实时定量聚合酶链反应结合Taqman荧光探针JCV-P检测肾移植受者JC病毒

Detection of JC virus in urine and peripheral blood from renal transplant recipients by real-time PCR assay combined with TaqMan-JCV-P probe

目的：建立肾移植受者尿液和外周血JC病毒（JCV）感染实时荧光定量聚合酶链反应（PCR）检测方法。方法针对JCV基因组设计特异性引物JCV-F和JCV-R ，Taqman荧光探针JCV-P ，探针的5′端标记有荧光基团，在除5′端外的任意一个位置上标记有淬灭基团；然后处理待测样本，进行PCR反应，建立重组质粒标准曲线，评价特异性、灵敏度、重复性。结果将检测阳性的扩增产物进行基因测序，测序结果经BLAST 比对后证实JC V基因序列；利用上述方法对86份样本进行检测：20份JC V血清样本及20份JC V尿液样本检测均为阳性，有S型扩增曲线，动态范围测定显示103～1010拷贝/m L标准曲线具有良好的相关性；20份健康人尿液样本和20份血液样本及6份临床常见的其他病原体血液样本检测均为阴性，无S型扩增曲线。结论实时荧光定量PCR结合Taqman荧光探针JCV-P检测肾移植术后患者JCV方法可用于肾移植术后JCV感染的的早期诊断，对预防JCV感染造成的多瘤病毒相关性肾病（PVAN）和进行性多灶性脑白质病（PML）具有一定意义。

Objective To establish a real-time fluorescent quantitative PCR method for determining the JCV level of urine and peripheral blood in renal transplant recipient and to evaluate its clinical application .Methods According to the JC virus genome ,JCV-F ,JCV-R and Taqman fluorescent probe JCV-P were designed .5′ex-tremity of Taqman fluorescent probe JCV-P was labeled with fluorophores .Except 5′extremity ,other sites were marked quenching group ;the results of PCR reaction were obtained after detecting the samples .Meanwhile ,the standard curve was established .The specificity ,sensitivity and reproducibility of this assay were estimated system-atically .Results The positive PCR products were preformed with gene sequencing , the result confirmed by BLAST was the JC virus gene sequence ;Eighty-six samples were detected with this method ,twenty cases of JCV in serum samples and twenty cases of JCV in urine samples were positive and had a good S-type amplification curve .Dynamic range tests showed that there was a good correlation among the 103~1 010 copies/mL standard curves .Twenty urine samples and twenty blood samples from healthy individuals ,six blood samples of common pathogens in clinical were negative ,there was no S-type amplification curve .Conclusion The real time PCR assay combined with TaqMan-JCV-P probe established in this study could be applied to early diagnosis of JC virus and the prevention of JC virus associated nephropathy and progressive multifocal leukoenphalapathy .