title: Development of a 16S-23S rRNA intergenic spacer-based quantitative PCR assay for improved detection and enumeration of Lactococcus garvieae.
abstract: Lactococcus garvieae is an important foodborne pathogen causing lactococcosis associated with hemorrhagic septicemia in fish worldwide. A real-time quantitative polymerase chain reaction (qPCR) protocol targeting the 16S-23S rRNA intergenic spacer (ITS) region was developed for the detection and enum-eration of L. garvieae. The specificity was evaluated using genomic DNAs extracted from 66 cocci strains. Fourteen L. garvieae strains tested were positive, whereas 52 other strains including Lactococcus lactis ssp. lactis, Lactococcus lactis ssp. hordniae and Lactococcus lactis ssp. cremoris did not show a specific signal. The minimal limit of detection was 2.63 fg of purified genomic DNA, equivalent to 1 genome of L. garvieae. The optimized protocol was applied for the survey of L. garvieae in naturally contaminated fish samples. Our results suggest that the qPCR protocol using ITS is a sensitive and efficient tool for the rapid detection and enumeration of L. garvieae in fish and fish-containing foods.
authors.affiliation: Department of Microbiology, Chung-Ang University College of Medicine, Seoul, Korea.
authors.forename: Hien Dang
authors.initials: HD
authors.lastname: Thanh
authors.forename: Hee Kuk
authors.initials: HK
authors.lastname: Park
authors.forename: Wonyong
authors.initials: W
authors.lastname: Kim
authors.forename: Hyoung-Shik
authors.initials: HS
authors.lastname: Shin
mesh_terms: D000818:Animals; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics*; D021901:DNA, Intergenic; D033364:Decapoda / Q000382:microbiology; D005798:Genes, Bacterial; D017045:Lactococcus / Q000145:classification / Q000235:genetics* / Q000302:isolation & purification*; D057230:Limit of Detection; D012336:RNA, Ribosomal, 16S / Q000235:genetics*; D012338:RNA, Ribosomal, 23S / Q000235:genetics*; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D012474:Salmon / Q000382:microbiology; D017747:Seafood / Q000382:microbiology
chemical_list: D004269:DNA, Bacterial; D021901:DNA, Intergenic; D012336:RNA, Ribosomal, 16S; D012338:RNA, Ribosomal, 23S