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Abstract：
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.
摘要：
通过L1共有引物系统扩增人乳头瘤病毒（HPV）DNA（例如，MY09/11或GP5（+）/6（+））可以检测到生殖器样品中少至10至100分子的HPV靶标。然而,通过斑点印迹杂交确定基因型是费力的,并且需要至少27个单独的杂交以进行实质性的HPV类型鉴别.开发了一种反向印迹方法，该方法使用与固定的寡核苷酸探针阵列杂交的生物素标记的PCR产物。通过反向印迹分析，可以在单个杂交和洗涤循环中完成多种HPV类型的基因型区分。27种HPV探针混合物，两个对照探针浓度，将一条参考线固定在75×6毫米的尼龙条上。每个单独的探针线含有两种对独特HPV基因型特异的牛血清白蛋白缀合的寡核苷酸探针的混合物。在此条带上区分的基因型谱包括高风险，或者与癌症相关的，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)和低风险，或者与癌症无关的，基因型6、11、40、42、53、54、57、66和MM8(P155)。此外,两种浓度的β-珠蛋白探针允许在扩增后评估单个样本的充分性。我们已经相对于先前报道的斑点印迹格式（H.M.Bauer等人。,第132-152页，见C.S.赫灵顿和J.O.D.麦基(编辑。),诊断分子病理学：一种实用的方法，(1992),通过测试在Digene样本运输培养基中收集的328个宫颈拭子样本(DigeneDiagnostics，银泉,Md.).我们在两种检测格式之间表现出极好的一致性，HPV阳性的一致性为92%(κ=0.78，P<0.001)。几乎所有不同的HPV阳性样品都是由弱信号引起的，并且可以归因于来自低浓度（<1拷贝/微升）HPVDNA的样本的采样误差。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品中存在的HPV进行基因分型。最小化错误分类的可能性。
