同时检测人多瘤病毒和巨细胞病毒PCR技术的建立及在肾移植受者中的初步应用  

Establishment of A Real-time PCR Assay for Simultaneously Detecting Human BKV and CMV DNA and Its Application in Renal Transplantation Recipients

作　　者：张纯武[1] 陈孝倩[1] 白永恒[1] 潘晓东[1] 王斯璐[1] 蔡勇[1] 夏鹏[1] 吴存造[1] 陈必成[1] 

机构地区：[1]温州医学院附属第一医院,温州325000

出　　处：《病毒学报》2013年第4期410-414,共5页Chinese Journal of Virology

基　　金：浙江省重中之重外科学资助项目;浙江省自然科学基金(LY12H10004);温州市科技局基金(No.H20100070)

摘　　要：建立同时检测人多瘤病毒(BKV)和巨细胞病毒(CMV)载量的实时定量荧光PCR技术(FQ-PCR),并探讨其在肾移植术后感染监测中的应用。选择BKV和CMV核酸保守性序列作为靶基因,PCR扩增靶片段与质粒pcD-NA3.1(+)连接。通过测序技术对进行克隆筛选。提取质粒并采用10倍梯度稀释(5×103～107拷贝/mL)作为BKV和CMV核酸序列的标准品,并评价其灵敏度;采用对比正常人DNA、BKV阳性质控和CMV阳性质控评价其特异性;通过对标准品DNA分别进行批内和批间各20次重复实验,以其循环阈值(Ct)的变异系数(CV)评价其精密度。对480例肾移植受者外周血样本,进行FQ-PCR检测BKV和CMV DNA拷贝数,检测FK506血药浓度,并对两者进行相关性分析。重组质粒经测序检测显示含有靶BKV和CMV核酸序列。所建立的FQ-PCR方法,其最低检测下限均为5.0×103拷贝/mL的DNA标准品。正常人DNA的BKV和CMV拷贝数检测为阴性,而阳性对照能够发现荧光值显著变化,证实为针对靶DNA的特异性扩增;重复性检测显示批内差异分别为3.44%(BKV)和2.23%(CMV),批间差异分别为4.98%(BKV)和3.76%(CMV)。肾移植患者CMV感染的阳性率为27.08%(130/480),明显高于BKV的阳性率(13.33%,64/480,P<0.05),并且感染程度与免疫抑制剂FK506用量呈正相关。建立的同时检测BKV和CMV两种病毒载量的FQ-PCR方法具有快速、重复性好、灵敏的优点,可为临床监测肾移植术后病毒感染提供技术平台。To establish a fluorescent quantitative PCR method （FQ-PCR） with TaqMan probe for simultaneous detection of polyomavirus （BKV） and cytomegalovirus （CMV） and to evaluate its clinical application in the renal transplantation recipients. The conservative sequences of BKV and CMV were targeted and amplified by nested PCR technique. The PCR products were cloned into the plasmids pcDNA3.1 （＋）. The recombinant plasmid containing target sequences of BKV and CMV were constructed as external stand- ards. The TaqMan-based assay was optimized. For evaluating the assay, the sensitivity was determinated by diluted standard （S×10^3-10^7copies/mL）, and the specificity was verified by negative control and positive control, and the precision was assessed by intra-assay coefficient of variation （ICV） through detecting standard repeatedly （20 times）. A total of 480 blood samples of renal transplantation recipients were used to detect BKV and CMV DNA simultaneously with FQ-PCR, and the concentrations of FK506 were measured by ELISA. The association of DNA copy and concentrations of FK506 was analyzed. The cloned target BKV and CMV DNA was confirmed by sequencing and analysis. The sensitivity of the FQ-PCR assay reached 5 × 10^3 copies/ml in detecting BKV or CMV DNA. Control DNA verified the assay specifically detecting target DNA. The precision of the assay to quantif target DNA copies was acceptable （Intra-assay CV was 3.44% for BKV and 2.23% for CMV; Inter-assay CV was 4.98% for BKV and 3.76% for CMV;）. Of 480 samples, 130 samples （27.08%） were CMV DNA positive, significantly higher than the BKV DNA positive （13.33%, 64/480, P〈0.05）. The positive BKV or CMV DNA was found to be asso- ciated with high concentrations of FK506 （P〈0.05）. In conclusion, the developed real-time PCR assay for detecting both CMV and BKV DNA simultaneously was s high sensitive, precise and time-effectiveand could be applied in the monitoring of the CMV and BKV infection in the renal transplantation recipie

关 键 词：巨细胞病毒 多瘤病毒 荧光定量PCR 肾移植 

分 类 号：R373.9[医药卫生—病原生物学]