Detection of genetically modified crops using multiplex asymmetric polymerase chain reaction and asymmetric hyperbranched rolling circle amplification coupled with reverse dot blot

卷:173

Wang, Xiumin ; Teng, Da ; Guan, Qingfeng ; Tian, Fang ; Wang, Jianhua

关键词: Hyperbranched rolling circle amplification;    Reverse dot blot;    Padlock probe;    Genetically modified crops;   

DOI  :  10.1016/j.foodchem.2014.10.126

学科分类：食品科学和技术

【 摘 要 】

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 (Led, Hmg and Sad1), event-specific targets (RRS-5C, RRS-3C, Bt176-3C and MON810-3C), screening targets (35S promoter and NOS terminator), and control targets (18S and PLX). 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 mu g/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. (C) 2014 Elsevier Ltd. All rights reserved.