实时定量PCR技术与短时培养法检测尿样本HCMV的比较  

Comparison between real-time PCR technique and rapid culture assay for the quantitative detection of human cytomegalovirus in urine

作　　者：向稚丹[1] 方峰[1] 甄宏[1] 徐翼[1] 刘隽[2] 聂兴草[1] 

机构地区：[1]华中科技大学同济医学院同济医院儿科病毒研究室,湖北武汉430030 [2]华中科技大学同济医学院协和医院干细胞研究中心

出　　处：《临床儿科杂志》2005年第7期434-436,443,共4页Journal of Clinical Pediatrics

摘　　要：目的建立实时定量PCR系统定量检测尿样本中HCMV并与短时培养快速诊断半定量法比较。方法根据HCMVul123基因保守区设计特异性引物和taqman探针,以GAPDH为内参照校准所检测样本的细胞数,建立实时定量PCR检测系统,定量检测132份尿标本中HCMV拷贝数与短时培养结果比较,并同时分析19例接受更昔洛韦治疗前后尿排毒量的变化。结果132份尿标本实时定量PCR结果:短时培养(-)组HCMVDNA为(83±155)拷贝/5×105细胞(n=16例),(+)组为(571±231)拷贝/5×105细胞(n=26例),(++)组为(792±303)拷贝/5×105细胞(n=35例),(+++)组为(1126±416)拷贝/5×105细胞(n=16例),(++++)组为(2201±1043)拷贝/5×105细胞(n=9例);19例38份更昔洛韦治疗前后尿样本中3例治疗前后短时培养结果无改变,而实时定量结果明显改变。结论实时定量PCR法比短时培养法更适于评价抗病毒疗效,而短时培养法判断HCMV是否为活动感染优于实时定量PCR法。Objective To establish a newly designed real-time PCR technique to detect quantitatively HCMV in urine specimen and to compare it with the rapid culture assay (shell vial assay). Methods Uponthe design of two sets for species-specific primers and taqman probes based on the conserved regions of HCMV ul 123 gene and the using of GAPDH as an internal amplification control,we developed a real-time PCR system, which could quantitatively detect HCMV.One hundred and thirty-two urine specimens including 38 urine specimens of 19 cases who were treated with Ganciclovir were detected by this assay and compare it with rapid culture meanwhile. Results The copy numbers of HCMV DNA of the 132 samples in urine specimens detected by real-time PCR were classified into five groups according to the results of the rapid culture assay.The mean CMV DNA copy number per 5×5 cells were 83±155 for samples in (-) group (n=16),571±231 for samples in (+) group (n=26),792±303 for samples in (++) group (n=35),1126±416 for samples in(+++) group (n=16)and 2201±1043 for samples in (++++) group (n=9),respectively.Furthermore, among 38 urine specimens obtained from 19 cases before and after Ganciclovir treatment,3 cases showed no change by rapid culture assay while obvious change by real-time PCR. ConclusionsThe real-time PCR assay is more suitable to evaluate the effect of antiviral therapy than that of rapid culture,while the rapid culture have superiority in diagnosis of active HCMV infection.

关 键 词：实时定量PCR 短时培养法 人巨细胞病毒 尿样本 

分 类 号：R446.5[医药卫生—诊断学]