DAO 102:225-235 (2013)  -  DOI: https://doi.org/10.3354/dao02546

Development of a sensitive and specific LAMP PCR assay for detection of fish pathogen Lactococcus garvieae

M.-A. Tsai1, P.-C. Wang1,*, T. Yoshida2, L.-L. Liaw3, S.-C. Chen1,4,*

1Department of Veterinary Medicine, National Pingtung University of Science and Technology, Pingtung, Taiwan, ROC
2Department of Fisheries, Faculty of Agriculture, Miyazaki University, Miyazaki, Japan
3Bioresource Collection and Research Center, Food Industry Research and Development Institute, Hsinchu, Taiwan, ROC
4Graduate Institute of Animal Vaccine, National Pingtung University of Science and Technology, Pingtung, Taiwan, ROC

ABSTRACT: Based on use of a loop-mediated isothermal amplification (LAMP) identification protocol, this study attempted to detect Lactococcus garvieae in fish by using primer sets designed from an L. garvieae alpha/beta fold family hydrolase gene. Reaction time and temperatures were optimized for 60 min at 60°C with the resulting amplicons visualized by adding SYBR Green I to the reaction tube. The assay specificity was assessed using 45 different bacterial strains. Positive results were observed in all 30 L. garvieae isolates from various aquatic animals. No false-positive results were observed in 15 non-L. garvieae strains. The detection limit of the LAMP assay was 10-fold more sensitive than the conventional polymerase chain reaction (PCR) targeting 16S rDNA when using purified L. garvieae DNA. The detection limit of the LAMP assay was approximately 300 colony-forming units (CFU) using crude bacterial lysates, 100-fold more sensitive than PCR. Furthermore, L. garvieae in spleen, kidney and brain of experimentally challenged tilapia and grey mullet were detected using this optimized LAMP assay. Results of this study demonstrate the effectiveness of LAMP in providing a rapid yet simple test for detecting L. garvieae in fish.


KEY WORDS: Lactococcosis · Alpha/beta fold family · Hydrolase gene · Loop-mediated isothermal amplification · Polymerase chain reaction · SYBR Green I


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Cite this article as: Tsai MA, Wang PC, Yoshida T, Liaw LL, Chen SC (2013) Development of a sensitive and specific LAMP PCR assay for detection of fish pathogen Lactococcus garvieae. Dis Aquat Org 102:225-235. https://doi.org/10.3354/dao02546

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