探索实时定量PCR有效检测尿液中BK病毒的方法-第三军医大学学报.docVIP

实时定量PCR检测BK病毒时尿样本的优化处理 林春兰1，卢育洪2*，陈少华3，李扬秋3，朱康儿2，张涛2，陈洁2 （暨南大学1医学院生化教研室，2附属第一医院血液科，3医学院血液研究所 广东 广州510632） [摘要] 目的：建立有效样本处理方法提高实时荧光定量PCR检测尿液中BK病毒载量的敏感性和精确性。方法：随机选出16份标本，每一样本进行4种不同的处理：原尿、原尿病毒DNA提纯、1：10稀释尿液、1：100稀释尿液，实时荧光定量PCR检测各组样本BK病毒载量，所测数据用SPSS 11.0进行统计学分析。结果：四种不同的处理方法，对实时荧光定量PCR检测尿液中BK病毒载量有明显的影响：原尿液假阴性3/16例，病毒载量低值占75%，1：100稀释尿液假阴性1/16例，病毒载量在四组中的最高值占69%，但中位值和1：10稀释组差异无显著性；DNA纯化组与1：10稀释尿液病毒检测率相当为100%，但目标基因的流失量较明显。结论：1：10稀释尿液在实时荧光定量PCR检测BK病毒时DNA损失小，效率高，且处理过程简便、成本低，可做为临床检测尿液中BK病毒载量检测时尿样本处理的首选。 [关键词]：BK病毒，尿样本，实时荧光定量PCR，病毒载量 Optimization of urinary sample preparation process for BK virus detection by real-time PCR LIN Chun-lan1, LU Yu-hong2, CHEN Shao-hua3, LI Yang-qiu3, ZU Kang-er2, ZHANG Tao2, CHEN Jie2. (1Department of biochemistry of medical college, 2Hematological station of the first affiliated clinical hospital, 3Hematology Institute of medical college, Jinan University, Guangzhou 510632, China) [Abstract] Objective: to develop an effective preparing process to improve the sensitivity and accuracy of BK virus’s (BKV’s) load detection in urine by real-time PCR. Methods: 16 samples were enrolled randomly in this study. The candidate samples are prepared in 4 approaches respectively: unprocessed urine, BKV’s DNA [作者简介] 林春兰，女，广东惠来人，汉族，硕士，实验师，研究方向：医学分子生物学 基金项目：广东省自然科学基金自由项目（项目编号。 *通讯作者：卢育洪，联系电话：20Email:yhl2006jn@163.com extracted from urine, 1:10 diluted urine, 1:100 diluted urine and then are tested by real-time PCR to obtain viral load. Results: comparison of the assay outcome shows the different preparation process of urinary specimens impact significantly on sensitivity and accuracy of quantitative BKV detection by real-time PCR. In the unprocessed urine group, 3/16 samples are false-negatives and 75% samples have the lowest viral load values comparing with the other 3 groups. In the 1:100 diluted urines group, 1/16 sample is false-negatives and 69% have the highest viral load values. In the DNA extracti