Genotyping of 27 human papillomavirus types by using L1 consensus PCR products by a single-hybridization, reverse line blot detection method
Genotyping of 27 human papillomavirus types by using L1 consensus PCR products by a single-hybridization, reverse line blot detection method
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DOI：
10.1128/jcm.36.10.3020-3027.1998
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发表时间：
1998-10-01
影响因子：
9.4
通讯作者：
Wheeler, CM
中科院分区：
文献类型：
--
作者：
Gravitt, PE;Peyton, CL;Wheeler, CM
通过L1共有引物系统（例如，MY 09/11或GP 5（+）/6（+））扩增人乳头瘤病毒（HPV）DNA可以从生殖器样品中检测到少至10至100个分子的HPV靶分子。然而，通过斑点印迹杂交进行基因型测定是费力的，并且需要至少27次单独的杂交来进行实质性的HPV类型区分。建立了一种反向杂交方法，将生物素标记的PCR产物与固定化寡核苷酸探针阵列杂交，通过反向杂交条分析，在一次杂交和洗涤循环中即可完成多种HPV基因型的鉴别。将27种HPV探针混合物、两种对照探针浓度和一条参考线固定在75 × 6 mm尼龙条上。每个单独的探针线含有对独特的HPV基因型特异的两种牛血清白蛋白缀合的寡核苷酸探针的混合物。在该条上辨别的基因型谱包括高风险或癌症相关的HPV基因型16、18、26、31、33、35、39、45、51、52、55、56、58、59、68（ME 180），MM 4（W13 B）、MM 7（P291）和MM 9（P238 A）以及低风险或非癌症相关的基因型6、II、40、42、53、54、57、66和MM 8（P155）。此外，P-珠蛋白探针的最大浓度允许在扩增后评估个体标本的充分性，我们已经评估了船舶方法的性能相对于以前报道的斑点印迹格式（H。RI. Bauer等人，第132-152页，载于C，S，Herrington和J.O. D，McGee（编辑），诊断分子病理学：a Practical Approach，（1992），通过测试在Digene样本转运培养基（Digene Diagnostics，银Spring，MD）中收集的328个宫颈拭子样品，我们发现两种检测方式之间具有很好的一致性，BPV阳性的一致性为92%（kappa = 0.78，P < 0.001）。几乎所有不一致的HPV阳性样本都是由弱信号引起的，并且可以归因于低浓度（< 1拷贝/μ l）HPV DNA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本中存在的HPV进行基因分型，从而最大限度地减少错误分类的可能性。
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, II, 40, 42, 53, 54, 57, 66, and MM8 (P155), In addition, mo concentrations of P-globin probes allowed for assessment of individual specimen adequacy following amplification, We have evaluated the performance of the ship method relative to that of a previously reported dot blot format (H. RI. 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 BPV 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/mu l) 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.