Hong Sun Pyo, Shin Soo-Kyung, Lee Eun Hee, Kim Eun Ok, Ji Seung Il, Chung Hyun Jae, Park Sun Nie, Yoo Wangdon, Folk William R, Kim Soo-Ok
R&D Center, GeneMatrix Inc, Yongin 446-913, Korea.
Nat Protoc. 2008;3(9):1476-84. doi: 10.1038/nprot.2008.136.
We describe a matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry (MS)-based assay for human papillomavirus (HPV) genotyping--the restriction fragment mass polymorphism (RFMP) assay, which is based on mass measurement of genotype-specific oligonucleotide fragments generated by TypeIIS restriction endonuclease cleavage after recognition sites have been introduced by PCR amplification. The use of a TypeIIS restriction enzyme makes the RFMP assay independent of sequence and applicable to a wide variety of HPV genotypes, because these enzymes have cleavage sites at a fixed distance from their recognition sites. After PCR amplification, samples are subjected to restriction enzyme digestion with FokI and BtsCI and desalting using Oasis purification plates, followed by analysis by MALDI-TOF MS. Overall, the protocol is simple, takes approximately 4-4.5 h and can accurately detect and identify at least 74 different HPV genotypes.
我们描述了一种基于基质辅助激光解吸/电离飞行时间（MALDI-TOF）质谱（MS）的人乳头瘤病毒（HPV）基因分型检测方法——限制性片段质量多态性（RFMP）检测法，该方法基于对PCR扩增引入识别位点后由IIS型限制性内切酶切割产生的基因型特异性寡核苷酸片段的质量测量。IIS型限制性酶的使用使得RFMP检测法不依赖于序列，适用于多种HPV基因型，因为这些酶在距其识别位点固定距离处具有切割位点。PCR扩增后，样品用FokI和BtsCI进行限制性酶切，并用Oasis净化板进行脱盐，然后进行MALDI-TOF MS分析。总体而言，该方案简单，耗时约4 - 4.5小时，能够准确检测和鉴定至少74种不同的HPV基因型。
