Multiplex PCR assay for the accurate and rapid detection and differentiation of Lactococcus garvieae and L. petauri
Investigation article published in Journal of Fish Diseases
November 1st, 2024
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
Ustaoglu D., Ozturk RC., Ture M., Colussi S., Pastorino P., Vela AI., Kotzamanidis C., Volpatti D., Acutis PL. and Altinok I.
| Surmene Faculty of Marine Sciences. Karadeniz Technical University (KTU). | |
| Aquatic Animal Health and Molecular Genetic Lab. Department of Fisheries Technology Engineering. Karadeniz Technical University (KTU). | |
| Department of Fish Health. Central Fisheries Research Institute (SUMAE). | |
 | Istituto Zooprofilattico Sperimentale del Piemonte, Liguria e Valle d`Aosta (IZSPLV). |
 | Servicio de Identificación y Caracterización Microbiana (ICM). Centro de Vigilancia Sanitaria Veterinaria (VISAVET). Universidad Complutense (UCM). |
| Departamento de Sanidad Animal. Facultad de Veterinaria. Universidad Complutense (UCM). |
| Veterinary Research Institute. Elgo-Dimitra Thessaloniki. | |
| Department of Agricultural, Food, Environmental and Animal Sciences. Università degli Studi di Udine (UNIUD). | |
| Surmene Faculty of Marine Sciences. Karadeniz Technical University (KTU). | |
