基于R6G-ddATP/SNaPShot-凝胶荧光法的高危型人乳头瘤病毒基因型微量检测

  目的  使用R6G-ddATP作为双脱氧荧光底物建立单碱基末端延伸（SNaPShot）-凝胶荧光法快速检测3种高危型人乳头瘤病毒（high risk human papillomavirus，HR-HPV）（HPV18、HPV33、HPV35）基因型。  方法  使用HPV质控品作为样本，R6G-ddATP双脱氧荧光试剂作为底物，首先利用通用引物对HPV进行扩增，得到第一轮扩增产物，经纯化后作为后续SNaPShot反应的模板；然后利用特异性的一步延伸引物进行SNaPShot反应，生成带有R6G荧光标记的DNA延伸产物；产物经过琼脂糖凝胶电泳，在凝胶成像仪下观察电泳结果，通过不同的一步延伸引物对HPV进行分型。每个样本均重复检测3次，并与DNA测序结果进行比较。  结果  优选的SNaPShot反应的退火温度为55℃；仅需3 h即可对HPV进行分型；在该最适条件下使用R6G-ddATP/SNaPShot-凝胶荧光法检测3种HPV基因型，检测结果与测序结果一致。  结论  成功建立了3种HR-HPV基因型的微量检测方法——R6G-ddATP/SNaPShot-凝胶荧光法，可用于HPV基因型的快速检测。

Microassay of High-Risk Human Papillomavirus Genotype Based on R6G-ddATP/SNaPshot-Gel Fluorescence Method

  Objective  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) (HPV18, HPV33 and HPV35) genotypes.  Methods  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.  Results  The preferred annealing temperature for SNaPShot reaction is 55 ℃. Three HPV genotypes were examined by R6G-ddATP/SNaPShot gel fluorescence assay under optimal conditions, and the results were consistent with DNA sequencing results.  Conclusion  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 HPV genotypes.