Bead-based multiplex genotyping of human papillomaviruses
Bead-based multiplex genotyping of human papillomaviruses
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DOI：
10.1128/jcm.44.2.504-512.2006
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发表时间：
2006-02-01
影响因子：
9.4
通讯作者：
Waterboer, T
中科院分区：
文献类型：
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作者：
Schmitt, M;Bravo, IG;Waterboer, T
人类乳头瘤病毒(HPV)的DNA杂交分型方法，如反向线杂交(RLB)，是一种灵敏且有效的方法。然而，这些分析方法在高通量分析中的应用是有限的。在这里，我们描述了多重人乳头瘤病毒基因分型(MPG)的发展，这是一种定量和灵敏的高通量程序，用于在单一反应中识别多种高危和低危生殖器HPV基因型。MPG的基础是通过通用引物聚合酶链式反应(GP5+/6+)扩增HPV DNA，然后用连接到荧光标记聚苯乙烯珠上的特定类型的寡核苷酸探针检测产物(Luminex悬浮阵列技术)。MPG可同时检测多达100种不同的HPV类型，具有快速、省力的特点。我们检测了所有22种HPV类型，具有高度的特异性和重复性(中位数板间变异系数低于10%)。不同HPV类型的检测限在100至800 pg的聚合酶链式反应产物之间。我们将MPG的性能与建立的基于94个临床样本的GP5+/6+-PCR产物的RLB分析进行了比较。评价结果显示一致性很好(kappa=0.922)，但也表明mpg具有较高的敏感性。总而言之，MPG似乎非常适合大规模流行病学研究和疫苗接种试验以及常规诊断目的。
Typing of human papillomaviruses (HPV) by DNA hybridization procedures, such as reverse line blot (RLB) assay, is sensitive and well validated. However, the application of these assays to high-throughput analyses is limited. Here, we describe the development of multiplex human papillomavirus genotyping (MPG), a quantitative and sensitive high-throughput procedure for the identification of multiple high- and low-risk genital HPV genotypes in a single reaction. MPG is based on the amplification of HPV DNA by a general primer PCR (GP5+/6+) and the subsequent detection of the products with type-specific oligonucleotide probes coupled to fluorescence-labeled polystyrene beads (Luminex suspension array technology). Up to 100 different HPV types can be detected simultaneously with MPG, and the method is fast and labor saving. We detected all 22 HPV types examined with high specificity and reproducibility (the median interplate coefficient of variation was below 10%). Detection limits for the different HPV types varied between 100 and 800 pg of PCR products. We compared the performance of MPG to an established RLB assay on GP5+/6+-PCR products derived from 94 clinical samples. The evaluation showed an excellent agreement (kappa = 0.922) but also indicated a higher sensitivity of MPG. In conclusion, MPG appears to be highly suitable for large-scale epidemiological studies and vaccination trials as well as for routine diagnostic purposes.