Rapid and sensitive detection of Streptococcus iniae by loop-mediated isothermal amplification (LAMP)

Abstract

Keywords: diagnosis, loop-mediated isothermal ampli-fication, phosphoglucomutase gene, Streptococcus iniae.Streptococcus iniae is a beta-haemolytic, Gram-positive coccus, which affects a broad range offreshwater and marine fish species, causing sub-stantial economic losses in the aquaculture industryworldwide (Agnew & Barnes 2007). For diagnosisof S. iniae infection, PCR assays have been devel-oped (Berridge, Fuller, Azavedo, Low, Bercovier F Lau, Woo, Tse, Leung, Wong Y Mata, Mar Blanco, Domi´nguez, Fern-a´ndez-Garayzabal & Gibello 2004). However, useof a thermal cycler in the field as a routinediagnostic tool is difficult because of the expensiveequipment and materials and laboratory trainingrequired (De Franchis, Cross, Foulkes & Cox 1988;Berridge et al. 1998; Mata et al. 2004). Loop-mediated isothermal amplification (LAMP) hasbeen developed to amplify DNA with high speci-ficity and simplicity (Notomi, Okayama, Masubu-chi, Yonekawa, Watanabe, Amino & Hase 2000).In this study, we developed a LAMP method basedon the phosphoglucomutase gene (Buchanan, Stan-nard, Lauth, Ostland, Powell, Westerman & Nizet2005) for the rapid detection of S. iniae infection.Five S. iniae-specific LAMP primers weredesigned to target the phosphoglucomutase gene(pgm; GenBank accession no. AY846302) usingthe LAMP primer support software program(PrimerExplorer V3) (http://primerexplorer.jp/elamp3.0.0/index.html). We compared the pgmsequences of S. iniae and that of S. parauberisstrains (GenBank accession no. FJ004970), which isthe most closely related bacterial fish pathogen. Theconsensus sequence between S. iniae and S. para-uberis was elucidated, and S. iniae-specific LAMPprimers were designed using the alignment analysis(Fig. 1).We initially optimized and standardized theLAMP assay for S. iniae detection using a DNAtemplate from the S. iniae BS9 strain. BacterialDNA was extracted using a genomic DNA purifi-cation kit (Promega), according to the manufac-turers instructions. S. iniae pgm-LAMP wasaccomplished with a Loopamp DNA amplificationkit (Eiken Chemical Co. Ltd). LAMP was carriedout in a 25 lL total reaction mixture containing40 pmol each of inner primers FIP (F1c-F2) andBIP (B1c-B2), 5 pmol each of outer primers F3 andB3, 20 pmol of the LF primer (loop primer),12.5 lLof2· reaction mixture (40 mm Tris–HCl[pH 8.8], 20 mm KCl, 16 mm MgSO