荧光定量PCR检测鼻咽部脱落细胞内EBV-DNA  

A fluorescence quantitative PCR detecting EBV-DNA in nasopharyngeal brush biopsies

作　　者：张明[1] 童明庆[1] 潘世扬[1] 黄珮珺[1] 赵旺胜[1] 

机构地区：[1]南京医科大学第一附属医院临床检验中心,南京210029

出　　处：《临床检验杂志》2002年第2期87-89,共3页Chinese Journal of Clinical Laboratory Science

摘　　要：目的　建立荧光定量PCR(FQ PCR)检测鼻咽部脱落细胞中EBV DNA的方法。方法　以患者鼻咽部脱落细胞内DNA作为样本 ,以EB病毒基因序列中EBNA1(核抗原 1)基因片段作为扩增的靶DNA ,同时以人 β 珠蛋白DNA片段作为内参照 ,合成了两对引物 ,并设计了两个特异的荧光探针 ,优化了FQ PCR反应体系 ,同时对这两个片段进行扩增 ,每一标本得到两个Ct值 :CtE和Ctβ ,计算CtE/Ctβ ,得到单位细胞EB病毒的相对量。 结果　临床标本 2 9例中 ,阳性 2 2例 ,阴性 7例 ,临床资料证实阳性者全部为鼻咽癌 (NPC) ,阴性者全部为非鼻咽癌。结论　建立的FQ PCR检测鼻咽部脱落细胞中EBV DNA的方法 ,能反映单位细胞病毒的复制情况 ,可用于NPC的筛查 。Objectives To establish a real time polymerase chain reaction system to detect EBV DNA in the specimen nasopharyngeal brush biopsies.Methods Using DNA in cells of nasopharyngeal brush biopsies as sample, one set of primers for the target DNA segment in the Epstein Barr nuclear antigen(EBNA1) and a second set of primers for the internal control DNA segment of human β globin were synthesised and specific probs were designed and synthesised.The amplification system was optimized to amplify the two segments simultaneously. And then, each sample was analyzed by FQ PCR and got a set of data, CtE and Ctβ, the value of CtE/Ctβ is corresponsive with the quantity of EBV DNA of unit cell. Results Twenty nine specimens of nasopharyngeal brush biopsies(22 specimens from nasopharyngeal carcinoma,7 normal nasopharyngeal bush biosies)was analyzed, the result showed that EBNA1 sequences were positive in all NPC samples and none of the normal samples and human β globin sequences were positive in all the samples. Conclusions A FQ PCR system to detect EBV DNA in the sample nasopharyngeal brush biopsies was established With a internal control, from which the quantitative of Epstein Barr virus in unit cell can be manifested. This approach merits the diagnosis of NPC and may be applied to the evaluation of malignant risk and the observation of curative effect of NPC.

关 键 词：荧光定量PCR 细胞内参照 Β-珠蛋白 EBV-DNA 鼻咽癌 

分 类 号：R739.63[医药卫生—肿瘤] R730.43[医药卫生—临床医学]