实时荧光定量PCR检测肾移植患者术后BK病毒感染

A real-time fluorescent quantitative PCR method for determination of BK virus in renal transplant recipients

目的：建立肾移植术后患者尿液和外周血BK病毒（BKV）感染负荷的实时荧光定量PCR检测方法,并初步探讨其临床应用价值.方法：构建含有BKV VP1蛋白保守区核苷酸序列片段的质粒作为外参照品,采用TaqMan荧光探针检测技术,建立定量检测BKV DNA方法;并对112例肾移植术后患者,40例正常体检者的尿液和外周血标本BKV含量进行检测.结果：本研究建立的BKV实时荧光定量PCR检测方法灵敏度、特异度及重复性较好,定量PCR方法最低检测下限为8×10^2copy/ml,测量批内差异为0.54%～3.78%,批间差异为0.62%～4.58%.112例肾移植术后患者尿液和外周血BKV DNA的检测阳性率分别为27.7%和11.6%,BKV DNA阳性者尿液和外周血BKV中位数水平分别为8.2×10^4copy/ml和2.4×10^3copy/ml.40例正常人BKV尿液和外周血阳性率为2.5%和0%;与正常体检者相比,肾移植术后患者尿液及外周血BKVDNA阳性率及水平明显升高（P＜0.01）.尿液BKV阳性率较外周血明显升高（P=0.02）,但尿液和外周血中BKV含量无明显相关性.结论：实时荧光定量PCR检测肾移植患者术后BKV感染方法简便、可靠、准确,为进一步研究BKV感染与肾移植术后移植物丢失的关系奠定了基础.

Objective：To establish a real-time fluorescent quantitative PCR method for determining the BK virus（BKV） level in renal transplant recipients,and to evaluate its clinical application. Methods： Plasmids containing part of BKV VP1 gene conservative region were constructed as external standards, and a TaqMan probe technique was used to establish a quantitative method for determination of BKV. Urine and peripheral blood（PB） samples from 112 renal transplant recipients were assayed for BK virus levels, and the results were compared with those of 40 healthy controls. Results： The real-time fluorescent quantitative PCR assay established in this study had good sensitivity, specificity, and reproducibility. The minimal detectable level was 8 × 10^2 copy/ml, intro-experiment variation was 0. 54 %-3.78 %, and intra-experiment variation was 0.62 %-4. 58 %. BKV was detected in 27.7% urine samples and 11.6% PB samples from patients. The median level of BKV in urine and PB were 8.2 × 10^4 copy/ml and 2.4 × 10^3 copy/ml, respectively. BKV positive rate of 40 healthy population in urine and PB samples were 2.5 % and 0 %, respectively. The positive rate and level of BKV in renal transplant recipients were both significantly higher than those in normal cohort （both P〈0.01）. The positive rate of BKV in urine samples were significantly higher than that in PB samples（P=0.02）, but the BKV load in urine samples was not related to that in PB samples. Conclusion： The real-time fluorescent quantitative PCR assay in this study is simple, reliable, and precise, which lays a foundation for future study of the relationship between BKV infection and renal graft loss.