in the different sample which indicated its precision high and repeatability good. The follow-up study will be clinical application.
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