Dataset Information

Development of a rapid real-time PCR method as a tool to quantify viable Photobacterium phosphoreum bacteria in salmon (Salmo salar) steaks.

ABSTRACT: A specific real-time PCR quantification method combined with a propidium monoazide sample treatment step was developed to determine quantitatively the viable population of the Photobacterium phosphoreum species group in raw modified-atmosphere-packed salmon. Primers were designed to amplify a 350-bp fragment of the gyrase subunit B gene (gyrB) of P. phosphoreum. The specificity of the two primers was demonstrated by using purified DNA from 81 strains of 52 different bacterial species. When these primers were used for real-time PCR in pure culture, a good correlation (R(2) of 0.99) was obtained between this method and conventional enumeration on marine agar (MA). Quantification was linear over 5 log units as confirmed by using inoculated salmon samples. On naturally contaminated fresh salmon, the new real-time PCR method performed successfully with a quantification limit of 3 log CFU/g. A correlation coefficient (R(2)) of 0.963 was obtained between the PCR method and classic enumeration on MA, followed by identification of colonies (290 isolates identified by real-time PCR or by 16S rRNA gene sequencing). A good correlation with an R(2) of 0.940 was found between the new PCR method and an available specific conductance method for P. phosphoreum. This study presents a rapid tool for producing reliable quantitative data on viable P. phosphoreum bacteria in fresh salmon in 6 h. This new culture-independent method will be valuable for future fish inspection, the assessment of raw material quality in fish processing plants, and studies on the ecology of this important specific spoilage microorganism.

SUBMITTER: Mace S

PROVIDER: S-EPMC3623209 | biostudies-literature | 2013 Apr

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Development of a rapid real-time PCR method as a tool to quantify viable Photobacterium phosphoreum bacteria in salmon (Salmo salar) steaks.

Macé Sabrina S Mamlouk Kelthoum K Chipchakova Stoyka S Prévost Hervé H Joffraud Jean-Jacques JJ Dalgaard Paw P Pilet Marie-France MF Dousset Xavier X

Applied and environmental microbiology 20130208 8

A specific real-time PCR quantification method combined with a propidium monoazide sample treatment step was developed to determine quantitatively the viable population of the Photobacterium phosphoreum species group in raw modified-atmosphere-packed salmon. Primers were designed to amplify a 350-bp fragment of the gyrase subunit B gene (gyrB) of P. phosphoreum. The specificity of the two primers was demonstrated by using purified DNA from 81 strains of 52 different bacterial species. When these ...[more]

PMID: 23396343

Similar Datasets

Project description:BackgroundAs farming of Atlantic salmon is growing as an aquaculture enterprise, the need to identify the genomic mechanisms for specific traits is becoming more important in breeding and management of the animal. Traits of importance might be related to growth, disease resistance, food conversion efficiency, color or taste. To identify genomic regions responsible for specific traits, genomic large insert libraries have previously proven to be of crucial importance. These large insert libraries can be screened using gene or genetic markers in order to identify and map regions of interest. Furthermore, large-scale mapping can utilize highly redundant libraries in genome projects, and hence provide valuable data on the genome structure.ResultsHere we report the construction and characterization of a highly redundant bacterial artificial chromosome (BAC) library constructed from a Norwegian aquaculture strain male of Atlantic salmon (Salmo salar). The library consists of a total number of 305,557 clones, in which approximately 299,000 are recombinants. The average insert size of the library is 188 kbp, representing 18-fold genome coverage. High-density filters each consisting of 18,432 clones spotted in duplicates have been produced for hybridization screening, and are publicly available 1. To characterize the library, 15 expressed sequence tags (ESTs) derived overgos and 12 oligo sequences derived from microsatellite markers were used in hybridization screening of the complete BAC library. Secondary hybridizations with individual probes were performed for the clones detected. The BACs positive for the EST probes were fingerprinted and mapped into contigs, yielding an average of 3 contigs for each probe. Clones identified using genomic probes were PCR verified using microsatellite specific primers.ConclusionIdentification of genes and genomic regions of interest is greatly aided by the availability of the CHORI-214 Atlantic salmon BAC library. We have demonstrated the library's ability to identify specific genes and genetic markers using hybridization, PCR and fingerprinting experiments. In addition, multiple fingerprinting contigs indicated a pseudo-tetraploidity of the Atlantic salmon genome. The highly redundant CHORI-214 BAC library is expected to be an important resource for mapping and sequencing of the Atlantic salmon genome.

Project description:The high-affinity/low-capacity system Slc15a2 (PepT2) is responsible for the reuptake of di/tripeptides from the renal proximal tubule, but it also operates in many other tissues and organs. Information regarding PepT2 in teleost fish is limited and, to date, functional data are available from the zebrafish (Danio rerio) only. Here, we report the identification of two slc15a2 genes in the Atlantic salmon (Salmo salar) genome, namely slc15a2a and slc15a2b. The two encoded PepT2 proteins share 87% identity and resemble both structurally and functionally the canonical vertebrate PepT2 system. The mRNA tissue distribution analyses reveal a widespread distribution of slc15a2a transcripts, being more abundant in the brain and gills, while slc15a2b transcripts are mainly expressed in the kidney and the distal part of the gastrointestinal tract. The function of the two transporters was investigated by heterologous expression in Xenopus laevis oocytes and two-electrode voltage-clamp recordings of transport and presteady-state currents. Both PepT2a and PepT2b in the presence of Gly-Gln elicit pH-dependent and Na+ independent inward currents. The biophysical and kinetic analysis of the recorded currents defined the transport properties, confirming that the two Atlantic salmon PepT2 proteins behave as high-affinity/low-capacity transporters. The recent structures and the previous kinetic schemes of rat and human PepT2 qualitatively account for the characteristics of the two Atlantic salmon proteins. This study is the first to report on the functional expression of two PepT2-type transporters that operate in the same vertebrate organism as a result of (a) gene duplication process(es). KEY POINTS: Two slc15a2-type genes, slc15a2a and slc15a2b coding for PepT2-type peptide transporters were found in the Atlantic salmon. slc15a2a transcripts, widely distributed in the fish tissues, are abundant in the brain and gills, while slc15a2b transcripts are mainly expressed in the kidney and distal gastrointestinal tract. Amino acids involved in vertebrate Slc15 transport function are conserved in PepT2a and PepT2b proteins. Detailed kinetic analysis indicates that both PepT2a and PepT2b operate as high-affinity transporters. The kinetic schemes and structures proposed for the mammalian models of PepT2 are suitable to explain the function of the two Atlantic salmon transporters.

Dataset Information

Development of a rapid real-time PCR method as a tool to quantify viable Photobacterium phosphoreum bacteria in salmon (Salmo salar) steaks.

Publications

Development of a rapid real-time PCR method as a tool to quantify viable Photobacterium phosphoreum bacteria in salmon (Salmo salar) steaks.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets