目的:以R6G-ddATP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(HR-HPV)基因型(HPV18、HPV33和HPV35)。
方法:以HPV质控品作为样品,以R6G-ddATP双脱氧荧光试剂为底物。首先,用通用引物扩增HPV,获得第一轮扩增产物,将其纯化并用作随后的SNaPShot反应的模板。然后,使用特异性的一步延伸引物进行SNaPShot反应以产生R6G荧光标记的DNA延伸产物。将产物进行琼脂糖凝胶电泳,其结果在凝胶成像仪下观察,用不同的一步法延伸引物进行HPV基因分型。每个样品测试三次,并将结果与DNA测序结果进行比较。
结果:SNaPShot反应的优选退火温度为55℃。在最佳条件下通过R6G-ddATP/SNaPShot凝胶荧光测定法检测了三种HPV基因型,结果与DNA测序结果一致。
结论:成功建立了R6G-ddATP/SNaPShot-gel荧光法微量检测三种HR-HPV基因型的方法,可用于HPV基因型的快速检测。
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.
CONCLUSIONS: 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.