尿标本池检测法筛选新生儿先天性人巨细胞病毒感染

摘要

目的 探讨敏感性强、特异性好、简便易行并能降低试剂和人力成本的适于大规模人巨细胞病-毒(HCMV)感染筛查的方法.方法 收集日龄＜14 d的住院新生儿尿标本200例,每例标本分成2份,1份标本采用临床常规实时荧光定量PCR法(RT-QPCR)检测HCMV-DNA,1份尿标本进行尿池预混和三级筛检,即采用相同的操作方法、仪器和试剂进行20份/池的筛检,阳性池再进一步分为5份/池进行筛检,再阳性者进行单个标本检测.结果 采用的RT-QPCR的最低检出量为10 copies/mL.在200例新生病儿尿标本中,分别用标准法和尿池法检测,均有4例相同的标本为HCMV-DNA阳性.多份样本混合在同一检测池中未发现抑制物质对检测的影响.尿池法检测患者标本节约近80％的试剂成本.结论 尿池法除具有标准RT-QPCR法的优点外,极大地降低了试剂和人力成本,适合于大规模新生儿HCMV先天性感染的筛查.%Objective To explore a sensitive,specific,simple and suitable method for large-scale screening of human cytomegalovirus (HCMV) congenital infection in newborns.Methods A total of 200 urine specimens were collected from newborns with age less than 14 days.The specimens were divided into two portions,one for HCMV DNA detections by routine real-time fluorescence quantitative PCR (RT-QPCR),and the other one for urine specimen pools method.Specimen pools were prepared by mixing 50 μL of each urine samples.Each pool was tested by RT-PCR first.The specimens of those positive in the firt round test were further divided into 4 pool (5 specimens in each pool) and analyzed as above.Then,the positive pool was tested individually.Results The sensitivity of the RT-PCR assay used in this study was 10 copies per milliliter.Among the 200 urine specimens of newboms,4 cases were HCMV-DNA Positive which were detected by both standard method and the urine specimen pools assay.No inhibitory substance to PCR reaction was detected when samples mixed in urine specimen pools.The urine pools assay saved nearly 80％ of the reagent costs.Conclusion This method represents a valid option for large-scale screening the HCMV congenital infection in newborns with a significant reduction in both terms of labor and cost of testing materials but without losing sensitivity.