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

作者：Wang, Xiumin; Teng, Da; Guan, Qingfeng; Tian, Fang; Wang, Jianhua^*

来源：Food Chemistry, 2015, 173: 1022-1029.

DOI：10.1016/j.foodchem.2014.10.126

• Hyperbranched rolling circle amplification
• Reverse dot blot
• Padlock probe
• Genetically modified crops

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.

• 出版日期2015-4-15
• 单位内蒙古科技大学包头医学院; 中国农业科学院