巢式荧光定量RT-PCR检测中枢神经系统HCV方法的构建  

Development of the nest fluorescent quantitative RT-PCR for detecting HCV infection of central nervous system

作　　者：冯裕星[1,2] 何丰[1,2] 孙后超[1,2] 徐红波[1,2] 胡子成[1,2] 展群岭[1,2] 徐鸣明[1,2] 张亮[1,2] 胡永波[1,2] 李雷雷[2] 谢鹏[1,2] 

机构地区：[1]重庆医科大学附属第一医院神经内科,重庆400016 [2]重庆医科大学神经科学研究中心,重庆400016

出　　处：《中国微生态学杂志》2009年第11期975-981,共7页Chinese Journal of Microecology

基　　金："十一五"国家高技术研究发展计划(863计划)项目(2006AA02Z196)

摘　　要：目的建立丙型肝炎病毒5′NCR基因的巢式荧光定量RT-PCR检测方法,用于中枢神经系统感染超低浓度HCV的准确和快速定量检测。方法选择高度保守区5′NCR基因片段,设计并合成相应的特异性引物和探针,建立巢式荧光定量RT-PCR检测中枢神经系统感染样本中的超低浓度HCV正负链RNA的方法。并对32例血清HCV抗体检查阴性的病毒性脑炎患者脑脊液有核细胞和外周血单个核细胞(PBMC)进行检测。结果可特异性地检出脑脊液有核细胞及PBMC中HCV正负链RNA,最低检出浓度均可达7.85 cop ies/μl,与其他单股正链RNA病毒如登革热病毒(DEV)无交叉反应。32例血清HCV抗体检查阴性样本脑脊液有核细胞和PBMC中HCV5′NCR正链片段阳性的分别为1例(1/32)和2例(2/32),负链片段阳性的分别为1例(1/32)和0例(0/32)。结论本方法的构建适用于中枢神经系统感染超低浓度HCV正负链RNA检测,且快速有效、敏感性和特异性较高,不易出现假阳性,可有效提高检出率,进一步完善目前临床常规检测HCV的方法。Objective To establish a method of nest fluorescent quantitative RT-PCR according for 5′NCR gene of hepatitis C virus to detect the ultra-low concentration of HCV in central nervous system accurately and rapidly.Method The highly conserved region of 5′NCR gene fragments was solected to design and synthesize the corresponding specific primers and probe to establish the nest fluorescent quantitative RT-PCR for detecting the ultra-low concentration of HCV positive and negative-strand RNA in the samples of the central nervous system infections. Nucleated cells in cerebrospinal fluid and peripheral blood mononuclear cells （PBMCs） were detected in 32 serum HCV antibody negative patients with viral encephalitis. Result The method was capable specifically of detecting HCV positive and negative-strand nucleated cell RNA in cerebrospinal fluid and PBMCs and the detectable ultra-low concentration reached to 7.85copies/μl. There was no cross-reaction with other single-positive-strand RNA viruses such as dengue virus （DEV）. One positive case （1/32） of 5＇NCR positive-strand fragment was detected in nucleated cells in cerebrospinal fluid and two （2/32） in PBMC while one positive case （1/32） and none （0/32） in negative-strand fragment in the 32 cases. Conclusion The method is suitable for detecting ultra-low concentration of HCV positive and negative-strand RNA in central nervous system infection. It was rapid, efficient, highly sensitive, specific and less false-positive, improving the detection for the clinical routine tests effectively.

关 键 词：丙型肝炎病毒 荧光定量RT-PCR 外周血单个核细胞 脑脊液有核细胞 

分 类 号：R373.2[医药卫生—病原生物学]