Gravitt P E, Peyton C L, Apple R J, Wheeler C M
Department of Human Genetics, Roche Molecular Systems, Inc., Alameda, California 94501, USA.
J Clin Microbiol. 1998 Oct;36(10):3020-7. doi: 10.1128/JCM.36.10.3020-3027.1998.
Amplification of human papillomavirus (HPV) DNA by L1 consensus primer systems (e.g., MY09/11 or GP5(+)/6(+)) can detect as few as 10 to 100 molecules of HPV targets from a genital sample. However, genotype determination by dot blot hybridization is laborious and requires at least 27 separate hybridizations for substantive HPV-type discrimination. A reverse blot method was developed which employs a biotin-labeled PCR product hybridized to an array of immobilized oligonucleotide probes. By the reverse blot strip analysis, genotype discrimination of multiple HPV types can be accomplished in a single hybridization and wash cycle. Twenty-seven HPV probe mixes, two control probe concentrations, and a single reference line were immobilized to 75- by 6-mm nylon strips. Each individual probe line contained a mixture of two bovine serum albumin-conjugated oligonucleotide probes specific to a unique HPV genotype. The genotype spectrum discriminated on this strip includes the high-risk, or cancer-associated, HPV genotypes 16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 55, 56, 58, 59, 68 (ME180), MM4 (W13B), MM7 (P291), and MM9 (P238A) and the low-risk, or non-cancer-associated, genotypes 6, 11, 40, 42, 53, 54, 57, 66, and MM8 (P155). In addition, two concentrations of beta-globin probes allowed for assessment of individual specimen adequacy following amplification. We have evaluated the performance of the strip method relative to that of a previously reported dot blot format (H. M. Bauer et al., p. 132-152, in C. S. Herrington and J. O. D. McGee (ed.), Diagnostic Molecular Pathology: a Practical Approach, (1992), by testing 328 cervical swab samples collected in Digene specimen transport medium (Digene Diagnostics, Silver Spring, Md.). We show excellent agreement between the two detection formats, with 92% concordance for HPV positivity (kappa = 0.78, P < 0.001). Nearly all of the discrepant HPV-positive samples resulted from weak signals and can be attributed to sampling error from specimens with low concentrations (<1 copy/microliter) of HPV DNA. The primary advantage of the strip-based detection system is the ability to rapidly genotype HPVs present in genital samples with high sensitivity and specificity, minimizing the likelihood of misclassification.
通过L1通用引物系统（如MY09/11或GP5(+)/6(+)）扩增人乳头瘤病毒（HPV）DNA，可从一份生殖器样本中检测到低至10到100个HPV靶分子。然而，通过斑点杂交进行基因型测定既费力，又需要至少27次单独杂交才能对HPV各型进行有效区分。于是开发了一种反向杂交方法，该方法采用生物素标记的PCR产物与固定化寡核苷酸探针阵列杂交。通过反向杂交条带分析，在一次杂交和洗涤循环中就能完成多种HPV型别的基因型区分。将27种HPV探针混合物、两种对照探针浓度和一条单一参考线固定在75×6毫米的尼龙条上。每条单独的探针线都包含两种牛血清白蛋白偶联的寡核苷酸探针的混合物，这些探针特异于一种独特的HPV基因型。在这条带上区分出的基因型谱包括高危型或与癌症相关的HPV基因型16、18、26、31、33、35、39、45、51、52、55、56、58、59、68（ME180）、MM4（W13B）、MM7（P291）和MM9（P238A），以及低危型或与癌症无关的基因型6、11、40、42、53、54、57、66和MM8（P155）。此外，两种浓度的β-珠蛋白探针可用于评估扩增后各个样本的充足性。我们通过检测在Digene样本运输培养基（Digene诊断公司，马里兰州银泉）中收集的328份宫颈拭子样本，评估了这种条带法相对于先前报道的斑点杂交法（H.M.鲍尔等人，第132 - 152页，载于C.S.赫林顿和J.O.D.麦吉编著的《诊断分子病理学：实用方法》，1992年）的性能。我们发现这两种检测方法具有极佳的一致性，HPV阳性的一致性为92%（kappa = 0.78，P < 0.001）。几乎所有不一致的HPV阳性样本都是由于信号较弱导致的，这可归因于HPV DNA浓度低（<1拷贝/微升）的样本的取样误差。基于条带的检测系统的主要优点是能够以高灵敏度和特异性快速对生殖器样本中存在的HPV进行基因分型，将错误分类的可能性降至最低。
