将跨越式滚环等温扩增技术(SRCA)与荧光技术相结合,建立一种实时荧光SRCA技术检测蜂蜜掺伪大米糖浆的方法.对DNA提取方案进行评估,以大米的特异性基因(PDL、SPS、rbcL、GOS9)为靶序列设计引物,筛选适宜的引物,优化扩增反应条件结合荧光技术建立检测蜂蜜掺伪大米糖浆的方法,并对该方法进行评价.结果表明,建立的实时荧光SRCA方法检测大米DNA的灵敏度为8.45×101 fg/μL,经特异性评价证实其特异性良好,在人工模拟掺伪检测中建立掺伪比例的对数与Ct值的线性关系,线性方程为y=6.618x+7.651(R2=0.993),可准确检出蜂蜜中低至1%的大米糖浆成分.该方法灵敏度高,检出限低,能够快速、准确检测蜂蜜掺伪大米糖浆,为蜂蜜掺伪的快速检测提供了新思路.
In this study,It combined saltatory rolling circle amplification(SRCA)with fluorescence technology to estab-lish a real-time fluorescence SRCA technique for detection of honey adulterated rice syrup.Three DNA extraction proto-cols were evaluated,four specific genes of rice(PDL,SPS,rbcL,GOS9)were used as target sequences to design primers,suitable primers were screened,amplification reaction conditions were optimized,and a method for detection of honey adulterated rice syrup was established in combination with fluorescence technology.The results showed that the sensitivity of the established real-time fluorescence SRCA method for the detection of rice DNA was 8.45×101 fg/μL,and its good specificity was confirmed by the specificity evaluation.The linear relationship between the logarithm of the pro-portion of adulteration and the Ct value was established in the manual simulated adulteration detection test,and the lin-ear equation was y=6.618x+7.651(R2=0.993),which can accurately detect the components of rice syrup in honey down to 1%.The method is sensitive,with low detection limit,and can quickly and accurately quantify the adulteration of honey with rice syrup,which provides a new idea for the rapid quantitative detection of honey adulteration.