A self-probing primer PCR method for detection of very short DNA fragments

作者：Zhang, Li; Wu, Yu-Hua; Li, Jun; Li, Wei; Liu, Biao^*; Wu, Gang^*

来源：Analytical Biochemistry, 2016, 514: 55-63.

DOI：10.1016/j.ab.2016.09.001

• Short DNA fragment
• Self-probing primer
• Fluorescence resonance energy transfer
• Quantitative detection
• Polymerase chain reaction

A novel self-probing primer method that based on the fluorescence resonance energy transfer principle is designed to detect DNA fragments of approximately 40 bp. Four self-probing primer reaction systems were developed to target a maize endogenous reference gene (HMG), a soybean endogenous reference gene (Lectin), a rapeseed endogenous reference gene (CruA) and an exogenous gene 5-enolpyruvylshikimate-3-phosphate synthase (ctp2-cp4epsps). These four primer systems were confirmed to have a high level of inter-species specificity and good intra-species stability. The limit of detection was estimated to be 10 copies of haploid genomes for all four assays. The validation results demonstrated that the self-probing primer methods are able to quantify the DNA amount in the different samples with good sensitivity and precision. When highly processed food products were assayed, the self-probing primer method produced better results than the TagMan probe method. Overall, the self probing primer method is suitable for qualitative and quantitative detection of very short DNA targets in samples of different sources.

• 出版日期2016-12-1
• 单位环境保护部南京环境科学研究所; 中南民族大学; 中国农业科学院