Objective For implementation of labeling regulations,an event-specific real-time polymerase chain reaction (PCR) method for the detection of genetically modified AquAdvantage salmon was established in this study.Methods Primers and TaqMan probe were designed based on the event-specific sequence of AquAdvantage salmon.The specificity,sensitivity and repeatability of the developed method were examined,respectively.Results The specificity test of this method showed it was specific to AquAdvantage salmon.The 600 000-60 copies range showed a good linear relationship with Ct values,and its linear regression equation was y =-3.2194x + 40.805 (R2 =0.997).The limit of quantification (LOQ) was 60 copies and the repeatability was good.Conclusion This event-specific real-time PCR method was suitable for the identification of genetically modified AquAdvantage salmon.

根据转基因低木质素苜蓿草品系KK179-25'端外源插入序列与苜蓿草基因组DNA之间的邻接区序列设计引物和探针,建立了KK179-2品系特异性实时荧光PCR检测方法,并对其特异性、灵敏度及可重复性进行了测定.结果表明:建立的定量实时荧光PCR检测方法特异性良好;标准曲线线性相关系数(R2)为0.99,扩增效率E为105%;检测限为20拷贝,定量限为200拷贝.重复性实验显示本方法的标准偏差(SD)及相对标准偏差(RSD)都在可接受范围内.综上,建立的KK179-2品系特异性定量PCR检测方法适于快速、准确、稳定地对转基因苜蓿草KK179-2品系进行检测.

目的 建立同时检测单增李斯特菌(Listeria monoc ytogenes,Lm)溶血素基因hlyA、内化素基因inlA和磷脂酶C基因plcB的TaqMan-MGB三重实时荧光PCR检测方法,并对101株单增李斯特菌的hlyA、inlA和plcB基因进行了检测,分析3个毒力基因在多位点序列分型(Multilocus sequence typing,MLST)聚类群组中的分布情况.方法 根据单增李斯特菌毒力基因hlyA、inlA和plcB的保守序列设计引物和小沟结合物(minor groove binder,MGB)探针建立三重荧光PCR方法,对其特异性、灵敏度和可重复性进行了测试,并对分离的101株单增李斯特菌进行三重PCR检测.结果 建立的三重实时荧光PCR方法特异于单增李斯特菌的检测,重复性良好,组内Ct值变异系数均小于1.5％,灵敏度可达100 CFU/mL.对101株单增李斯特菌的hlyA、inlA和plcB的检出率分别为98％、75％和98％.在可被MLST分型的89株菌株中,groupⅠ的30株菌株有20株均缺失inlA基因;groupⅡ的57株菌株中有56株三个基因均检出;groupⅢ的菌株hlyA、inlA和plcB三个基因均未检出.结论 本文建立的三重实时荧光PCR方法能准确、快速、稳定的检测单增李斯特菌,101株单增李斯特菌的hlyA、inlA、plcB三个毒力基因的检出情况与单增李斯特菌的MLST分型聚类有较一致的关系,对分析单增李斯特菌毒力的强弱有重要参考意义.