Automated high throughput DNA isolation for detection of human papillomavirus in oral rinse samples
Automated high throughput DNA isolation for detection of human papillomavirus in oral rinse samples
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DOI：
10.1016/j.jcv.2010.12.005
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发表时间：
2011-04-01
影响因子：
8.8
通讯作者：
Gillison, Maura L.
中科院分区：
文献类型：
--
作者：
Broutian, Tatevik R.;He, Xin;Gillison, Maura L.
背景：口腔 HPV 感染会增加口咽癌的风险，但其自然史尚不清楚。自然史研究需要验证口腔冲洗样品 (ORS) 中 HPV 基因组 DNA 检测的自动化、高通量方法。 目标：比较基于磁珠的自动化平台处理的 ORS 中口腔 HPV 检测与之前的金标准手动蛋白沉淀方法的一致性。将一致性与重复采样和重复 HPV 检测的一致性进行比较。研究设计：HIV 感染者 (n = 100) 提供两次 ORS，间隔 15 分钟收集。通过基于蛋白质沉淀的方法（Puregene，Qiagen）或基于磁珠的方法（QIAsymphony (TM) SP，Qiagen）从等份中分离DNA。通过共有引物 PCR 和反向线印迹杂交检测 HPV DNA 并确定类型。 kappa 统计用于评估总体一致性 (OA) 和阳性检测一致性 (Ps+)。 结果：DNA 纯化方法对于将个体分类为 HPV 感染（OA = 0.95；Ps+ = 0.94）以及在 ORS 中检测 HPV 类型特异性感染（OA = 0.99；Ps+ = 0.88）具有非常高的一致性。 HPV 类型特异性感染检测的一致性大于重复口腔冲洗取样观察到的一致性（OA = 0.99，Ps+ = 0.76），但与测定间一致性（OA = 1.00，Ps+ = 0.90）相当。结论：使用基于磁珠的自动化平台处理的 ORS 中的 HPV 检测将促进口腔 HPV 感染的大型自然史研究，这对于评估口腔 HPV 检测在口腔癌筛查中的潜在用途是必要的。 (C) 2011 Elsevier B.V. 保留所有权利。
Background: Oral HPV infection elevates risk of oropharyngeal cancer, but its natural history is unknown. Natural history studies necessitate validation of an automated, high-throughput method for HPV genomic DNA detection in oral rinse samples (ORS).Objectives: To compare agreement of oral HPV detection in ORS processed by a magnetic-bead based automated platform to a previous gold-standard, manual protein-precipitation method. Agreement was compared to that of repeat sampling and repeat HPV testing.Study design: HIV-infected individuals (n = 100) provided two ORS collected 15 min apart. DNA was isolated from equal aliquots by either a protein-precipitation based (Puregene, Qiagen) or magnetic bead-based (QIAsymphony (TM) SP, Qiagen) method. HPV DNA was detected and type-specified by consensus primer PCR and reverse line blot hybridization. The kappa statistic was used to assess overall agreement (OA) and agreement on a positive test (Ps+).Results: The DNA purification methods had very high agreement for categorizing an individual as HPV infected (OA = 0.95; Ps+ = 0.94) as well as for detection of HPV type-specific infection (OA = 0.99; Ps+ = 0.88) in ORS. Agreement for detection of HPV type-specific infection was greater than that observed with repeat oral rinse sampling (OA = 0.99, Ps+ = 0.76) but comparable to inter-assay agreement (OA = 1.00, Ps+ = 0.90).Conclusions: HPV detection in ORS processed with a magnetic-bead based automated platform will facilitate large natural history studies of oral HPV infection necessary to evaluate the potential use of oral HPV detection in oral cancer screening. (C) 2011 Elsevier B.V. All rights reserved.