基于 SPF10 引物和 INNO-LiPA HPV Genotyping Extra assay 的实时 PCR 方法用于检测和分型人乳头瘤病毒。
A real-time PCR approach based on SPF10 primers and the INNO-LiPA HPV Genotyping Extra assay for the detection and typing of human papillomavirus.
机构信息
Applied Molecular Biology Research group, Laboratory of Cell Biology and Histology, University of Antwerp, Groenenborgerlaan 171, Antwerp, Belgium.
出版信息
J Virol Methods. 2013 Jan;187(1):166-71. doi: 10.1016/j.jviromet.2012.09.013. Epub 2012 Sep 24.
The SPF10 PCR targets a conserved 65bp region of the HPV L1 gene for broad-spectrum amplification. The LiPA assay allows subsequent genotyping of the HPV amplicons. This study aims to develop a SPF10 real-time PCR to achieve simultaneous amplification and detection of the HPV target. That way, LiPA analysis of the HPV-negative samples can be avoided, reducing workload and cost. The real-time PCR shows an analytical sensitivity of 29.7 copies for HPV 6, 16, 18 and 31 and an HPV-specific melting peak. Thirty-one HPV DNA plasmids were genotyped correctly using the SPF10 real-time PCR in combination with the LiPA. Here, the LiPA assay was performed at an increased hybridisation temperature (49.5°C) in combination with a reduced amplicon volume (1μl) to avoid cross-reactivity. In conclusion, the SPF10 real-time PCR proves to be very sensitive and generates amplicons, which are compatible with the LiPA.
SPF10 PCR 针对 HPV L1 基因的保守 65bp 区域进行广谱扩增。LiPA 检测法可对 HPV 扩增子进行后续基因分型。本研究旨在开发一种 SPF10 实时 PCR 以实现 HPV 靶标的同时扩增和检测。这样，就可以避免对 HPV 阴性样本进行 LiPA 分析，从而减少工作量和成本。实时 PCR 显示 HPV 6、16、18 和 31 的分析灵敏度为 29.7 拷贝，并且具有 HPV 特异性的熔解峰。使用 SPF10 实时 PCR 结合 LiPA 可正确对 31 种 HPV DNA 质粒进行基因分型。在此，将 LiPA 检测法在增加的杂交温度（49.5°C）下与减少的扩增子体积（1μl）一起使用，以避免交叉反应。总之，SPF10 实时 PCR 证明非常敏感，并产生与 LiPA 兼容的扩增子。
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