Nguyen T L, Lim Y J, Kim D-H, Austin B
Department of Aquatic Life Medicine, College of Fisheries Science, Pukyong National University, Busan, South Korea.
Institute of Aquaculture, Pathfoot Building, University of Stirling, Stirling, UK.
J Fish Dis. 2016 Jan;39(1):31-9. doi: 10.1111/jfd.12322. Epub 2014 Oct 27.
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.
副乳房链球菌对韩国牙鲆（Paralichthys olivaceus Temminck & Schlegel）水产养殖的威胁日益增加。我们开发了一种使用TaqMan探针法的实时聚合酶链反应（PCR）方法，通过靶向gyrB基因序列来检测和定量副乳房链球菌，该基因序列对链球菌属的分子分析有效。我们的实时PCR检测方法每次反应能够检测到10 fg的基因组DNA。测定内和测定间的变异系数（CV）值范围为0.42 - 1.95%，表明该检测方法具有良好的重复性。对海豚链球菌或其他链球菌/乳球菌属鱼类病原体，如无乳链球菌和格氏乳球菌，均无交叉反应，表明该检测方法对副乳房链球菌具有高度特异性。实时PCR检测结果与接种组织匀浆中副乳房链球菌的传统培养检测结果高度吻合（r = 0.957；P < 0.05）。因此，这种灵敏且特异的实时PCR是临床样本中副乳房链球菌诊断定量的宝贵工具。
