肾移植后BK病毒感染者实时荧光定量PCR检测

BK virus infection detected by real-time fluorescent quantitative PCR method after renal transplantation

背景:对于BK病毒感染、BK病毒相关性肾病的认识缺乏规范的实验室诊断程序和标准化的无创性检验方法。目的:建立肾移植后患者尿液和外周血BK病毒感染负荷实时荧光定量PCR检测方法。方法:针对BK病毒基因组,自主设计特异性引物BKV-F和BKV-R,Taqman荧光探针BKV-P,Taqman荧光探针BKV-P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。结果与结论:将检测阳性的扩增产物进行基因测序,测序结果经BLAST比对后证实为BK病毒基因序列;利用上述方法对56份样本进行检测,其中,20份BK病毒血清样本及20份BK病毒尿液样本检测均为阳性,有S型扩增曲线。动态范围测定显示在103～1010copies/mL之间标准曲线具有良好的相关性。5份健康人尿液样本,5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。结果表明该方法可进行定性、定量检测,特异性好,反应快速,一般30min即可得到反应结果,并且成本低、假阳性少。

BACKGROUND：For the BK virus（BKV） and BK virus-associated nephropathy,there lack of standardized laboratory diagnostic procedures and non-invasive testing methods.OBJECTIVE：To establish a real-time fluorescent quantitative PCR method for determining the BKV level of urine and peripheral in renal transplant recipient and to evaluate its clinical application.METHODS：According to the BK virus genome,BKV-F,BKV-R and Taqman fluorescent probe BKV-P were designed by us.5＇ extremity of Taqman fluorescent probe BKV-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.RESULTS AND CONCLUSION：The positive PCR products were preformed with gene sequencing,the result confirmed by BLAST was the BK virus gene sequence;56 samples were detected with this method,20 cases of BKV in serum samples and 20 cases of BKV 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 103-1010 copies/mL standard curves.Five urine samples from healthy individuals,five blood samples and six blood samples of common pathogens in clinical were negative,there was no S-type amplification curve.The real-time fluorescent quantitative PCR assay established in this study was qualitative and quantitative,and has the ability of sensitivity and specificity,low-cost and less false-positive.The general determining results can be obtained in 30 minutes and suitable for large-scale clinical application.