摘要
为建立CpTI基因定性PCR检测方法,通过数据库查询和测序获得CpTI基因序列,设计特异性定性PCR引物,对引物、PCR反应条件和反应体系进行优化,并对检测方法的特异性、灵敏度等进行分析试验。结果表明,CpTI基因特异性定性PCR检测方法选择退火温度为58℃,引物浓度为0.4μmol/L较好,该方法能够特异性检测样品中CpTI基因,检测灵敏度达0.05%(质量比)。该方法符合农业转基因生物产品成分检测标准对特异性、灵敏度、重复性的要求,可为抗虫转CpTI基因植物生物安全管理提供技术支撑。
To develop the qualitative PCR assay for detection cowpea trypsin inhibitor（CpTI） gene,a series of gene-specific qualitative PCR primers were designed according to the sequences of CpTI gene from GenBank and Sanger sequencing.The annealing temperature of primers,PCR reaction system and PCR amplification conditions were optimized,and the specificity and sensitivity of reactions were tested.The results showed that the optimal annealing temperature was 58 ℃ and the optimal primer concentration was 0.4 μmol/L.The CpTI gene could be distinguished specifically among the samples by this method,and the limit of detection sensitivity was 0.05%（w/w）,which accorded with the request of the genetically modified organisms（GMO） testing standard for specificity,sensitivity and repeatability.This detection method provided the corresponding technology support to the genetically modified organisms safety management of insect-resistant plant with CpTI gene.
出处
《河南农业科学》
CSCD
北大核心
2012年第5期56-60,共5页
Journal of Henan Agricultural Sciences
基金
科技部国际合作项目(2006DFA32380)
