Next generation sequencing for human papillomavirus genotyping
Next generation sequencing for human papillomavirus genotyping
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DOI：
10.1016/j.jcv.2013.07.013
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发表时间：
2013-10-01
影响因子：
8.8
通讯作者：
Dillner, Joakim
中科院分区：
文献类型：
--
作者：
Arroyo, L. Sara;Smelov, Vitaly;Dillner, Joakim
背景：使用下一代测序（NGS）进行人乳头瘤病毒（HPV）基因分型可能有助于研究HPV变异特异性流行病学，包括在引入HPV疫苗接种计划后监测可能出现的新的HPV变异。目的：我们希望设计并验证一种在临床样本中快速检测、分型和测序的方法。研究设计：将15种不同HPV类型的质粒混合并在每个样本中按1至100份的浓度在人类DNA中连续稀释，使用HPV通用PCR引物对PGMY进行扩增，并使用454技术进行测序。采用基于ngs的方法和基于与荧光珠结合的类型特异性探针（Luminex）进行基因分型的比较方法对60例宫颈样本进行了检测。采用NGS法对33份临床样本进行重复性检测。结果：基于ngs的方法在100拷贝/样本中正确识别了所有15种混合HPV类型，在10拷贝/样本中正确识别了13115种混合HPV类型。对36160份宫颈样本，NGS和Luminex基因分型结果相同。对于12160个样品，NGS方法比Luminex测试更敏感，其余的大部分差异可以通过分析的不同类型覆盖来解释。重复性测试发现30133个样本完全或部分一致。结论：NGS是一种灵敏、准确的HPV基因分型方法。扩增子序列的获得对于研究病毒变异的流行病学和监测HPV疫苗接种具有重要意义。(C) 2013 Elsevier B.V.版权所有
Background: Human papillomavirus (HPV) genotyping using next generation sequencing (NGS) could be useful to study the HPV variant-specific epidemiology, including monitoring for possible emergence of new HPV variants after introduction of HPV vaccination programs.Objectives: We wished to design and validate a method for rapid HPV detection, typing and sequencing in clinical samples.Study design: Plasmids of 15 different HPV types were mixed and serially diluted in human DNA in concentrations ranging from 1 to 100 copies per sample, amplified using the HPV general PCR primer pair PGMY and sequenced using 454 technology. Sixty cervical samples were tested both with the NGS-based method and with a comparison method based on genotyping using type-specific probes bound to fluorescent beads (Luminex). Thirty-three clinical samples were repeat tested using NGS to evaluate reproducibility.Results: The NGS-based method correctly identified all 15 mixed HPV types when present in 100 copies/sample and 13115 types when present in 10 copies/sample. For 36160 cervical samples genotyping results using NGS and Luminex were identical. For 12160 samples the NGS method was more sensitive than the Luminex test and most of the remaining discrepancies could be explained by the different type coverage of the assays. Reproducibility testing found complete or partial concordance in 30133 samples.Conclusions: NGS provides a sensitive and accurate method for genotyping of HPV. The fact that also the amplimer sequence is obtained could be important for studying the epidemiology of viral variants and monitoring of HPV vaccination. (C) 2013 Elsevier B.V. All rights reserved.