Isolation of an unusual strain ofEdwardsiella tardafrom turbot and establish a PCR detection technique with thegyrBgene

Abstract

Aims: The aim of this study was to report an unusual Edwardsiella tarda and develop an effective method to identify this bacterium. Methods and Results: During the spring and summer of 2006, an epizootic occurred among cultured turbot (Scophthalmus maximus) in Qingdao, China. A gram‐negative, rod‐shaped bacterium (designated as LTB‐4) was isolated from the infected fish, and was proved to be virulent to turbot. Based on the 16S rDNA sequencing and phenotypic tests, the bacterial pathogen was identified as E. tarda. Unlike those commonly described E. tarda strains, no flagellate was observed. Partial gyrB genes were amplified from E. tarda using the universal primers of gyrB genes and sequenced. The polymerase chain reaction (PCR) primers for the gyrB gene were designed specific to E. tarda. It revealed positive amplification of the gyrB fragment in E. tarda, whereas other bacterial species were negative. In addition, the technique enabled the recognition of E. tarda from diseased fish. Conclusions: The isolate was identified as E. tarda without flagellate and an effective method was developed to identify E. tarda based on using the gyrB gene as a taxonomic marker. Significance and Impact of the Study: The unusual E. tarda was first reported in China and the PCR allowed the rapid and sensitive detection of E. tarda.

Evaluation of four polymerase chain reaction primer pairs for the detection of Edwardsiella tarda in turbot

“…The obtained results demonstrated that only the primer pair etfD was specific for E. tarda detection. The lack of amplification in all strains employed in the present study when the primers gyrBF1 and gyrBR1 were used is explained by the fact that the sequences of these primers, published by Lan et al (2008), show 4 and 2 mismatches, respectively, with the gyrB sequence reported for E. tarda NCIMB2034 (GenBank accession number EU259314.1). These mismatches together would be enough to prevent amplification of the gyrB gene in E. tarda strains whose sequence differs from that of the unusual strain LTB-4.…”

“…In the present study, we evaluated in parallel the effectiveness of the primers tardaF and tardaR from Chen & Lai (1998), 2 selected sets of primers (etfA and etfD) from Sakai et al (2007) and the pair of primers gyrBF1/ gyrBR1 from Lan et al (2008) in order to assess the best PCR protocol to identify and detect Edwardsiella tarda from both pure and mixed cultures, as well as in fish and environmental samples. For this, we employed a collection of E. tarda strains with a wide range of host and geographical origins, as well as a collection of related and unrelated bacterial isolates.…”

“…All PCR amplifications were performed employing commercial Ready-To-Go PCR beads (Amersham Pharmacia Biotech), which included all the reagents needed for the PCR reactions with the exception of the specific primers and DNA template. Four species-specific primer pairs described by Chen & Lai (1998), Sakai et al (2007) and Lan et al (2008) were synthesized by Sigma-Genosys and employed in this work for the identification of Edwardsiella tarda (see Table 2). …”

“…Only 2 primer sets (etfA and etfD) showed an ability to detect E. tarda. Recently, Lan et al (2008), designed a set of primers (gyrBF1 and gyrBR1) based on the sequence of the divergent region of the partial gyrB gene of an unusual E. tarda strain isolated from turbot in China. This set of primers generated a specific PCR product of 415 bp.…”

Detection of Quorum Sensing Signal Molecules in Edwardsiella ictaluri Ei-151

“…The 16S rDNA of bacterial isolates from diseased fish was amplified by PCR with bacterial universal primers P11: 5 0 -AGAGTTTGATCCTGGCTCAG-3 0 and P12: 5 0 -GGTT ACCTTGTTACGACTT-3 0 ) [16]. After purification, the 16S rDNA fragment (1,503 bp) was cloned into pUC vector and sequenced (Sangon, Shanghai).…”