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

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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