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

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 Resale or republication not permitted without written consent of the publisherDis Aquat Org 102: [225][226][227][228][229][230][231][232][233][234][235] 2013 swimming. However, similar clinical signs are also found in Streptococcus sp. infections (Eldar & Ghittino 1999).Molecular detection methods of Lactococcus garvieae have been developed, including polymerase chain reaction (PCR) (Zlotkin et al. 1998, Aoki et al. 2000, Pu et al. 2002 and 16S rRNA and sodA sequence analysis (Fihman et al. 2006). However, although considered rapid and specific methods for detection, these require expensive equipment and other costly materials (Gunimaladevi et al. 2005).A method of amplifying nucleic acid sequences with high specificity, sensitivity and rapidity under isothermal conditions (60 to 65°C), loop-mediated isothermal amplification (LAMP) has been developed more recently (Notomi et al. 2000) and applied to fish pathogen detection (Caipang et al. 2004, Savan et al. 2004, Yeh et al. 2005, Itano et al. 2006. The assay uses a set of 4 or 6 primers that create a targetspecific stem loop DNA structure during initial amplification steps; subsequent LAMP auto-cycling is performed by the Bst DNA polymerase large fragment (Notomi et al. 2000). Since it does not require complicated equipment, the LAMP method provides a cost-effective, simple, and rapid (producing a result within 60 min) DNA amplification method.Recently, a fosmid library of Lactococcus garvieae from grey mullet has been constructed and many new genes of L. garvieae were cloned (M. A. Tsai et al. unpubl. data). The clone flg08-6 contained an alpha/beta fold family hydrolase gene that is also...

A novel and rapid diagnostic method for discriminating between feces of sika deer and Japanese serow by loop-mediated isothermal amplification

“…3), indicating that the LAMP method can detect the presence of target DNA in a crude extract of DNA solution obtained by the toothpick method. Although we tested PCR only with one primer set and one taq PCR kit, similar sensitivity as LAMP was not observed for the PCR method, thus corroborating previous reports that the LAMP method is superior to the PCR method in sensitivity (Kikuchi et al 2009;Nakao et al 2010;Thekisoe et al 2005;Tsai et al 2013).…”

Development of the Novel Loop Mediated Isothermal Amplification (LAMP) of IS711 Sequence for Rapid Detection of Brucella Species

“…5 b, c, d). Previous studies reported that sensitivity of LAMP was higher such as 10 times for the detection of fish pathogen [25], 20 times for the rapid detection of Avian Leucosis virus from culture isolates and clinical samples [26], 50 times for the detection of M. tuberculosis, M. avium, M. intracellulare for the diagnosis of pulmonary tuberculosis in microscopy Fig. 3 a- centre of developing countries [27,28] and 100 times more sensitive than conventional PCR [29] for the detection of yellow mosaic virus.…”

Development of a Loop-Mediated Isothermal Amplification Assay for Rapid Detection of Nocardia salmonicida, the Causative Agent of Nocardiosis in Fish

“…Moreover, the results of the LAMP assay can be interpreted using a simplified optical system or even with the naked eye. By taking advantage of this technology, the LAMP assay has been successfully applied to the diagnosis of some fish diseases [1,2,16,17,26]. The intergenic spacer region between the 16S-23S rRNA genes is an excellent candidate for the identification of bacterial species.…”