摘要
目的建立1种检测16型乳头状瘤病毒(HPV16)7862nt上CpG位点非甲基化的方法.方法培养宫颈癌SiHa和CaSki细胞系,提取基因组DNA,通过亚硫酸氢钠修饰将未甲基化的胞嘧啶转化成尿嘧啶,PCR法预扩增包含该位点的HPV序列,针对该CpG位点5'端序列设计并合成延伸引物,再在同时含ddTrP和ddCTP的体系中进行延伸反应,用变性高效液相色谱法同时分离检测2种延伸产物.结果在SiHa细胞的延伸产物中仅能够检测到代表未甲基化位点的ddTTP延伸产物(保留时间6.7 min),而在CaSki细胞的延伸产物中不仅能够检测到ddTTP的延伸产物,还能检测到代表甲基化位点的ddCTP的延伸产物(保留时间6.3 min).结论所建立的引物延伸-变性高效液相色谱联合检测法能够同时检出感染细胞中非甲基化活化和甲基化失活的HPV病毒拷贝,灵敏度达1/500(0.2%).
Objective To establish a sensitive assay to detect methylation status of the critical 7862nt CpG site related to transcription of HPV16 E6 and E7 genes.Methods Genomic DNA of two HPV16-infected cell line SiHa and CaSki was extracted and modifed by sodium bisulfite to convert the unmethylated Cs to Us(Ts in PCR products).The target sequence of HPV16 including the 7862nt CpC site was pre-amplified by PCR Then,the methylation status of the 7862nt site was diferentiated in a primer extension reaction with an HPV16-specific primer,and separated by DHPLC at 80 C.Results The primers without extension and with extension,whether matched to CpG or TpG,could be separated by DHPLC completely.The peak for ddTTP extension products correspo nding to the demethylated CpC site was observed at retention time 6.7 min in both cell lines.However,the peak for ddCTP-extension products representing the methylated CpG site could be detected at retention time 6.3 min in CaSki cell line only,which integrated with 499 methylated and one demethylated HPV16 copies Conclusion The established DHPLC-primer extension assay can be used to detect methylated and demethylated HPV16 copies simultaneously with a sensitivity up to 1/500(0.2%).
作者
白桦
邓大君
BAI Hua;DENG Da-jun(Peking University School of Oncology and Bejing Institute for Cancer Research,Beijing 100036,China)
出处
《中华预防医学杂志》
CAS
CSCD
北大核心
2007年第S01期81-83,共3页
Chinese Journal of Preventive Medicine
基金
国家“973”计划资助项目(2005CB522403)
关键词
乳头状瘤病毒
人
甲基化
色谱法
高效液相
Papillomavirus,human
Methylation
Chromatography,high pressure liquid
