A real-time PCR targeted to the upstream regions of HlyB for specific detection of Edwardsiella tarda

Specific and Rapid Diagnosis of Edwardsiella tarda by a Novel Loop‐Mediated Isothermal Amplification Targeting the Upstream Region of hlyb Gene

“…The objective of this study was to develop a novel LAMP method for species-specific detection of E. tarda. The primer sets were designed from the upstream regions of the gene hlyb of the hemolysin activator HlyB domain protein, in which this nucleotide sequence homology between E. tarda and E. ictaluri is only 66.5% through DNA sequencing analysis and which showed a good genus-and species-specific property based on our previous results obtained from a real-time PCR assay (Xie et al 2012a).…”

“…However, these procedures have some disadvantages, such as pathogen isolation, sophisticated technical skill, time consumption, specific antibody needed, and poor sensitivity. Assays based on the polymerase chain reaction (PCR) for rapid detection of E. tarda have been developed and seem to be more rapid and sensitive than the classical methods (Chen and Lai 1998;Sakai et al 2007;Lan et al 2008;Chang et al 2009;Sakai et al 2009;Castro et al 2010;Li et al 2011;Xie et al 2012a). Unfortunately, LAMP ASSAY FOR DETECTION OF E. TARDA 111 these PCR assays required expensive laboratory facilities for the amplification of the targeting sequence and detection of the amplified product, which makes these methods inaccessible under the conditions of most aquaculture farms.…”

“…Here we describe a novel and species-specific LAMP method for the detection of E. tarda, which was designated as UH-LAMP. A set of six primers, including two loop primers, was designed based on the amplification of the upstream regions of gene hlyb of the hemolysin activator HlyB domain protein of E. tarda, which showed a good genus-and species-specific property according to our previously reported real-time PCR for detection of this pathogen (Xie et al 2012a). The UH-LAMP reaction conditions, including Mg 2 + concentration, the reaction temperature, and the reaction time, were optimized.…”

Identification and Characterization of the Replication Region of the Virulence Plasmid pEIB202 in Edwardsiella piscicida

“…PCR primers were designed by Primer Premier 5.0 software. The glyceraldehyde-3-phosphate dehydrogenase gene gap, a single-copy gene in the genome of E. coli, and hemolysin activator HlyB domain gene hlyB[17], single-copy in E. piscicida, were used as the reference genes. The rep-F/R were designed for detection of pEIB202 derivates.…”