Development of a TaqMan Probe Fluorescence PCR Assay for Detection of Red Fox Fur Based on COX3 Gene
Abstrct To rapidly detect red fox fur,we conducted an experiment to design specific primers and a TaqMan-MGB probe based on the conserved region of the red fox fur mitochondrial COX3 gene.Through optimizing reaction conditions,we developed a real-time PCR detection method for red fox fur.Results show that the assay is 100%specific for the red fox fur without cross-reactions with rabbit fur,goat fur,sheep fur,mink fur and raccoon fur.The optimal linear detection range for the plasmid standard control(PUC-VV-)is 4.8×101-4.8×108 copies/μL,with a standard curve equation of Y = 3.4752X + 40.118,a correlation coefficient(r2)of 0.9897,and a detection limit of 48 copies/μL.The intra-assay and inter-assay tests were conducted for three different concentrations of pCR-GPTV-ITR.The coefficient of variation for repeated experiments at each concentration,as indicated by the Ct values,was consistently below1.5%,indicating excellent reproducibility.Application of this method for detecting two red fox fur samples yileded positive results while the other 10 animal fur samples tested negative.In conclusion,the development of this assay provides a rapid and accurate technical means for detecting red fox fur.