摘要
目的建立寡核苷酸基因芯片检测 HBV 基因分型的方法并对 HBV 携带者基因型进行临床分析。方法根据 Genbank 中已发表的128株 HBV 全基因组序列系统进化分析结果设计了A～H不同 HBV 基因型的特异探针及巢式 PCR 引物。利用反向杂交原理,先将不同 HBV 基因型特异的寡核苷酸探针固定在芯片上,再与地高辛(Dig)标记的扩增产物杂交从而检测 HBV 基因型。本研究检测了200例 HBV DNA 阳性患者,为了验证基因芯片检测的结果,随机选取其中40份样本进行直接测序。结果在200份血清样本中检测到 B、C 基因型以及 BC 混合型,依次占11.5%(23/200),80.5%(161/200),8%(16/200)。在与测序方法进行对比的40份样本中,除3份例混合型用测序方法不能检出混合感染外,利用测序结果进行系统发育分析得到的结果与基因芯片检测结果一致。进一步的克隆分析发现这3份样本均为混合感染。结论基因芯片是进行 HBV 基因分型的方便可靠的工具,并能更好地检测不同基因型混合感染的情况。本研究中,HBV基因型以 C 型为主,与 B 型相比,C 型 HBV 感染者病情较为严重。
Objective To develop a new method for the detection of HBV genotpe with oligonucleotide chip assay and investigate the clinical significance of HBV genotpe. Methods The specific probes and primers for eight HBV genotype were designed according to the phylogenetic analysis of 128 complete HBV genome sequences in Genbank. This method is based on the principle of reverse hybridization with Dig-labeled amplicons hybridizing to type-specific oligonucleotide probes that are immobilized on slides, 200 HBV DNA positive sera were tested with oligonucleotide chips. The results of 40 randomly chosen sera were confirmed by direct sequencing. Results HBV genotype B, C and mixed genotype were detected in 200 serum samples, accounting for 11.5% （n = 23） , 80. 5% （ n = 161 ） , 8% （ n = 16） of total specimens respectively. The evaluation of the oligonucleotide chip assay showed good concordance with the amplicon phylogenetic analysis except 3 mixed genotype infections undetected by sequencing. Clonal analysis confirmed that all three specimens were mixed genotype. Conclusions The oligonucleotide chip is a reliable and convenient tool for the detection of HBV genotype especially for the mixed genotype. The major genotype in the research is genotype C which induces more severe liver diseases, including hepatocellular carcinoma （HCC）, than genotype B.
出处
《中华检验医学杂志》
CAS
CSCD
北大核心
2007年第10期1148-1152,共5页
Chinese Journal of Laboratory Medicine
基金
中国科学院知识创新工程资助项目(KSCX-06)
关键词
肝炎病毒
乙型
基因型
芯片分析技术
Hepatitis B virus
Genotype
Microchip analytical procedures
