Department of Fisheries Technology Engineering, Faculty of Marine Sciences, Karadeniz Technical University, Trabzon, Turkey.
Aquatic Animal Health and Molecular Genetic Lab, Karadeniz Technical University, Trabzon, Turkey.
J Fish Dis. 2024 Nov;47(11):e14004. doi: 10.1111/jfd.14004. Epub 2024 Aug 4.
Lactococcosis is a common bacterial fish disease caused by Lactococcus garvieae, L. petauri and L. formosensis. Although there are different PCR-based techniques to identify the etiological agent, none of these can differentiate these two bacteria without sequencing PCR-amplified fragments. In the present study, we developed a multiplex PCR assay for simultaneous detection and differentiation of L. garvieae and L. petauri. The specificity of the primers was validated against the bacterial DNA of the targeted and non-targeted bacteria. The sizes of the PCR amplicons were obtained as 204 bp for the DUF1430 domain-containing protein gene of L. garvieae, 465 bp for the Lichenan permease IIC component gene of L. petauri, and 302 bp for the teichoic acid biosynthesis protein F gene of both L. garvieae and L. petauri. The PCR amplicons were clearly separated by agarose gel electrophoresis. The multiplex PCR assay did not produce any amplification products with the DNA of the non-targeted bacteria. The multiplex PCR detection limits for L. garvieae and L. petauri were 5 and 4 CFU in pure culture and 50 and 40 CFU/g in spiked tissue samples, respectively. It takes less than 2 h from plate-cultured bacteria and 3 h from tissue samples to get results. In conclusion, the developed multiplex PCR assay is a rapid, specific, accurate, and cost-effective method for the detection and differentiation of L. garvieae and L. petauri and is suitable to be used for routine laboratory diagnosis of L. garvieae and L. petauri.
乳球菌病是一种由格氏乳球菌、巴氏乳球菌和弗氏乳球菌引起的常见细菌性鱼类疾病。虽然有不同的基于 PCR 的技术可以鉴定病原体，但如果不进行测序，这些技术都无法区分这两种细菌。在本研究中，我们开发了一种用于同时检测和区分格氏乳球菌和巴氏乳球菌的多重 PCR 检测方法。引物的特异性通过针对靶向和非靶向细菌的细菌 DNA 进行验证。PCR 扩增子的大小为格氏乳球菌的 DUF1430 结构域蛋白基因的 204bp，巴氏乳球菌的岩藻糖甘露聚糖渗透酶 IIC 成分基因的 465bp，以及格氏乳球菌和巴氏乳球菌的磷壁酸生物合成蛋白 F 基因的 302bp。琼脂糖凝胶电泳可清晰分离 PCR 扩增子。多重 PCR 检测方法未与非靶向细菌的 DNA 产生任何扩增产物。纯培养物中格氏乳球菌和巴氏乳球菌的多重 PCR 检测限分别为 5 和 4 CFU，添加组织样本中的检测限分别为 50 和 40 CFU/g。从平板培养细菌到组织样本的检测结果需要不到 2 小时和 3 小时。总之，开发的多重 PCR 检测方法是一种快速、特异、准确且具有成本效益的方法，可用于格氏乳球菌和巴氏乳球菌的检测和区分，适用于格氏乳球菌和巴氏乳球菌的常规实验室诊断。
