Species-specific PCR detection of the fish pathogen, Vibrio anguillarum, using the amiB gene, which encodes N-acetylmuramoyl-l-alanine amidase

Abstract

Vibrio anguillarum is the causative agent of the fish disease vibriosis and is the most intensely studied species of Vibrio. In the present study, specific primers and a PCR assay were designed to detect V. anguillarum. The primers were designed to amplify a 429-bp internal region of the V. anguillarum amiB gene, which encodes the peptidoglycan hydrolase N-acetylmuramoyl-L-alanine amidase. PCR specificity was demonstrated by successful amplification of DNA from V. anguillarum and by the absence of a PCR product from 25 other Vibrio strains and various enteric bacteria. The PCR produced a 429-bp amplified fragment from as little as 1 pg of V. anguillarum DNA. The limit of detection for this PCR technique was c. 20 bacterial colonies in 25 mg of infected flounder tissue. These results suggest that this PCR system is a sensitive and species-specific detection method, and is possible to use as a diagnostic tool to detect V. anguillarum.

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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.…”

“…This is a sufficient value comparedwith those of previous studies using PCR for the detection of pathogenic bacteria. (Hong et al, 2007;Kimet al, 2008;Tehet al, 2010;Payattikul et al, 2015). In summary, the PCR method described in the present study allows for the specific detection of V. anguillarum from the tissue of diseased fish, rapidly and with sufficient sensitivity, and without isolating disease-causing bacteria using culture method.…”

“…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.…”

Are type IV pili involved in <i>Vibrio anguillarum</i> virulence towards sea bass (<i>Dicentrarchus labrax</i> L.) larvae?

“…These results suggest that the PilA type IV pilus is not a crucial virulence factor in V. anguillarum VIB15. To confirm that mortality was caused by the tested V. anguillarum strains and not by contamination, the identity of the inoculated strains was confirmed by isolating the bacteria again at the end of the experiment (DAH 12) followed by PCR using the V. anguillarum-specific primers van-ami8/van-ami417 and the pilA-specific primers PilAF/PilAR [43].…”