A self-probing primer PCR method for detection of very short DNA fragments.
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 TaqMan 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.
Key Laboratory of Oil Crop Biology of the Ministry of Agriculture, Oil Crops Research Institute of the Chinese Academy of Agricultural Sciences, No. 2 Xudong Second Road, Wuhan 430062, PR China; Hubei Provincial Key Laboratory for Protection and Application of Special Plants in Wuling Area of China, College of Life Science, South-Central University for Nationalities, 182 Min-Yuan Road, Wuhan 430074, PR China.
Li
L
Zhang
Key Laboratory of Oil Crop Biology of the Ministry of Agriculture, Oil Crops Research Institute of the Chinese Academy of Agricultural Sciences, No. 2 Xudong Second Road, Wuhan 430062, PR China.
Yu-Hua
YH
Wu
Key Laboratory of Oil Crop Biology of the Ministry of Agriculture, Oil Crops Research Institute of the Chinese Academy of Agricultural Sciences, No. 2 Xudong Second Road, Wuhan 430062, PR China.
Jun
J
Li
Key Laboratory of Oil Crop Biology of the Ministry of Agriculture, Oil Crops Research Institute of the Chinese Academy of Agricultural Sciences, No. 2 Xudong Second Road, Wuhan 430062, PR China.
Wei
W
Li
Nanjing Institute of Environmental Sciences, Ministry of Environmental Protection of China, Nanjing 210042, PR China. Electronic address: 85287064@163.com.
Biao
B
Liu
Key Laboratory of Oil Crop Biology of the Ministry of Agriculture, Oil Crops Research Institute of the Chinese Academy of Agricultural Sciences, No. 2 Xudong Second Road, Wuhan 430062, PR China. Electronic address: wugang@caas.cn.
Gang
G
Wu
D017931:DNA Primers; D018744:DNA, Plant / Q000032:analysis*; D031541:Fluorescence Resonance Energy Transfer; D017343:Genes, Plant; D030821:Plants, Genetically Modified / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods*; D015203:Reproducibility of Results; D013025:Glycine max / Q000235:genetics; D013045:Species Specificity; D003313:Zea mays / Q000235:genetics
D017931:DNA Primers; D018744:DNA, Plant
Fluorescence resonance energy transfer; Polymerase chain reaction; Quantitative detection; Self-probing primer; Short DNA fragment