FQ-PCR与IMSA检测转基因豆奶外源基因的比较研究

Comparison of FQ-PCR and IMSA for Detecting Exogenous Genes in Genetically Modified Soymilk

为比较实时荧光定量PCR(FQ-PCR)与等温多自配引发扩增(IMSA)技术检测转基因豆奶中外源基因(CaMV35S、NOS、EPSPS)的灵敏度和特异性。合成CaMV35S、NOS以及EPSPS基因的质粒,通过FQ-PCR与IMSA检测定量的质粒,比较质粒灵敏度;按0. 50%、0. 10%、0. 05%以及0. 01%的比例制作转基因豆奶,通过FQ-PCR与IMSA检测转基因大豆标准品制成的豆奶,比较样品灵敏度和特异性。结果表明:IMSA检测CaMV35S、NOS、EPSPS基因的质粒灵敏度分别为31. 01,31. 01,3. 10 cps·μL^(-1),FQ-PCR检测CaMV35S、NOS、EPSPS基因的质粒灵敏度皆为31. 01cps·μL^(-1); IMSA检测CaMV35S、NOS、EPSPS基因的样品灵敏度分别为0. 05%、0. 05%、0. 01%,FQ-PCR检测CaMV35S、NOS、EPSPS基因的样品灵敏度皆为0. 10%; FQ-PCR非特异性检出DP356043的NOS以及MON87701的CaMV35S与EPSPS。说明IMSA方法检测转基因豆奶外源基因(CaMV35S、NOS、EPSPS)的灵敏度与特异性优于FQ-PCR。

In order to compare the sensitivity and specificity between real-time fluorescent quantitative PCR （FQ-PCR） and isothermal multiple self-matching-initiated amplification （IMSA） detecting exogenous gene （CaMV35S, NOS, EPSPS） of ge- netically modified soymilk. The sensitivity of plasmids which were constructed with CaMV35S, NOS and EPSPS gene between FQ-PCR and IMSA were compared. The genetically modified soymilk was made at a ratio of 0. 50%, 0. 10%, 0. 05% and 0. 01% to compare sample detection sensitivity and specificity between FQ-PCR and IMSA. The results showed that the plas- mid sensitivity of CaMV35S, NOS and EPSPS gene in genetically modified soymilk detected by IMSA were 31. 01, 31.01, 3.10 cps·μL^-1 respectively. And the plasmid sensitivity of the three genes detected by FQ-PCR was 31.01 cps·μL^-1. The sample sensitivity of CaMV35S, NOS and EPSPS gene in genetically modified soymilk detected by IMSA were 0. 05%, 0. 05%, 0. 01% respectively. And the sample sensitivity of the three genes detected by FQ-PCR was 0. 1%. False positive of NOS in DP305423 and CaMV35S, EPSPS in MON87701 by FQ-PCR. It indicated that the sensitivity and specificity of IMSA on detecting exogenous gene （C, aMV35S, NOS, EPSPS） of genetically modified soymilk was much better than FQ-PCR.