转基因食物中常见外源基因PCR检测方法的建立
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摘要
通过对CTAB法、胍-氯仿法和试剂盒法三种植物基因组提取方法进行比较,筛选出回收率最大的CTAB法用于本实验样品基因组的提取。其次,以转基因作物中9种常见的外源基因序列为模板,设计出9对引物,拟扩增片段皆小于200bp以适用于深加工食品的检测,并进行了引物的特异性、灵敏度、适用性验证试验。最后,将所建立的普通PCR检测方法应用到了转基因样品的实际检测中。结果表明,所建立的针对转基因作物中9种常见外源基因的PCR检测方法特异性强、灵敏度高,相对检测限可达0.1%～0.5%,约18～90拷贝的基因组。因此,该方法可用于转基因食品及加工品中常见基因的筛选检测。
In this study, the three plant genome extraction methods of CTAB method, guanidine-chloroform method and extraction kit were compared with each other,and the CTAB extraction method with the highest recovery rate was selected for extraction of the genome of experiment samples. Then,9 pairs of primers were designed based on 9 common exogenous gene sequences in genetically modified crops. The amplicon size was less than 200bp,which was suitable for the detection of processed foods due to DNA degradation,and the specificity,sensitivity and applicability of the primers were tested. Finally,these assays were applied to the actual detection of transgenic samples. The results show that the assays for 9 common exogenous genes in GMC have high specificity and sensitivity,of which the limits were 0.1%-0.5%,approximately 18-90 copies of the genome. Therefore,these assays can be used for screening and detection of common genes in genetically modified foods and processed products.
In this study, the three plant genome extraction methods of CTAB method, guanidine-chloroform method and extraction kit were compared with each other,and the CTAB extraction method with the highest recovery rate was selected for extraction of the genome of experiment samples. Then,9 pairs of primers were designed based on 9 common exogenous gene sequences in genetically modified crops. The amplicon size was less than 200bp,which was suitable for the detection of processed foods due to DNA degradation,and the specificity,sensitivity and applicability of the primers were tested. Finally,these assays were applied to the actual detection of transgenic samples. The results show that the assays for 9 common exogenous genes in GMC have high specificity and sensitivity,of which the limits were 0.1%-0.5%,approximately 18-90 copies of the genome. Therefore,these assays can be used for screening and detection of common genes in genetically modified foods and processed products.
引文
[1]国际农业生物技术应用服务组织.2016年全球生物技术/转基因作物商业化发展态势[J].中国生物工程杂志,2017,37(4):1-8.
[2]周延培,孙海新,赵美丽,等.一种转基因马铃薯PCR鉴别方法的建立[J].粮油食品科技,2018,26(3):67-70.
[3]王东,宋君,叶先林,等.转基因大豆外源基因NOS终止子定量测定的不确定度分析[J].大豆科学,2013,32(5):601-605.
[4]Cottenet G,Blancpain C,Sonnard V,et al.Development and validation of a multiplex real-time PCRmethod to simultaneously detect 47 targets for the identifcation of genetically modifed organisms[J].Anal Bioanal Chem,2013,405(21):6831-6844.
[5]王东,宋君,叶先林,等.转基因大豆GTS40-3-2定量测定的不确定度分析[J].山西农业科学,2013,41(9):919-923.
[6]金红.转基因农产品标识管理和检测技术研究进展[J].天津农业科学,2002,8(4):48-53.
[7]郭云鹏,俞淑芳,张凤红,等.大豆中转基因成分的定性PCR检测[J].中国饲料,2013,20(3):40-42.
[8]张建中,刘轶男,张海波,等.转基因生物核酸检测新技术研究进展[J].上海农业学报,2011,27(1):117-120.
[9]Delano J,Anna-Mary S,Erika W,et al.Reliable detection and identifcation of genetically modifed maize,soybean,and canola by multiplex PCR analysis[J].J Agric Food Chem,2003,51(20):5829-5834.
[10]Guo J,Chen L,Liu X,et al.A multiplex degenerate PCR analytical approach targeting to eight genes for screening GMOs[J].Food Chem,2012,132(6):1566-1573.
[11]Huber I.,Block A.,Sebah D.,et al.Development and validation of duplex,triplex,and pentaplex real-time PCR screening assays for the detection of genetically modified organisms in food and feed[J].Journal of Agriculture and Food Chemistry,2013,61(6):10293-10301.
[12]Mano J.,Hatano S.,Futo S.,et al.Development of direct real-time PCR system applicable to a wide range of foods and agricultural products[J].Food Hygiene and Safety Science,2014,55(3):25-33.
[13]Junichi M,Yasuyuki N,Yosuke K,et al.Quantification of DNA fragmentation in processed foods using real-time PCR[J].Food Chemistry,2017,226(5):149-155.
[14]Liang L,Mei D,Na A,et al.A novel reference plasmid for the qualitative detection of genetically modified rice in food and feed[J].BioMed Research International,2015,22(4):1-6.
[15]中华人民共和国山东出入境检验检疫局.GB/T19495.3转基因产品检测核酸提取纯化方法[S].北京:中国标准出版社,2004.
[16]中华人民共和国上海出入境检验检疫局.GB/T19495.4转基因产品检测核酸定性PCR检测方法[S].北京:中国标准出版社,2004.
[17]潘琳,褚春旭,李梓文,等.BKR基因启动子鉴定及表达调控[J].长春理工大学学报:自然科学版,2017,40(4):129-132.
[18]宋英林,赵玉环,褚春旭,等.人血清淀粉样蛋白A1原核表达及胶体金检测方法的建立[J].长春理工大学学报:自然科学版,2016,39(10):134-143.
[2]周延培,孙海新,赵美丽,等.一种转基因马铃薯PCR鉴别方法的建立[J].粮油食品科技,2018,26(3):67-70.
[3]王东,宋君,叶先林,等.转基因大豆外源基因NOS终止子定量测定的不确定度分析[J].大豆科学,2013,32(5):601-605.
[4]Cottenet G,Blancpain C,Sonnard V,et al.Development and validation of a multiplex real-time PCRmethod to simultaneously detect 47 targets for the identifcation of genetically modifed organisms[J].Anal Bioanal Chem,2013,405(21):6831-6844.
[5]王东,宋君,叶先林,等.转基因大豆GTS40-3-2定量测定的不确定度分析[J].山西农业科学,2013,41(9):919-923.
[6]金红.转基因农产品标识管理和检测技术研究进展[J].天津农业科学,2002,8(4):48-53.
[7]郭云鹏,俞淑芳,张凤红,等.大豆中转基因成分的定性PCR检测[J].中国饲料,2013,20(3):40-42.
[8]张建中,刘轶男,张海波,等.转基因生物核酸检测新技术研究进展[J].上海农业学报,2011,27(1):117-120.
[9]Delano J,Anna-Mary S,Erika W,et al.Reliable detection and identifcation of genetically modifed maize,soybean,and canola by multiplex PCR analysis[J].J Agric Food Chem,2003,51(20):5829-5834.
[10]Guo J,Chen L,Liu X,et al.A multiplex degenerate PCR analytical approach targeting to eight genes for screening GMOs[J].Food Chem,2012,132(6):1566-1573.
[11]Huber I.,Block A.,Sebah D.,et al.Development and validation of duplex,triplex,and pentaplex real-time PCR screening assays for the detection of genetically modified organisms in food and feed[J].Journal of Agriculture and Food Chemistry,2013,61(6):10293-10301.
[12]Mano J.,Hatano S.,Futo S.,et al.Development of direct real-time PCR system applicable to a wide range of foods and agricultural products[J].Food Hygiene and Safety Science,2014,55(3):25-33.
[13]Junichi M,Yasuyuki N,Yosuke K,et al.Quantification of DNA fragmentation in processed foods using real-time PCR[J].Food Chemistry,2017,226(5):149-155.
[14]Liang L,Mei D,Na A,et al.A novel reference plasmid for the qualitative detection of genetically modified rice in food and feed[J].BioMed Research International,2015,22(4):1-6.
[15]中华人民共和国山东出入境检验检疫局.GB/T19495.3转基因产品检测核酸提取纯化方法[S].北京:中国标准出版社,2004.
[16]中华人民共和国上海出入境检验检疫局.GB/T19495.4转基因产品检测核酸定性PCR检测方法[S].北京:中国标准出版社,2004.
[17]潘琳,褚春旭,李梓文,等.BKR基因启动子鉴定及表达调控[J].长春理工大学学报:自然科学版,2017,40(4):129-132.
[18]宋英林,赵玉环,褚春旭,等.人血清淀粉样蛋白A1原核表达及胶体金检测方法的建立[J].长春理工大学学报:自然科学版,2016,39(10):134-143.