HRM分析方法中不同扩增片段、染料和仪器的比较

摘要

High resolution melting curve ( HRM ) technology is a new type of technology for genotyping and mutation scanning developing in recent years based on the technology of real⁃time fluorescence quantitative PCR.This study compared several causative agents of animal diseases,including Classical swine fever virus/Ei⁃meria species/Avain leukemia virus.The effects of different amplified regions of pathogens’ genomes(C202/ALV450/ En573 / C965),different fluorescent dyes(SYBR GreenⅠ/ LC Green / SYTO9)and different instruments(LightScanner96(Idaho) / LightCycler480(Roche) / Rotor⁃Gene Q(Qiagen)) on the HRM⁃based genotyping.Theexperimental data showed that HRM analysis was mainly affected by different amplified fragments and fluorescentdyes with different properties:nucleic acids with multiple melting range had higher specificity than that of singlenucleotide polymorphism(SNP)and were more suitable for differentiation analysis; difference in Tm on multiplemelting range influenced HRM results; saturated fluorescent dyes(LC Green and SYTO9) had higher resolutionthan unsaturated dye(SYBR GreenⅠ); concentration of fluorescent dye had a greater influence on the value ofTm; there was no significant difference in the HRM results from different PCR⁃HRM analyzers.%高分辨率熔解曲线技术（ High resolution melting，HRM）是近年发展起来的基于实时荧光定量技术分析核苷酸突变而进行基因分型的一种新型技术。比较了几种动物疫病病原（猪瘟病毒、禽白血病病毒、鸡艾美耳球虫株）的不同扩增片段（ C202、ALV450、En573和C965）、不同荧光染料（ LC Green、SYTO9和SYBR GreenⅠ）和不同仪器[LightScanner96（Idaho）， LightCycler480（Roche）和Rotor－Gene Q（Qiagen）]在HRM基因分型上的异同，揭示了HRM技术对病原体进行分型时，不同扩增片段和不同性质荧光染料对其分型结果影响较大：拥有多个熔解区间的核酸序列比单独1个SNP位点特异性高，更适合于分型；核酸相邻熔解区间Tm差值影响实际HRM分型结果；饱和荧光染料（LC Green和SYTO9）比非饱和染料（SYBR GreenⅠ）分辨率更高，更适合用于HRM分析，并且不同浓度荧光染料对Tm值有一定的影响；不同HRM仪器之间HRM结果无明显差异。