高通量流式荧光微球悬液芯片检测乙肝病毒基因分型  

Development of High-throughput Detection of HBV Genotypes Using a Fluid Fluorescent Microsphere Suspension Array

作　　者：刘永兰[1] 包晗[1] 赵锦荣[1] 向安[1] 郭晏海[1] 雷小英[1] 汪钦[1] 颜真[1] 

机构地区：[1]第四军医大学药学院药物基因组学教研室全军基因诊断技术研究所,陕西西安710032

出　　处：《现代生物医学进展》2012年第35期6805-6809,共5页Progress in Modern Biomedicine

基　　金：国家"十一五"重大传染病专项(2009ZX10004-311);国家重点基础研究发展规划(973)项目(2010CB933900);西安市科技计划(SF09027)

摘　　要：目的:建立HBV基因分型高通量液相芯片检测技术,并探讨其应用价值。方法:对GenBank中收录的明确分型的HBV基因序列进行分析,选择preS2-S区设计引物和A、B、C和D型特异性探针。与荧光编码微球偶联的特异型探针与一条引物生物素标记的PCR产物直接杂交反应,然后结合亲和素标记的藻红蛋白,用流式检测仪(Bio-Plex 200)检测荧光信号。检测182份阳性乙肝患者血清DNA,其中35份样品检测结果与测序法比较。用B、C型质粒DNA倍比稀释及混合样品检测灵敏度来评估该方法。结果:建立了HBV基因分型的快速高通量液相芯片检测方法。182份患者血清检测结果为:B型占24.2%(44/182),C型占71.4%(130/182),D型为6.6%(12/182),BC混合型4.4%(8/182)。其中35份样本与测序法比较,除3份混合型测序法未检出外,其它32例结果均相同。本方法的灵敏度检测下线为1×103copies/mL。结论:应用悬液芯片技术进行乙肝病毒的基因分型分析,具有较好的特异性和较高的灵敏度,并有简便、灵活和高通量等优势。该检测系统不仅在科研中有广泛的前景,也有望成为临床推广的多重分子诊断和基因分型的新方法。Objective： A rapid and high-throughput method has been developed to detect multiple HBV genotypes by a Fluorescent-coded Microsphere Suspension Array,and the application value has been discussed.Methods： According to the sequence with known genotypes of HBV in GenBank,the primers and specific genotyping probes from A to D were designed in preS2-S of HBV genome.The type-specific oligonucleotide probes coupled to fluorescence-labeled polystyrene beads,subsequently hybridized directly to biotinylated PCR products,then conjugated streptavidin-phycoerythrin,and analyzed by the flow cytometer detector（Bio-plex 200）.We detected 182 serum samples from HBV DNA-positive patients,and 35 cases were compared with sequencing analysis.The sensitivity of the method was evaluated by using serial diluted Plasmids of genotype B and C,And the sensitivity for the detection of co-infections was examined by mixture of plasmids.Results： The suspension array system for rapid and high-throughput detection HBV genotypes was established.A total of 182 serum samples of patients were tested by the assay.Genotype B was detected in 24.2 %（44/182）,genotype C in 71.4 %（130/182）,genotype D in 6.6%（12/182） and genotype B/C mixture in 4.4 %（8/182）.Hepatitis virus genotyping in 35 cases by Suspension Array were compared with the sequences,there were 32 serum samples except 3 B/C mixed samples were concordant.Further studies showed suspension Array could detect 10% of genotype B and genotype C among mixed genotype samples at HBV DNA ≥3×103copies/mL.The sensitivity of the detecting limit for the HBV was 1000 copies/ml.Conclusion： Suspension array technology for HBV genotyping analysis has good specificity and high sensitivity,and simple,flexible and high throughput advantage.The detection system is expected to be clinically popularized as a new method for multiple molecular diagnostics and identification of genotyping.

关 键 词：荧光微球 悬液芯片 乙肝病毒 基因分型 

分 类 号：R373.2[医药卫生—病原生物学] R446.119[医药卫生—基础医学]