Design, Preparation And Clinincal Application Of HBV Specific DNA Microarray

IntroductionHepatitis B virus (HBV) infection is a major cause of liver disease around the world. HBV genome can be divided into several subtypes, among which one is serologic subtype according to the amino acids sequence in S protein of HBV and another is genetic subtype according to the nucleotides sequence. The genotypes of HBV are correlated with HBV clinical syndromes and therapeutic efficacy, so the genotyping of HBV had become more and more important. In traditional clinic the genotype of HBV was confirmed by ELISA method, the new genotyping products are being studied.Genechip provides a new promising means for HBV genotyping. In this study we want to establish a integrated method genechip design for HBV examination , including probe design, slide decoration and manipulation, and do some test and analysis about the specificity and sensitivity of the chips and explore its clinical application.Materials and MethodsMaterials and equipments;Primers and probes of the genechips were synthesized by Shanghai Sangon Co. PCR checkbox was purchased from Huamei Co: Clinical sera were offered by the first affiliated Hospital of China Medical University (CMU) and the second affiliated Hospital of CMU. Genechip scanner was purchased from Genomic Solution, Inc; MicroGrid I was purchased from BioRobotics Ltd. ; PCR system was Biometra Personal PCR system, Germany. Electrophoresis Mini II systemwas purchased from BIO - RAD, USA; Centrifugal was purchase from Sigma, Germany.Methods:1. The bioinformatics analysis and PCR primer design of HBV genome. HBV subtype was found in NCBI s Genbank, and the results were align-mented with ClustalW and their conservative and specific sequences were determined. Oligo6.0 and primer5.0 were used to design genotype probes and PCR primers, the reliability of the probes was analyzed.2. Slide decorationThe effects of different chemical reagents on slide were compared and slide fixation efficiency was detected by fluorescence probe; active chemical groups in slide were protected by acetal reaction.3. The best condition of oligonucleotide probesThe best condition of probe buffer, blocking reagent, and balancing time of the active groups reacted with the probes were tested.4. DNA sample and target sequence obtaining.DNA samples were obtained by different method and amplified with PCR, and the best method was determined. Asymmetrical amplifications were designed to find out best ratio of PCR primers.5. 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.6. Detection of clinical hepatitis B virus by DNA microarray.We have tested 360 serum samples that were collected from patients in the first Hospital of China Medical University ( CMU) and the second Hospital of CMU with our microarrays. We want to find if there is difference between the two methods by comparing the results obtained from genechips with those from ELISA. The sequence was analyzed and HBV subtypes were determined in NCBI by blast.7. The repetition of genechips test.50 random samples were tested and 3 repeat tests were done for each sam-pie and the results were analyzed.Results1. HBV genome bioinformatics analysis and chip probe and PCR primer design.We have found 40 HBV subtypes genome in Genbank, including 8 genotypes: A, B, C, D, E, F, G and H, which are classified by all the serotypes. Phylogenetic tree and the evolutionary relationships were analyzed and found that C subtype is not only a chain of the phylogenetic tree itself and is also distributed in the subtrees of H and G subtypes . PCR primers and 19 probes were designed according to the difference of the sequences and the conservative consequences at HBV S gene region. The product of PCR was 432bp. Blasting the probes in NCBIs Genbank and analyzing the first 50 sequences of the results, we found the accuracy of completely matching with our designed probes is 96%.2. Slide decorationWe analyzed the stability and signal intensity of decorated chip slide and found the selected TM552 is better than A1100 and A1200. The slides made by our lab, of which the intensity is two times stronger, are better than those purchased. The slides can be preserved longer when the chemical group in slide can be protected by methanol.3. The best condition of oligonucleotide probes.We found that when the probe buffer is 0.2M ,pH =9.0 and the concentration of probe is IOOjxM the signal of hybridization is good. The fixation reaction will be balanced when hydrating time is over 4 days. NaBH4 is better than BSA and dimethylamine as a blocking reagent.4. DNA sample and target sequence obtaining.No distinct difference in 3 methods in DNA sample obtaining was found. We selected the boiling method. When ratio of primers is 1:10, the signal of hybridization is best. The best hybridization temperature is 50^ , the best hybridization time is lh.5. Veracity and specification of Hybridization...