Designation, Preparation And Clinical Application Of HBV Specific DNA Microarrays

IntroductionHepatitis B virus(HBV)infection is a major cause of liver disease around the world,especially in China.HBV genome can be divided into several subtypes.According to the nucleotides sequence,HBV has been classified into 8 genotypes(A-H).The genotypes of HBV are correlated with HBV clinical syndromes and therapeutic efficacy,so the genotyping of HBV had become more and more important.Currently there were some diagnostic methods of genotype,including the complete genome sequence analysis or S gene sequencing,PCR-RFLP and so on.But they have the disadvantages of a high cost and time-consuming to carry out or the result lack of reliability,and they haven't the ability to detect many.targets simultaneously.Recently,as the progress of functional genomic techniques,microarray has been developed into an ideal tool for genotyping.In this study we want to establish a integrated method genechip design for HBV genotyping,and do some test and analysis about the specificity and repetition of the chips and explore its clinical application,And,to investigate the distribution of hepatitis B virus genotypes in Hangzhou area.Methods1.PCR primer and probe design of HBV genome.HBV genotypes were found in NCBI's Genbank,and the results were alignmented with MegAlign and their conservative and specific sequences were determined.Primer Primer 5.0 was used to design genotype probes and PCR primers,the reliability of probes was analyzed.2.Microarray fabricationThe oligonucleotide probes were stablized on the chip and each one spotted at a layout of 3×1 blocks to avoid possible hybridization failures.Six blocks of the HBV-Con probes targeting the conserved region of Pre-s monitor the PCR amplification,hybridization reactions and so on3.Microarray hybridization and detectionAfter DNA samples were obtained and amplified with PCR,the HBV PCR products were hybridized with oligonucleotide probes.The hybridized results were colorized by BCIP/NBT.According to the hybridization signal and the corresponding probe sequence,HBV genotype was determined.4.Veracity and specification of HybridizationRandom positive PCR products were sent for sequence analysis,and analysis results were compared with hybridization to test the veracity and specification of the genechips.5.The repetition of genechips test.20 samples,which HBV-DNA was positive,were tested and 3 repeat tests were done for each samples and the results were analyzed.Results1.PCR primer and chip probe designWe have found 17 HBV genotypes genome in Genbank,including7 genotypes:A,B,C,D,E,F and G.Phylogenetic tree and the evolutionary relationships were analyzed.PCR primers and 7 probes were designed according to the difference of the sequences and the conservative consequences at HBV pre-S gene region.The product of PCR was 340bp.Blasting the probes in NCBI'Genbank and analyzing the first 50 sequences of the results,we found the accuracy of completely matching with our designed probes is 97%.2.Genotyping of clinical samplesWe tested the clinical DNA samples,which genotypes were determined with genome sequence comparison method.The microarray hybridization assay displayed a complete match to the previously identified genotypes;106 clinical serum samples collected from HBV infection individuals in Hangzhou.Among them,37and69 samples were positive to genotypes B and C.3.Validation of microarray genotypingThe results of hybrizations were identical with those of sequencing analysis.4.The repetition and sensitiveness of genechips test.20 samples,which HBV-DNA was positive,were tested and 3 repeat tests were done for each samples.The results are all positive.Of the 106 samples,,which HBV-DNA was positive,the lowest HBV-DNA title was 2~*10~4copies/ml,so we beliver the sensitiveness of the chips was not under the lever.Conclusion1.We have established a method of oligonucliotide probe designation according to the analysis of bioinformatics of HBV genome.2.We have fabricated microarray and begun to apply the chips for clinical use.It is demonstrated that this assay is a promising method for virus genotyping.3.HBV genotype B and C exsited in Hangzhou,and genotype C is a predominated genotype.However,To determine the distribution of HBV genotypes,we should take further investigation for HBV genotypes by enlarging study population.4.The assay is a simple to perform and can be used as a rapid,accurate and high-throughput method.It has a high clinical value in detecting the HBV genotype.