摘要
目前应用比较多的采用反向点杂交/线性杂交方法的人乳头瘤病毒(HPV)基因分型检测方法,操作比较繁琐。该文通过设计针对29种HPV基因型的引物对和特异性探针,对HPV样品进行PCR扩增后,将反向引物生物素标记的PCR产物与固定在HPV基因芯片上的HPV DNA探针杂交、清洗和酶标显色,再对芯片进行图像分析转化为数字信号、确定HPV型别,建立HPV高通量基因分型方法(HPG)。HPV基因芯片大小为0.36 cm2,每个芯片可在96孔板内的小孔中进行杂交和显色。与HCII方法检测的203例样本比较,对13种高危型检测的结果表明,HPG和HCII方法的一致性较好(kappa值0.663),HPG的分析灵敏度更高。显示了高通量快速检测的在大规模流行病调查、疫苗试验和常规诊断的临床应用前景。
Genotyping of human papilomavirus( HPV) by DNA hybridization procedures,such as reverse dot / line bolt( RLB) assay,is well validated but limited because of its laborious process. Based on the primers and specific probes for twenty-nine HPV types,HPV sequences are detected by PCR amplification,subsequent typing by hybridization of the biotinylated PCR products to type-specific oligonucleotide probes coupled to nylon membrane followed by enzyme immunoassay,and final transformation from imaging analyses of DNA Chip to digital signals.The membrane DNA chip is fabricated in a miniaturized size up to 0. 36 centimeter square,so every hybridization can be performed in the individual well on 96 well plates. We compared the performance of HPG to an established HCII assay for thirteen high-risk HPV genotypes on the PCR products derived from 203 clinical samples. The evaluation showed a good agreement( kappa = 0. 663) but also indicated a higher sensitivity of HPG. In conclusion,HPG appears to be highly suitable for large-scale epidemiological studies and vaccination trials as well as for routine diagnostic purposes.
出处
《生物医学工程学进展》
CAS
2013年第3期157-164,167,共9页
Progress in Biomedical Engineering
