痕量及微量转基因大米成分半巢式PCR检测方法的建立  

Establishment of Semi-nested PCR Detection Method for Trace Transgenic Rice Materials

作　　者：闻伟刚[1] 盛蕾[1] 张吉红[1] 谭钟[1] 

机构地区：[1]宁波出入境检验检疫局,浙江宁波315012

出　　处：《食品科学》2008年第12期622-626,共5页Food Science

基　　金：宁波市科技计划项目(2005C100094)

摘　　要：采用半巢式PCR技术建立了食品中痕量及微量转基因大米成分的检测方法。根据大米内源SPS基因以及转基因大米Bt63含有的外源Cry1A(b)/Cry1A(c)融合基因和Btc基因,分别设计了6对普通与半巢式PCR引物。扩增结果显示,针对理论DNA浓度的检测灵敏度,普通PCR扩增灵敏度为1ng/μl左右,半巢式PCR扩增灵敏度达到10-3～10-4ng/μl,半巢式PCR比普通PCR方法提高了103～104倍;在质量百分比的检测灵敏度方面,普通PCR方法扩增灵敏度在0.1%～1%之间,半巢式PCR方法的检测灵敏度能够达到0.001%～0.01%,半巢式PCR比普通PCR方法要提高10倍以上。在实际样品的检测中,速冻汤圆、婴儿米粉及芝士小圆饼等食品经普通PCR扩增,其内源SPS基因条带模糊或未扩增到,进一步采用半巢式PCR扩增后,能够得到清晰明亮的特异性条带。所有样品均未扩增到外源Cry1A(b)/Cry1A(c)和Btc基因。A method for detection of trace transgenic rice materials was built by using semi-nested PCR. According to SPS, CrylA （byCrylA（c） and Btc genes in transgenic rice Bt63, six pairs of primers were designed for general PCR and semi-nested PCR. Results of amplification showed that detection sensitivity of general PCR and semi-nested PCR in accordance with DNA concentration are 1 ng/μl and 10^-3 to 10^4 ng/μ1, respectively. The latter is 10^3 to 10^4 times of the former. In the aspect of mass percentage, the detection sensitivity of general PCR is 0.1% to 1% and semi-nested PCR is 0.001% to 0.01%, the latter being at least 10 times of the former. In the transgenic detection of food samples, the DNA band of SPS gene could not be obtained or confused by general PCR from frozen rice dumpling, rice cereal and cheese pancake samples. Further amplification with semi-nested PCR, a distinct and unique DNA band of SPS gene could be observed from these samples. The other two genes, CryIA（b）/CrylA（c） and Btc are not obtained with general PCR and semi-nested PCR from all the food samples.

关 键 词：痕量及微量转基因大米 半巢式PCR 检测 

分 类 号：TS207.3[轻工技术与工程—食品科学]