Construction And Clinical Application Of HBV Genotyping Microarray

IntroductionHepatitis B virus (HBV) infection is a major cause of liver disease around the world. HBV genome can be divided into serologic subtype and genetic subtype . The genotypes are calssified into 8 subtypes according to the difference of nucleotides sequence. The traditional serotypes can't reflect the evolution and variation of HBV genome while the genotypes are correlated with the hotspot variation ,disease activity, clinical syndrome , therapeutic efficacy and prognosis, so the genotyping of HBV has become more and more important. A convenient and effective genotyping method should be opened up.Microarray provides a new promising means for HBV genotyping. At present , HBV microarray is not perfect enough to apply to clinic though many news about it were reported. Further more the sdudy of HBV genotype needs development in our country, so we want to explore the construction of HBV genotyping microarray, including probe design, optimum of hybridize conditions, analysis of repetition effect and reliability of the chips, and utilize our genotyping microarray to type 150 HBV patients to get the result of the distribution of HBV genotype in LiaoNing province.Materials and MethodsMaterials1. Clinical sera were offered by the second affiliated Hospital of China Medical University.2. PCR checkbox was purchased from Huamei Co.3. Primers and probes of the microarray (Shanghai Bioasia Co. )4. Biometra Personal PCR system, Germany5. Micro Grind H 600, Biorobtics Ltd, England6. Microarray scanner (Gene TAC LS IV,Genomic Solutions Inc. USA). Methods1. Genotype probes and PCR primers were designed respectively by Omiga software and primer 5.0 and the rationality of the probes were analyzed .2. Slices are decorated by a series of chemical reactions and probes are fixed on slices by spot, hydration and pyrogenation.3. DNA samples were obtained by boiled method and amplified with PCR reaction.4. Asymmetrical amplifications are designed to find out best ratio of PCR primers.■ 5. Research the optimal hybridization temperature and time by gradual contrast.6. Every samples are tested 4 times to detect the repetition of chips. Random positive PCR products are sent for sequence analysis, and the feedback results are compared with hybridization results to test the reliability of chips.7. Utilize genotyping microarray to type 150 HBV patients to get the result of the distribution of HBV genotype in LiaoNing province.Results1. probes have no homology with other virus or bacteria. Common — probe contains all genotypes of HBV. Each genotyping probe carries with it's corresponding genotype.2. The signal of single — strand DNA hybridization is better than double -strand DNA hybridization. When ratio of primers is 1:10, the signal of hybridization is best.3. The best hybridization temperature is 60X1, the best hybridization time is 2h.4. The repetition ratio of genotyping probe is 94. 6% and the repetition ratioof Common — probe and positive contrast is 100% .5. The genotype C has 103 cases, genotype B has 34 cases, BC hybrid has 13 cases;The ratio is 68.6% , 22. 6% and 8. 6% respectively.Conclusion1. The probes design is rational and the detection results are ideal.2. We have confirmed the repetition and reliability of the genotyping mi-croarray and begun to apply the chips to clinical use.3. There are both genotype B and genotype C in LiaoNing province and genotype C is in predominance.