title: Detection of genetically modified crops using multiplex asymmetric polymerase chain reaction and asymmetric hyperbranched rolling circle amplification coupled with reverse dot blot.
abstract: 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, 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 2h, 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.
authors.affiliation: Key Laboratory of Feed Biotechnology, Ministry of Agriculture, Beijing 100081, China; Gene Engineering Laboratory, Feed Research Institute, Chinese Academy of Agricultural Sciences, Beijing 100081, China.
authors.forename: Xiumin
authors.initials: X
authors.lastname: Wang
authors.affiliation: Key Laboratory of Feed Biotechnology, Ministry of Agriculture, Beijing 100081, China; Gene Engineering Laboratory, Feed Research Institute, Chinese Academy of Agricultural Sciences, Beijing 100081, China.
authors.forename: Da
authors.initials: D
authors.lastname: Teng
authors.affiliation: Key Laboratory of Feed Biotechnology, Ministry of Agriculture, Beijing 100081, China; Gene Engineering Laboratory, Feed Research Institute, Chinese Academy of Agricultural Sciences, Beijing 100081, China.
authors.forename: Qingfeng
authors.initials: Q
authors.lastname: Guan
authors.affiliation: Baotou City Health School, Baotou Medical College of Career Technical College, Baotou 014030, China.
authors.forename: Fang
authors.initials: F
authors.lastname: Tian
authors.affiliation: Key Laboratory of Feed Biotechnology, Ministry of Agriculture, Beijing 100081, China; Gene Engineering Laboratory, Feed Research Institute, Chinese Academy of Agricultural Sciences, Beijing 100081, China. Electronic address: 2681298635@qq.com.
authors.forename: Jianhua
authors.initials: J
authors.lastname: Wang
mesh_terms: D018556:Crops, Agricultural / Q000235:genetics*; D017931:DNA Primers; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D030821:Plants, Genetically Modified / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods*
chemical_list: D017931:DNA Primers
keywords: Genetically modified crops; Hyperbranched rolling circle amplification; Padlock probe; Reverse dot blot