Detection of genetically modified crops using multiplex asymmetric polymerase chain reaction and asymmetric hyperbranched rolling circle amplification coupled with reverse dot blot.
Detection of genetically modified crops using multiplex asymmetric polymerase chain reaction and asymmetric hyperbranched rolling circle amplification coupled with reverse dot blot.
复制标题
DOI：
10.1016/j.foodchem.2014.10.126
复制
发表时间：
2015-04
期刊：
影响因子：
8.8
通讯作者：
Xiumin Wang;D. Teng;Qingfeng Guan;Fang Tian;Jianhua Wang
中科院分区：
文献类型：
--
作者：
Xiumin Wang;D. Teng;Qingfeng Guan;Fang Tian;Jianhua Wang
为了满足日益增长的转基因作物检测需求，需要低成本、高通量和高准确度的检测方法。建立了一种新的多重不对称聚合酶链反应-不对称超支化滚环扩增-反向斑点杂交（RDB）检测GMCs的方法。设计了13个寡核苷酸探针，用于鉴定内源性靶标（Lec 1、HmgandSad 1）、事件特异性靶标（RRS-5C、RRS-3C、Bt 176 - 3CandMON 810 -3C）、筛选靶标（35 S启动子和NOS终止子）和对照靶标（18和PLX）。优化的条件如下：通过烘烤2小时将加尾杂交探针（1- 2pmol/l）固定在膜上，并且使用10：1比例的正向引物与反向引物。方法的检出限为0.1 μg/l的2%RRS和0.5 ng/l的转基因大豆DNA。这些结果表明，RDB方法可以快速、廉价地检测GMCs的多重靶基因。
To meet the ever-increasing demand for detection of genetically modified crops (GMCs), low-cost, high-throughput and high-accuracy detection assays are needed. The new multiplex asymmetric polymerase chain reaction and asymmetric hyper-branched rolling circle amplification coupled with reverse dot blot (RDB) systems were developed to detect GMCs. Thirteen oligonucleotide probes were designed to identify endogenous targets (Lec1,HmgandSad1), event-specific targets (RRS-5C,RRS-3C,Bt176-3CandMON810-3C), screening targets (35Spromoter andNOSterminator), and control targets (18SandPLX). Optimised conditions were as follows: tailed hybridization probes (1–2 pmol/l) were immobilized on a membrane by baking for 2 h, and a 10:1 ratio of forward to reverse primers was used. The detection limits were 0.1 μg/l of 2% RRS and 0.5 ng/l of DNA from genetically modified (GM) soybean. These results indicate that the RDB assay could be used to detect multiplex target genes of GMCs rapidly and inexpensively.