title: The detection of T-Nos, a genetic element present in GMOs, by cross-priming isothermal amplification with real-time fluorescence.
abstract: An isothermal cross-priming amplification (CPA) assay for Agrobacterium tumefaciens nopaline synthase terminator (T-Nos) was established and investigated in this work. A set of six specific primers, recognizing eight distinct regions on the T-Nos sequence, was designed. The CPA assay was performed at a constant temperature, 63 °C, and detected by real-time fluorescence. The results indicated that real-time fluorescent CPA had high specificity, and the limit of detection was 1.06 × 10(3) copies of rice genomic DNA, which could be detected in 40 min. Comparison of real-time fluorescent CPA and conventional polymerase chain reaction (PCR) was also performed. Results revealed that real-time fluorescent CPA had a comparable sensitivity to conventional real-time PCR and had taken a shorter time. In addition, different contents of genetically modified (GM)-contaminated rice seed powder samples were detected for practical application. The result showed real-time fluorescent CPA could detect 0.5 % GM-contaminated samples at least, and the whole reaction could be finished in 35 min. Real-time fluorescent CPA is sensitive enough to monitor labeling systems and provides an attractive method for the detection of GMO.
authors.affiliation: College of Biosystems Engineering and Food Science, Zhejiang University, Hangzhou, 310058, China.
authors.forename: Fang
authors.initials: F
authors.lastname: Zhang
authors.forename: Liu
authors.initials: L
authors.lastname: Wang
authors.forename: Kai
authors.initials: K
authors.lastname: Fan
authors.forename: Jian
authors.initials: J
authors.lastname: Wu
authors.forename: Yibin
authors.initials: Y
authors.lastname: Ying
mesh_terms: D016960:Agrobacterium tumefaciens / Q000201:enzymology* / Q000235:genetics / Q000302:isolation & purification; D000594:Amino Acid Oxidoreductases / Q000235:genetics*; D001483:Base Sequence; D045142:Cross-Priming; D017931:DNA Primers; D005453:Fluorescence; D008969:Molecular Sequence Data; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D012275:Oryza / Q000235:genetics* / Q000382:microbiology; D030821:Plants, Genetically Modified / Q000235:genetics* / Q000382:microbiology; D016133:Polymerase Chain Reaction; D060888:Real-Time Polymerase Chain Reaction; D012639:Seeds / Q000378:metabolism / Q000382:microbiology; D012689:Sequence Homology, Nucleic Acid
chemical_list: D017931:DNA Primers; D000594:Amino Acid Oxidoreductases; C021883:nopaline synthase