【摘要】 [目的]通过分析样品前处理对大米及米制品转基因成分荧光PCR检测结果(Ct值)准确性的影响,建立优化的前处理方法。[方法]样品DNA提取采用CTAB法,设置3种样品颗粒细度与3种样品DNA提取温育时间的组合共9种处理,应用双因素方差分析的F检验分析处理间Ct值的差异显著性,根据t检验结果,筛选出DNA提取与荧光PCR检测效果最佳的前处理样品细度与CTAB温浴时间组合。[结果]F检验结果:在9种处理组合中,处理组合之间的大米及米制品DNA提取浓度都存在极显著差异;不同处理对大米内源基因GOS与外源CaMV 35S基因的Ct值影响极显著;对于米制品白粿干与米粉干,CTAB温浴时间对内源基因GOS与CaMV35S基因Ct值的影响达到显著或极显著水准,而样品颗粒大小仅对白粿干的CaMV 35S基因Ct值有显著影响,对米粉干的GOS与CaMV 35S基因及白粿干GOS的Ct值的影响都不显著。t检验结果表明最佳处理组合为:大米颗粒细度>100目,CTAB缓冲液温浴时间>8h;白粿干样品颗粒细度>50目,CTAB缓冲液浸泡温浴时间≥4h;米粉干颗粒细度>50目,CTAB缓冲液浸泡温浴时间>8h。更多还原

【Abstract】 Through analyzing the effects of sample preparations on the accuracy of detection results of genetically modified ingredients of rice and its products by fluorescence PCR,optimized preparation methods were established.CTAB method was used for DNA extraction.Nine treatment combinations of 3 levels of sample particle size and 3 levels of CTAB immersion bath time during DNA extraction process were set up.The significant differences in the Ct values obtained from various treatment combinations was analyzed by F test of double factor variance analysis.The best treatment combinations for DNA extraction and real-time fluorescence PCR detection were selected by t test.The results showed that significant differences were found among treatment combinations for DNA concentration of rice and rice products in 9 treatment combinations mentioned above.The real-time fluorescence PCR detection results(Ct values) of rice endogenous gene GOS and exogenous gene CaMV35S were extremely significant among different treatment combinations.In rice cake and rice noodle,influence of CTAB immersion bath time on Ct value of GOS gene and CaMV35S gene was significant or extremely significant.Significant effect of sample particle size on Ct value of CaMV35S gene was only found in rice cake,and no significant effect of that on Ct value of GOS gene and CaMV35S gene in rice noodle,as well as on Ct value of GOS gene in rice cake,was found.The best treatment combination for DNA extraction and fluorescence PCR detection were determined as follow: sample particle size >100 mesh and CTAB buffer immersion warm bath time >8h during DNA extraction in rice sample,sample particle size >50mesh and CTAB buffer solution immersion warm bath time ≥4h in rice cake slice;sample particle size >50 mesh and CTAB buffer solutionimmersion warm bath time >8h in rice noodle.更多还原