Premium
gro EL is a suitable genetic marker for detecting Vibrio parahaemolyticus by loop‐mediated isothermal amplification assay
Author(s) -
Siddique M.P.,
Jang W.J.,
Lee J.M.,
Ahn S.H.,
Suraiya S.,
Kim C.H.,
Kong I.S.
Publication year - 2017
Publication title -
letters in applied microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.698
H-Index - 110
eISSN - 1472-765X
pISSN - 0266-8254
DOI - 10.1111/lam.12760
Subject(s) - loop mediated isothermal amplification , vibrio parahaemolyticus , groel , biology , microbiology and biotechnology , agarose , agarose gel electrophoresis , ethidium bromide , vibrio , polymerase chain reaction , gene , escherichia coli , dna , bacteria , biochemistry , genetics
A gro EL gene‐based loop‐mediated isothermal amplification ( LAMP ) assay was developed to detect Vibrio parahaemolyticus in contaminated seafood and water. The assay was optimized and conducted at 63°C for 40 min using Bacillus stearothermophilus ( Bst ) DNA polymerase, large fragment. Amplification was analysed via multiple detection methods, including opacity, formation of white precipitate, DNA intercalating dyes (ethidium bromide and SYBR Green I), metal ion‐binding indicator dye, calcein, and 2% agarose gel electrophoresis. A characteristic ladder‐like band pattern on agarose gel and the desired colour changes when using different dyes were observed in positive cases, and these were species‐specific for V. parahaemolyticus when compared with other closely related Vibrio spp. The limit of detection (LoD) of this assay was 100 fg per reaction, 100‐fold higher than that for conventional polymerase chain reaction ( PCR ). When tested on artificially contaminated seafood and seawater, the LoDs of the LAMP assay were 120 and 150 fg per reaction respectively, and those of conventional PCR were 120 and 150 pg per reaction respectively. Based on our results, the gro EL gene‐based LAMP assay is rapid, specific, sensitive, and reliable for detecting V. parahaemolyticus , and it could be used in field diagnosis. Significance and Impact of the Study The loop‐mediated isothermal amplification ( LAMP ) assay using gro EL gene (an abundant, highly conserved gene and member of the gro ESL chaperone gene family) provided rapid, species‐specific and highly sensitive method for detecting Vibrio parahaemolyticus , the leading causal agent of seafood‐borne diseases worldwide. Moreover, gro EL LAMP revealed high efficiency than conventional PCR assay for V. parahaemolyticus using template both from pure culture and artificially contaminated seafood and water, which indicated the applicability in the field and environmental screening purpose for the organism.