Department of Laboratory Medicine, Faculty of Medicine and Health, Örebro University, Örebro, Sweden.
Cell Oncol (Dordr). 2017 Oct;40(5):521-527. doi: 10.1007/s13402-017-0331-y. Epub 2017 Jul 26.
Human papilloma virus (HPV) infection is associated with several anogenital malignancies. Here, we set out to evaluate digital droplet PCR (ddPCR) as a tool for HPV 16, 18, 33 and 45 viral load quantification and, in addition, to compare the efficacy of the ddPCR assay for HPV 16 detection with that of quantitative real-time PCR (qPCR).
Clinical samples, positive for HPV genotypes 16, 18, 33 and 45 were analyzed for viral load using ddPCR. Sample DNA was cleaved before droplet generation and PCR. Droplets positive for VIC and FAM fluorescence were read in a QX200 Droplet reader™ (BIO-RAD) after which the viral load was calculated using Quantasoft software.
We found that DNAs extracted from formalin fixed paraffin embedded (FFPE) tissue samples yielded lower amplification signals compared to those obtained from liquid based cytology (LBC) samples, but they were clearly distinguishable from negative background signals. The viral limit of detection was 1.6 copies of HPV 16, 2.8 copies of HPV 18, 4.6 copies of HPV 33 and 1.6 copies of HPV 45. The mean inter-assay coefficients of variability (CV) for the assays ranged from 3.4 to 7.0%, and the mean intra-assay CV from 2.6 to 8.2%. The viral load in the different cohorts of tumor samples ranged from 154 to 340,200 copies for HPV 16, 244 to 31,300 copies for HPV 18 and 738 to 69,100 copies for HPV 33. One sample positive for HPV 45 contained 1331 viral copies. When comparing qPCR data with ddPCR copy number data, the qPCR values were found to be 1 to 31 times higher.
Separation of fragments in nanodroplets may facilitate the amplification of fragmented human and viral DNA. The method of digital droplet PCR may, thus, provide a new and promising tool for evaluating the HPV viral load in clinical samples.
人乳头瘤病毒（HPV）感染与几种肛门生殖器恶性肿瘤有关。在这里，我们着手评估数字液滴 PCR（ddPCR）作为 HPV 16、18、33 和 45 病毒载量定量的工具，并比较 ddPCR 检测 HPV 16 检测的功效与定量实时 PCR（qPCR）。
使用 ddPCR 分析临床样本，这些样本对 HPV 基因型 16、18、33 和 45 呈阳性，用于病毒载量分析。在生成液滴之前和 PCR 过程中对样品 DNA 进行切割。在 QX200 Droplet reader™（BIO-RAD）中读取阳性 VIC 和 FAM 荧光的液滴，然后使用 Quantasoft 软件计算病毒载量。
我们发现，与从液体基细胞学（LBC）样本中获得的 DNA 相比，从福尔马林固定石蜡包埋（FFPE）组织样本中提取的 DNA 产生的扩增信号较低，但与阴性背景信号明显不同。HPV 16 的病毒检测限为 1.6 拷贝，HPV 18 为 2.8 拷贝，HPV 33 为 4.6 拷贝，HPV 45 为 1.6 拷贝。检测的平均批间变异系数（CV）为 3.4%至 7.0%，平均批内 CV 为 2.6%至 8.2%。不同肿瘤样本队列中的病毒载量范围为 HPV 16 为 154 至 340,200 拷贝，HPV 18 为 244 至 31,300 拷贝，HPV 33 为 738 至 69,100 拷贝。一个 HPV 45 阳性样本含有 1331 个病毒拷贝。当将 qPCR 数据与 ddPCR 拷贝数数据进行比较时，发现 qPCR 值高 1 至 31 倍。
纳米液滴中的片段分离可能有助于扩增碎片化的人和病毒 DNA。因此，数字液滴 PCR 方法可能为评估临床样本中的 HPV 病毒载量提供一种新的有前途的工具。
