title: Development of real-time PCR for detection and quantitation of Streptococcus parauberis.
abstract: Streptococcus parauberis is an increasing threat to aquaculture of olive flounder, Paralichthys olivaceus Temminck & Schlegel, in South Korea. We developed a real-time polymerase chain reaction (PCR) method using the TaqMan probe assay to detect and quantify S. parauberis by targeting the gyrB gene sequences, which are effective for molecular analysis of the genus Streptococcus. Our real-time PCR assay is capable of detecting 10 fg of genomic DNA per reaction. The intra- and interassay coefficient of variation (CV) values ranged from 0.42-1.95%, demonstrating that the assay has good reproducibility. There was not any cross-reactivity to Streptococcus iniae or to other streptococcal/lactococcal fish pathogens, such as S. agalactiae and Lactococcus garvieae, indicating that the assay is highly specific to S. parauberis. The results of the real-time PCR assay corresponded well to those of conventional culture assays for S. parauberis from inoculated tissue homogenates (r = 0.957; P < 0.05). Hence, this sensitive and specific real-time PCR is a valuable tool for diagnostic quantitation of S. parauberis in clinical samples.
authors.affiliation: Department of Aquatic Life Medicine, College of Fisheries Science, Pukyong National University, Busan, South Korea.
authors.forename: T L
authors.initials: TL
authors.lastname: Nguyen
authors.affiliation: Department of Aquatic Life Medicine, College of Fisheries Science, Pukyong National University, Busan, South Korea.
authors.forename: Y J
authors.initials: YJ
authors.lastname: Lim
authors.affiliation: Department of Aquatic Life Medicine, College of Fisheries Science, Pukyong National University, Busan, South Korea.
authors.forename: D-H
authors.initials: DH
authors.lastname: Kim
authors.affiliation: Institute of Aquaculture, Pathfoot Building, University of Stirling, Stirling, UK.
authors.forename: B
authors.initials: B
authors.lastname: Austin
mesh_terms: D000818:Animals; D027081:DNA Gyrase / Q000235:genetics; D017931:DNA Primers / Q000592:standards; D004269:DNA, Bacterial / Q000032:analysis; D005393:Fish Diseases / Q000382:microbiology*; D005398:Fisheries; D005432:Flounder; D005456:Fluorescent Dyes / Q000592:standards; D018628:Gene Dosage; D007413:Intestinal Mucosa / Q000382:microbiology; D007668:Kidney / Q000382:microbiology; D060888:Real-Time Polymerase Chain Reaction / Q000592:standards / Q000662:veterinary*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013154:Spleen / Q000382:microbiology; D013290:Streptococcal Infections / Q000382:microbiology / Q000662:veterinary*; D013291:Streptococcus / Q000235:genetics / Q000254:growth & development / Q000302:isolation & purification*
chemical_list: D017931:DNA Primers; D004269:DNA, Bacterial; D005456:Fluorescent Dyes; D027081:DNA Gyrase
keywords: Real-time PCR; Streptococcus parauberis; TaqMan probe; gyrB; qPCR