title: Development of a real-time PCR assay with an internal amplification control for detection of Gram-negative histamine-producing bacteria in fish.
abstract: Prompt detection of bacteria that contribute to scombrotoxin (histamine) fish poisoning can aid in the detection of potentially toxic fish products and prevent the occurrence of illness. We report development of the first real-time PCR method for rapid detection of Gram-negative histamine-producing bacteria (HPB) in fish. The real-time PCR assay was 100% inclusive for detecting high-histamine producing isolates and did not detect any of the low- or non-histamine producing isolates. The efficiency of the assay with/without internal amplification control ranged from 96-104% and in the presence of background flora and inhibitory matrices was 92/100% and 73-96%, respectively. This assay was used to detect HPB from naturally contaminated yellowfin tuna, bluefish, and false albacore samples. Photobacterium damselae (8), Plesiomonas shigelloides (2), Shewanella sp. (1), and Morganella morganii (1) were subsequently isolated from the real-time PCR positive fish samples. These results indicate that the real-time PCR assay developed in this study is a rapid and sensitive method for detecting high-HPB. The assay may be adapted for quantification of HPB, either directly or with an MPN-PCR method.
authors.affiliation: Gulf Coast Seafood Laboratory, U.S. Food and Drug Administration, 1 Iberville Dr., Dauphin Island, AL 36528, USA. Kristin.Butler@fda.hha.gov
authors.forename: Kristin
authors.initials: K
authors.lastname: Bjornsdottir-Butler
authors.forename: Jessica L
authors.initials: JL
authors.lastname: Jones
authors.forename: Ronald
authors.initials: R
authors.lastname: Benner
authors.forename: William
authors.initials: W
authors.lastname: Burkhardt
mesh_terms: D000818:Animals; D003257:Consumer Product Safety; D004269:DNA, Bacterial / Q000235:genetics; D004275:DNA, Ribosomal / Q000235:genetics; D005399:Fishes / Q000382:microbiology*; D005506:Food Contamination / Q000032:analysis*; D005516:Food Microbiology; D005784:Gene Amplification; D006090:Gram-Negative Bacteria / Q000235:genetics / Q000254:growth & development / Q000302:isolation & purification* / Q000378:metabolism; D006632:Histamine / Q000096:biosynthesis / Q000506:poisoning*; D006640:Histidine Decarboxylase / Q000235:genetics; D006801:Humans; D016133:Polymerase Chain Reaction / Q000379:methods*; D017747:Seafood / Q000382:microbiology*; D012680:Sensitivity and Specificity
chemical_list: D004269:DNA, Bacterial; D004275:DNA, Ribosomal; D006632:Histamine; D006640:Histidine Decarboxylase