| According to the gene sequences of 16S rRNA gene in GenBank, one pair of specific primer was designed for amplifying the specific fragments of 16S rRNA gene. Then 16S rRNA gene amplified by PCR was cloned into pMD-18T vector and it was used as positive standard. After optimization of annealing temperature and primer concentration, a SYBR Green I Real-time Quantitative PCR was established for detection of Flavobacterium columnar. The melting curve analysis using SYBR Green I dye showed one specific peak, no primer-dimers peak was observed. No amplification was amplified from Aeromonas hydrophila, Edwardsiella tarda, Pseudomonas fluorescens, Citrobacter freundii by the SYBR Green I Realtime Quantitative PCR. The PCR kit was highly sensitive in 12 copies/μL DNA. The results revealed that the SYBR Green I Real-time Quantitative PCR kit was sensitive, specific and it could be used to detect F. columnar in clinical samples. |
