Biel S S, Held T K, Landt O, Niedrig M, Gelderblom H R, Siegert W, Nitsche A
Robert Koch-Institut, Charité Campus Virchow-Klinikum, Humboldt Universität zu Berlin, Germany.
J Clin Microbiol. 2000 Oct;38(10):3689-95. doi: 10.1128/JCM.38.10.3689-3695.2000.
A combined PCR assay was developed for the detection and typing of human polyomavirus (huPoV) in clinical samples, consisting of (i) a qualitative seminested PCR assay (snPCR) to discriminate between huPoV BK and JC and (ii) a high-throughput, quantitative TaqMan PCR assay (TM-PCR) for the general detection of huPoV. The TM-PCR detects huPoV DNA in a linear range from 10(7) to 10(1) copies per assay. In reproducibility runs, the inter- and intra-assay variabilities were < or =60 and < or =50%, respectively. The snPCR assay uses a set of four primers for the same region of the BK and JC viral genomes. In the first round of amplification, two general primers were used; in the second round, one of these general primers and two additional, BK- or JC-specific primers were used simultaneously to produce amplicons of different sizes specific for BK virus (246 bp) and JC virus (199 bp), respectively. We tested different urine dilutions in order to determine the inhibitory effects of urine on PCR amplification. Furthermore, we compared the use of native urine with DNA purified by different preparation procedures. Our results show, that a 1:10 dilution of the urine led to complete reduction of the amplification inhibition found with 6% of undiluted urine samples. In a clinical study including 600 urine specimens, our assay turned out to be fast, cheap, and reliable in both qualitative and quantitative aspects.
开发了一种用于临床样本中人类多瘤病毒（huPoV）检测和分型的联合PCR检测方法，该方法包括：（i）一种定性半巢式PCR检测方法（snPCR），用于区分huPoV BK和JC；（ii）一种高通量定量TaqMan PCR检测方法（TM-PCR），用于huPoV的常规检测。TM-PCR在每次检测中可检测10⁷至10¹拷贝的线性范围内的huPoV DNA。在重复性实验中，检测间和检测内的变异率分别≤60%和≤50%。snPCR检测方法使用一组针对BK和JC病毒基因组相同区域的四种引物。在第一轮扩增中，使用两种通用引物；在第二轮中，同时使用其中一种通用引物和另外两种BK或JC特异性引物，以分别产生针对BK病毒（246 bp）和JC病毒（199 bp）的不同大小的扩增子。我们测试了不同的尿液稀释度，以确定尿液对PCR扩增的抑制作用。此外，我们比较了天然尿液与通过不同制备程序纯化的DNA的使用情况。我们的结果表明，尿液1:10稀释可完全消除6%未稀释尿液样本中发现的扩增抑制。在一项包括600份尿液标本的临床研究中，我们的检测方法在定性和定量方面都快速、廉价且可靠。
