title: Quantification of viable Brochothrix thermosphacta in cooked shrimp and salmon by real-time PCR.
abstract: Brochothrix thermosphacta, a Gram-positive bacterium, is considered as the predominant spoilage microbiota of modified atmosphere packing (MAP) shrimp and fish. Traditional methods currently used to detect B. thermosphacta in foods are time-consuming and labour-intensive. The aim of this study was to develop a real-time PCR quantification method combined with a propidium monoazide (PMA) sample treatment step to monitor the population of B. thermosphacta in cooked shrimp and salmon. The specificity of the two primers MO405 and MO404 used to amplify a 70 bp fragment of the 16S rRNA gene was demonstrated by using purified DNA from 30 strains, among 21 bacterial species including 22 reference strains. Using these primers for real-time PCR and in pure culture, a good correlation was obtained between real-time PCR and the conventional plating method. Quantification was linear over 7-log units using artificially inoculated samples. The method performed successfully when tested on naturally contaminated cooked shrimp and fresh salmon, with a minimum threshold of 1.9×10² CFU/g for accurate quantification of B. thermosphacta. The correlation between the B. thermosphacta counts obtained by real-time PCR and plate counts on naturally contaminated shrimp and salmon was high (R²=0.895). Thus, this study presents a rapid tool for producing reliable quantitative data on B. thermosphacta in cooked shrimp and fresh salmon.
authors.affiliation: LUNAM Université, Oniris, UMR1014 Secalim, Nantes, F-44307, France.
authors.forename: Kelthoum
authors.initials: K
authors.lastname: Mamlouk
authors.forename: Sabrina
authors.initials: S
authors.lastname: Macé
authors.forename: Morgan
authors.initials: M
authors.lastname: Guilbaud
authors.forename: Emmanuel
authors.initials: E
authors.lastname: Jaffrès
authors.forename: Mounir
authors.initials: M
authors.lastname: Ferchichi
authors.forename: Hervé
authors.initials: H
authors.lastname: Prévost
authors.forename: Marie-France
authors.initials: MF
authors.lastname: Pilet
authors.forename: Xavier
authors.initials: X
authors.lastname: Dousset
mesh_terms: D000818:Animals; D001483:Base Sequence; D058640:Brochothrix / Q000254:growth & development / Q000302:isolation & purification*; D015169:Colony Count, Microbial; D003296:Cooking; D017931:DNA Primers / Q000235:genetics; D004269:DNA, Bacterial / Q000235:genetics / Q000302:isolation & purification; D005506:Food Contamination / Q000032:analysis; D005516:Food Microbiology / Q000379:methods; D008969:Molecular Sequence Data; D012336:RNA, Ribosomal, 16S / Q000235:genetics / Q000302:isolation & purification; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D012474:Salmon / Q000382:microbiology*; D017747:Seafood / Q000382:microbiology; D012758:Shellfish / Q000382:microbiology*
chemical_list: D017931:DNA Primers; D004269:DNA, Bacterial; D012336:RNA, Ribosomal, 16S