Abstract：
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.
摘要：
乳球菌病是由链球菌引起的一种常见的细菌性鱼病,l.petauri和L.formosensis。尽管有不同的基于PCR的技术来鉴定病原体，如果不对PCR扩增的片段进行测序，这些都不能区分这两种细菌。在本研究中,我们开发了一种多重PCR方法，用于同时检测和区分L.garvieae和L.petauri。针对靶向和非靶向细菌的细菌DNA验证了引物的特异性。对于L.garvieae的含DUF1430结构域的蛋白质基因，PCR扩增子的大小为204bp，petauri乳杆菌的Lichenan通透酶IIC成分基因为465bp，和302bp的钙磷壁酸生物合成蛋白F基因。通过琼脂糖凝胶电泳清楚地分离PCR扩增子。多重PCR测定不产生具有非靶向细菌的DNA的任何扩增产物。在纯培养物中，甘菊乳杆菌和petauri乳杆菌的多重PCR检测限为5和4CFU，在加标组织样品中分别为50和40CFU/g。分别。从平板培养的细菌需要不到2小时，从组织样品需要3小时才能得到结果。总之,开发的多重PCR检测是一种快速的，具体,准确,和经济有效的方法用于检测和区分L.garvieae和L.petauri，适用于常规实验室诊断L.garvieae和L.petauri。
