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: Macé, Sabrina, Mamlouk, Kelthoum, Chipchakova, Stoyka, Prévost, Hervé, Joffraud, Jean-Jacques, Dalgaard, Paw, Pilet, Marie-France, Dousset, Xavier
• Format: Article in Journal/Newspaper
• Language: English
• Published: American Society for Microbiology 2013
• Subjects: Salmo salar
• Online Access: http://dx.doi.org/10.1128/aem.03677-12; https://journals.asm.org/doi/pdf/10.1128/AEM.03677-12

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.