Development of a Rapid Real-Time PCR Method as a Tool To Quantify Viable Photobacterium phosphoreum Bacteria in Salmon (Salmo salar) Steaks

• Published in: Applied and Environmental Microbiology
• Main Authors: Mace, Sabrina, Mamlouk, Kelthoum, Chipchakova, Stoyka, Prévost, Herve, Joffraud, Jean-Jacques, Dalgaard, Paw, Pilet, Marie-France, Dousset, Xavier
• Other Authors: SECurité des ALIments et Microbiologie, Institut National de la Recherche Agronomique (INRA)-École nationale d'ingénieurs des techniques des industries agricoles et alimentaires (ENITIAA)-École nationale vétérinaire, agroalimentaire et de l'alimentation Nantes-Atlantique (ONIRIS), LUNAM Université Nantes Angers Le Mans, Institut Français de Recherche pour l'Exploitation de la Mer (IFREMER), Institut National de la Recherche Agronomique (INRA), Danmarks Tekniske Universitet = Technical University of Denmark (DTU), Bretagne region; Pays de la Loire region; Oniris (Nantes)
• Format: Article in Journal/Newspaper
• Language: English
• Published: HAL CCSD 2013
• Subjects: PATHOGENIC VIBRIO-PARAHAEMOLYTICUS; POLYMERASE-CHAIN-REACTION; PROPIDIUM MONOAZIDE; FISH PRODUCTS; BROCHOTHRIX-THERMOSPHACTA; LISTERIA-MONOCYTOGENES; QUANTITATIVE DETECTION; MODIFIED ATMOSPHERES; MICROBIAL SPOILAGE; RISK-ASSESSMENT; [SDV]Life Sciences [q-bio]; Salmo salar
• Online Access: https://hal.inrae.fr/hal-02649129; https://doi.org/10.1128/AEM.03677-12

International audience 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.