荧光定量PCR检测新生儿干血斑中巨细胞病毒DNA方法的建立及评价  

Establishment and evaluation of a fluorescence quantitative PCR assay for detection of Cytomegalovirus DNA from newborn dried blood spots

作　　者：董妞妞[1] 苏犁云[1] 叶丽静[2] 钟华清[1] 俞蕙[2] 曹凌峰[1] 董左权[1] 徐锦[1] 

机构地区：[1]复旦大学附属儿科医院临床检验中心,上海201102 [2]复旦大学附属儿科医院感染传染科,上海201102

出　　处：《中华检验医学杂志》2014年第4期261-265,共5页Chinese Journal of Laboratory Medicine

摘　　要：目的 建立荧光定量PCR检测新生儿干血斑中巨细胞病毒（CMV）DNA的实验方法,并评价方法的最低检测限、精密度、特异性和临床应用能力.方法 确立CMV-gB段的引物和探针,构建标准质粒,检测CMV毒株的浓度.荧光定量PCR法检测10倍梯度稀释的病毒株干血斑,获得方法的最低检测限和批内、批间精密度.2011年9月至2013年2月收集复旦大学附属儿科医院10份确诊先天性CMV感染以及120份普通人群中出生3d内的新生儿干血斑标本,以本方法进行验证.结果 CMV DNA的最低检测限为4.44×103拷贝/ml;检测灵敏度为2.66拷贝/25μl反应体系.CMV毒株浓度为4.44×106拷贝/ml时检测CMV DNA的批内、批间精密度分别为0.74％和1.39％;CMV毒株浓度为4.44 ×104拷贝/ml时批内、批间精密度分别为1.04％和1.84％.10份确诊先天性CMV感染的标本中,8份CMV DNA阳性;120份新生儿标本中,2份CMV DNA阳性.阳性标本经套式PCR复测以及测序验证均为CMV阳性.结论 成功建立了一种可用干血斑中微量CMV检测的荧光定量PCR法,该方法快速、灵敏、精密度高,适用于大规模新生儿先天性CMV感染的筛查.Objective To establish the method of fluorescence quantitative PCR for detection of Cytomegalovirus （CMV） DNA from newborn dried blood spots （DBS） and evaluate the limit of detection （LOD）,precision,specificity and clinical application.Methods Primers and probes were selected from CMV-gB segment and standard plasmids were constructed to detect concentration of CMV strains.The LOD,within-run and between-run precisions were obtained by detection of 10-fold serial dilutions of CMV strains in DBS with quantitative PCR.This assay was further evaluated by detecting CMV DNA from 10 DBS with confirmed congenital CMV infection newborns and 120 DBS from healthy infants who were born within three days from September 2011 to February 2013.Results The LOD of CMV DNA was 4.44 × 103 copies/ml and the sensitivity was 2.66 copies/25 μl reaction system.The within-run and between-run precisions were 0.74% and 1.39% at a CMV strain concentration of 4.44 × 106 copies/ml,respectively,and 1.04% and 1.84% at a CMV strain concentration of 4.44 × 104 copies/ml respectively.Eight of ten newborns with congenital CMV infection and two of 120 neonatal DBS from healthy population were CMV-positive.All the positive products were verified by nested PCR and sequencing.Conclusions A method of fluorescence quantitative PCR were established successfully for CMV test in dried blood spots.This assay displayed the advantages of rapidness,good sensitivity and precision and could be used for large-scale screening for CMV infections.

关 键 词：巨细胞病毒感染 巨细胞病毒 DNA 病毒 

分 类 号：R446.5[医药卫生—诊断学]