In this study the specificity and sensitivity of 2 primer pairs, MAR1-MAR2 and Mar1-Mar2, for the detection of Tenacibaculum maritimum were evaluated in parallel using 79 T. maritimum strains isolated from different fish species, as well as 53 representatives of related and unrelated bacterial species. Both primer pairs were species-specific for T. maritimum, since no amplification products were obtained from chromosomal DNA of the non-T. maritimum bacteria tested. However, whereas MAR1-MAR2 identified all the T. maritimum strains studied, producing a unique and clear PCR band of the expected 1088 bp length, the Mar1-Mar2 primer pair failed to amplify the 400 bp specific band in 3 sole isolates. To verify if these strains belonged to T. maritimum species, 2 endonucleases (PvuI and SacII) were selected as the most adequate enzymes to confirm the specificity of the MAR1-MAR2 amplified fragment. The digestion patterns obtained with both endonucleases supported the assignation of all the strains to T. maritimum. The sensitivity of both PCR detection methods was also different, showing a reduction of sensitivity in at least one order of magnitude of the Mar1-Mar2 primer pair in comparison with MAR1-MAR2. When the MAR-MAR2 PCR protocol was applied to different seeded turbot tissues, the detection limit was 10 2 to 10 4 T. maritimum cells per reaction. In addition, a nested PCR protocol for detection of this pathogens based on MAR1-MAR2 was developed, which increased the sensitivity by approximately 2 orders of magnitude, ranging from 1 to 250 T. maritimum cells per reaction depending on the tissue employed. The tissues that allowed the most easy detection of T. maritimum were the skin and mucus. Based on the findings reported here, we propose the nested PCR protocol as the most adequate for an accurate detection of T. maritimum in diagnostic pathology as well as in epidemiological studies of gliding bacterial disease of marine fish.
Brown Ring Disease (BRD) is a bacterial disease caused by Vibrio tapetis which affects cultured clams and causes heavy economic losses. In this study, 28 V. tapetis strains isolated from 5 different hosts were intraspecifically characterized by 3 different polymerase chain reaction-(PCR-) based typing methods: enterobacteria repetitive intergenic consensus (ERIC)-PCR, repetitive extragenic palindromic (REP)-PCR and randomly amplified polymorphic DNA (RAPD)-PCR. Cluster analysis of genetic profiles obtained from these molecular techniques clearly showed the existence of 3 genetic groups strongly correlated to the host origin. The first group was formed by 23 V. tapetis strains isolated from Manila clam Ruditapes philippinarum, 1 isolated from venus clam Venerupis aurea, and 1 isolated from common cockle Cerastoderma edule, all collected from France and Spain. The second group was formed by 2 strains isolated from carpet-shell clam R. decussatus cultured in the northwest of Spain. The third group was composed of 1 strain isolated from Atlantic halibut Hippoglossus hippoglossus from the UK. We concluded that the 3 typing methods based on PCR were useful for the intraspecific typing of V. tapetis strains, and that they can potentially be used as a fast and reliable tool for epidemiological studies in the future. KEY WORDS: Vibrio tapetis · RAPD · ERIC-PCR · REP-PCR · Typing Resale or republication not permitted without written consent of the publisherDis Aquat Org 69: [175][176][177][178][179][180][181][182][183] 2006 demonstrated some genetic variations among V. tapetis strains (Castro et al. 1997, Romalde et al. 2002, Chevalier et al. 2003.Repetitive element PCR is a group of techniques that generates DNA fingerprints which can be utilized for the discrimination of bacterial species and/or strains (Versalovic et al. 1991(Versalovic et al. , 1994. These methods involve the application of oligonucleotide primers based on families of short, highly conserved extragenic repetitive sequences, including the repetitive extragenic palindromic (REP) and the enterobacterial repetitive intergenic consensus (ERIC) sequences (Stern et al. 1984, Hulton et al. 1991. REP and ERIC sequences are present in species throughout the Enterobacteriaceae family (Versalovic et al. 1991, Bachellier et al. 1999. The use of appropriate outward-facing PCR primers directed at these repeated sequences generates multiple amplification products, which reflect distance polymorphisms between adjacent DNA repeats. PCR with primers based on ERIC and REP sequences has been successfully used to differentiate bacterial strains from diverse species (de Bruijn 1992, Bennasar et al. 2002, Bruant et al. 2003, Hahm et al. 2003.The RAPD assay is based on the use of short random sequence primers, 9 to 10 bases in length, which hybridize with sufficient affinity to chromosomal DNA sequences to render a pattern of bands that is used for fingerprinting bacterial strains (Welsh & McClelland 1990, Williams et al. 1990. A number of studies have reported...
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