Application of the rpoS gene for the detection of Vibrio anguillarum in flounder and prawn by polymerase chain reaction

Abstract

Vibrio anguillarum, an opportunistic fish pathogen, is the main species responsible for vibriosis, a disease that affects feral and farmed fish and shellfish, and causes considerable economic losses in marine aquaculture. In this study, we used polymerase chain reaction (PCR) to detect V. anguillarum. PCR specificity was evaluated by amplifying the rpoS gene, a general stress regulator, in six strains of V. anguillarum and 36 other bacterial species. PCR amplified a species-specific fragment (689 bp) from V. anguillarum. Furthermore, the PCR assay was sensitive enough to detect rpoS expression from 3 pg of genomic DNA, or from six colony-forming units (CFU) mL(-1) of cultured V. anguillarum. However, the assay was less sensitive when genomic DNA from the infected flounder and prawn was used (limit of detection, 50 ng and 10 ng g(-1) tissue, respectively). These data demonstrate that PCR amplification of the rpoS gene is a sensitive and species-specific method to detect V. anguillarum in practical situations.

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“…The detection limit of a single PCR is 4.0 pgof V. anguillarum DNA per PCR tube, comparable to previously reported data (Hong et al, 2007;Kim et al, 2008).The nested PCRswere at least 100 times more sensitive than single PCRs and achieved good performance in field samples using gill washings of ayu. Thus, the result of the nested PCRs in this study is more sensitive than the PCR method previouslyreported (Hong et al, 2007;Kim et al, 2008), it is a useful method for detecting V. anguillarum.Furthermore, assuming the length of V. anguillarum chromosomal DNA is 4.0-4.1Mbp (Naka et al,2011),the limit of sensitivity of nested PCR amplification is calculated to be approximately 10 cells per PCR reaction tube.…”

“…Thus, the result of the nested PCRs in this study is more sensitive than the PCR method previouslyreported (Hong et al, 2007;Kim et al, 2008), it is a useful method for detecting V. anguillarum.Furthermore, assuming the length of V. anguillarum chromosomal DNA is 4.0-4.1Mbp (Naka et al,2011),the limit of sensitivity of nested PCR amplification is calculated to be approximately 10 cells per PCR reaction tube. This is a sufficient value comparedwith those of previous studies using PCR for the detection of pathogenic bacteria.…”

“…This method is desirable because PCR is a simple, sensitive, and efficient method for the detection of pathogenic bacteria from domesticated or wild animals including fish. The targeted genomic regions they used as primers were 16S rDNA (Kita-Tsukamoto et al, 1993;Urakawa et al, 1997),rpoN gene (Gonzalez et al, 2003), hemolysin gene (Hirono et al, 1996;Rodkhum et al, 2006),amiB gene (Hong et al, 2007), rpoS gene (Kim et al, 2008), empA gene (Xiaoet al, 2009). However, it is generally believed that the evolutionary rate of non-protein-coding regions, such as 16S rDNA, is slower than that of protein-coding regions and that the phylogenetic resolution of 16S rDNA is sometimes not sufficient to design specific PCR primers (Yamamoto and Harayama, 1998;Küpferet al, 2006).In addition,V.…”

Isolation of the groESL cluster from Vibrio anguillarum and PCR detection targeting groEL gene

“…However, none of these targets has been proven to be suitable for the high values of identity, gene mutation, and reduced sensitivity from environmental samples [8][9][10][11][12][13][14][15][16][17]. Therefore, we considered using a target gene that is abundant and that is influenced by environmental stress conditions and in particular stages of the cell cycle.…”

“…PCR methods for the detection of V. anguillarum have been developed based on 16S rRNA, recA, hemolysin, empA, rpoN, amiB, and rpoS genes [8][9][10][11][12][13][14][15][16][17]. However, none of these targets has been proven to be suitable for the high values of identity, gene mutation, and reduced sensitivity from environmental samples [8][9][10][11][12][13][14][15][16][17].…”