Feng Chuan-Rui, Yang Sen, Wang Yue-Qi, Liu Ying-Ke, Chen Xiu-Yuan, Zeng Shu-Shu, Luo Ya-Xiong, Gao Xiu-Feng
Department of Biochemistry and Molecular Biology, West China School of Basic Medical Sciences and Forensic Medicine, Sichuan University, Chengdu 610041, China.
West China School of Basic Medical Sciences and Forensic Medicine, Sichuan University, Chengdu 610041, China.
Sichuan Da Xue Xue Bao Yi Xue Ban. 2021 Jan;52(1):98-103. doi: 10.12182/20210160107.
R6G-ddATP was used as a dideoxy fluorescence substrate to establish the single base end extension (SNaPShot)-gel fluorescence method for the rapid detection of the genotypes of three high-risk human papillomaviruses (HR-HPV) ( 18, 33 and 35) genotypes.
HPV quality control products were used as as samples, and R6G-ddATP dideoxy fluorescence reagent was used as substrate. Firstly, HPV was amplified by using universal primers to obtain the first round of amplified products, which were purified and used as templates for subsequent SNaPShot reactions. Then, specific one-step extension primers were used to perform SNaPShot reaction to generate R6G-fluorescence-labeled DNA extension products. The product was subjected to agarose gel electrophoresis, the results of which were observed under a Gel Imager, and the HPV genotyping was done with different one-step extension primers. Each sample was tested three times and the results were compared with DNA sequencing results.
The preferred annealing temperature for SNaPShot reaction is 55 ℃. Three genotypes were examined by R6G-ddATP/SNaPShot gel fluorescence assay under optimal conditions, and the results were consistent with DNA sequencing results.
The R6G-ddATP/SNaPShot-gel fluorescence method for the micro-detection methods of three HR-HPV genotypes was successfully established and can be used for rapid detection of genotypes.
以罗丹明6G -双脱氧三磷酸腺苷（R6G-ddATP）作为双脱氧荧光底物，建立单碱基延伸（SNaPShot）-凝胶荧光法，用于快速检测3种高危型人乳头瘤病毒（HR-HPV）（18、33和35型）基因型。
以HPV质控品为样本，采用R6G-ddATP双脱氧荧光试剂作为底物。首先，用通用引物扩增HPV，获得第一轮扩增产物，经纯化后作为后续SNaPShot反应的模板。然后，使用特异性一步延伸引物进行SNaPShot反应，生成罗丹明6G荧光标记的DNA延伸产物。产物进行琼脂糖凝胶电泳，在凝胶成像仪下观察结果，采用不同的一步延伸引物进行HPV基因分型。每个样本检测3次，并将结果与DNA测序结果进行比较。
SNaPShot反应的最佳退火温度为55℃。在最佳条件下，采用R6G-ddATP/SNaPShot凝胶荧光分析法检测3种基因型，结果与DNA测序结果一致。
成功建立了用于3种HR-HPV基因型微量检测的R6G-ddATP/SNaPShot-凝胶荧光法，可用于基因型的快速检测。
