doi:

DOI: 10.3724/SP.J.1231.2013.38798

Journal of Fisheries of China (水产学报) 2013/37:12 PP.1829-1837

Development of an SYBR Green I real-time PCR assay for detection of Edwardsiella tarda and its application


Abstract:
According to the sequenced gyrB gene sequence of Edwardsiella tarda,a pair of primers was designed for establishing an SYBR Green I real-time fluorescence quantitative PCR method.A 207 bp gene fragment was amplified from chromosomal DNA of E.tarda from different sources,and no positive reaction was detected in 9 other bacteria species using conventional PCR,which indicated that the primer pair has good inter-species specificity and intra-species commonality.Recombinant plasmid containing gyrB gene of E.tarda was constructed and used to construct the standard curve.The standard curves was y=-3.32x+39.38,the correlation coefficient was 0.998 and the amplification efficiency was 1.00,which indicated that it had a good linear relationship between initial templates and Ct values.The melting curve has only one specific peak when annealing temperature was 63 ℃.The detection limit of the assay was 60 copies per reaction.Turbot samples infected by E.tarda artificially were detected using the real-time PCR assay.All the three samples were positive,which had good agreement with bacteriological analysis by isolation and culture.The results showed that the developed SYBR Green I real-time PCR assay had the advantages of specificity,sensitivity,rapidity and quantification,and would be helpful for E.tarda diagnosis and epidemiology investigation.

Key words:gyrB gene,Edwardsiella tarda,real-time PCR,SYBR Green I

ReleaseDate:2015-04-19 11:27:15



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