Journal
ANALYTICAL AND BIOANALYTICAL CHEMISTRY
Volume 406, Issue 13, Pages 3069-3078Publisher
SPRINGER HEIDELBERG
DOI: 10.1007/s00216-014-7735-6
Keywords
Genetically modified; Cross-priming amplification; Isothermal amplification; T-Nos; Silica-coated magnetic particles
Funding
- National Natural Science Foundation of China [31271617]
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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 A degrees 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 x 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.
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