摘要
建立高效、快速、准确的抗虫转基因玉米MIRl62转化体特异性实时荧光PCR（real—timePCR）检测方法。采用SYBRGreenI和Taqman探针2种定量方法对抗虫转基因玉米MIRl62进行了检测研究。SYBRGreenI法MIRl62转化体特异性片段和内标基因zSSIlb的熔解曲线均表现为单一峰，扩增特异性强；二者的扩增效率均在90％100％之间，标准曲线相关系数帮均大于0．998。2种定量方法的检测限（LOD）均为24个拷贝，定量限旺D0为48个拷贝，灵敏度较高。重复测试结果表明，SYBRGreenI法的标准偏差（㈣变化范围为0．05～0．13，相对标准偏差（RSD）范围为0．17％～0．40％，Taqman探针法的SD变化范围为0．03～0．14，RSD范围为0．11％～0．44％，说明2种定量检测方法具有良好的重复性和精密度。对MIRl62含量为3％、1％和0．5％的混合样品的定量结果为：3．13％、1．09％、0．53％（SYBRGreenI法）和3．07％、1．02％、0．50％（Taqman探针法），RSD均小于10％，T检验分析表明2种定量方法定值结果差异不显著。本研究建立的2种实时荧光定量PCR均可有效对转基因玉米MIRl62进行定量检测，可根据不同实际需求及实验室条件进行选择相应的检测方法。
The aim was to build efficient, rapid and accurate event-specific quantitative real-time PCR detection methods of insect-resistant genetically modified maize MIR162. SYBR Green I and TaqMan Probe quantitative PCR methods were established in this study. The melting curves of MIR162 event-specific and endogenous reference gene zSSIIb were both single peak which mean the primers were specific. The recovery rates of SYBR Green I and TaqMan were between 90%-100% and their R2 values were both above 0.998. The limits of detection （LOD） and limits of quantitation （LOQ） of these two methods were 24 and 48 copies, respectively, which suggested that the two quantitative systems had an acceptable and high sensitivity. Moreover, the standard deviations （SD） and relative standard deviations （RSD） of repeatability ranged from 0.05 to 0.13 and 0.17% to 0.40% for SYBR Green I; 0.03 to 0.14 and 0.11% to 0.44% for TaqMan. Thereafter, three mixed maize samples （3%, 1%, 0.5%） were quantified to be 3.13%, 1.09%, 0.53% （SYBR Green I） and 3.07%, 1.02%, 0.50% （Taqman） respectively. T-test showed the quantification results of these two methods were not significantly different. The two event-specific quantitative PCR systems established in the studies wereacceptable and suitable for MIR162 identification and quantification, and the users might select corresponding method based on actual demand and laboratory conditions.
出处
《中国农学通报》
CSCD
2014年第6期101-107,共7页
Chinese Agricultural Science Bulletin
基金
转基因生物新品种培育重大专项"农业生态风险监测与控制技术"(2013ZX08012-004)
