Development of a SYBR Green I real-time PCR for quantitative detection of Vibrio alginolyticus in seawater and seafood

Aims: To develop a SYBR Green quantitative real‐time PCR protocol enabling detection and quantification of a fish probiotic and two turbot pathogenic Vibrio spp. in microcosms. Methods and Results: Phaeobacter 27‐4, Vibrio anguillarum 90‐11‐287 and Vibrio splendidus DMC‐1 were quantified as pure and mixed cultures and in presence of microalgae (Isochrysis galbana), rotifers (Brachionus plicatilis), Artemia nauplii or turbot (Psetta maxima) larvae by real‐time PCR based on primers directed at genetic loci coding for antagonistic and virulence‐related functions respectively. The optimized protocol was used to study bioencapsulation and maintenance of the probiont and pathogens in rotifers and for the detection and quantification of Phaeobacter and V. anguillarum in turbot larvae fed rotifers loaded with the different bacteria in a challenge trial. Conclusions: Our real‐time PCR protocol is reproducible and specific. The method requires separate standard curve for each host organism and can be used to detect and quantify probiotic Phaeobacter and pathogenic Vibrio bioencapsulated in rotifers and in turbot larvae. Significance and Impact of the Study: Our method allows monitoring and quantification of a turbot larvae probiotic bacteria and turbot pathogenic vibrios in in vivo trials and will be useful tools for detecting the bacteria in industrial rearing units.

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“…Similarly to previous studies (Cai et al. 2006; Zhou et al. 2007), we estimated a higher Vibrio concentration by real‐time PCR than by culturing.…”

Section: Discussionmentioning

confidence: 74%

Real-time PCR detection and quantification of fish probioticPhaeobacterstrain 27-4 and fish pathogenicVibrioin microalgae, rotifer,Artemiaand first feeding turbot (Psetta maxima) larvae

Prol

Bruhn

Pintado

et al. 2009

Journal of Applied Microbiology

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“…In the present study, 14 strains were identified by their 16S rRNA gene sequence, but the other 4 strains cannot be identified. In that case, new standards, such as gyrB genes, can be used for strain identification (Zhou et al 2007, Garnier et al 2008). …”

Section: Discussionmentioning

confidence: 99%

Phenotypic and genetic characterization of bacteria isolated from diseased cultured sea cucumber Apostichopus japonicus in northeastern China

Li¹,

Qiao²,

Gu³

et al. 2010

Dis. Aquat. Org.

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During the winter-spring from 2004 to 2006 in northeastern China cultured Japanese sea cucumber Apostichopus japonicus suffered from a serious disease. Clinical signs included swollen mouth, skin ulceration and massive mortality. Clinical samples taken during this period were studied. Thirty-one bacterial samples were isolated from diseased sea cucumbers and identified through biochemical tests, 16S rRNA gene sequence analysis and PCR amplification, followed by pathogenicity determination. The results showed that the 31 isolates belonged to the genera Vibrio (64.5%), Shewanella (12.9%), Serratia (12.9%), Pseudoalteromonas (6.4%) and Flavobacterium (3.2%). The 3 prominent strains were Vibrio splendidus (41.9%), Shewanella (12.9%) and Serratia odorifera biogroup I (12.9%). Pathogenicity tests demonstrated that 13 out of 31 isolates were pathogenic, including 8 strains of V. splendidus, 3 strains of Shewanella sp. and 2 strains of Pseudoalteromonas tetraodonis. The pathogenic V. splendidus showed the highest frequency of appearance. Median lethal dose (LD 50 ) values (14 d) of V. splendidus, Shewanella sp. and P. tetraodonis were 1.74 × 10 7 , 7.76 × 10 6 , 7.24 × 10 7 CFU g -1 body weight of sea cucumber, respectively. The virulences differed by species: Shewanella sp. > V. splendidus > P. tetraodonis. This is the first report of Shewanella sp. virulence in sea cucumber. KEY WORDS: Sea cucumber · Apostichopus japonicus · Bacterial pathogen · Vibrio splendidus · Shewanella sp. · Pseudoalteromonas tetraodonis Resale or republication not permitted without written consent of the publisherDis Aquat Org 91: [223][224][225][226][227][228][229][230][231][232][233][234][235] 2010 gradient gel electrophoresis (DGGE) (Becker et al. 2004). Six isolates obtained from the lesions of diseased sea cucumbers were identified as Vibrio sp. but the pathogenicity assays of isolates were not successful (Becker et al. 2004). It is commonly believed that this disease of sea cucumbers is due to bacterial infections; however, the agent that initiates the skin ulcerations has not been identified (Becker et al. 2004). Most of the studies mainly focused on isolation and identification of pathogens.No information is available for the distribution, diversity and appearance of pathogens in cultured sea cucumbers in China. The objective of this study was to carry out a precise characterization of bacterial pathogens responsible for outbreaks that affected cultured sea cucumbers during the winter-spring period from 2004 to 2006 in northeastern China. MATERIALS AND METHODSSampling and processing. Seventy-three sea cucumbers were collected from 11 farms located in the suburbs of Dalian in northeastern China during winterspring from 2004 to 2006 whenever a disease case was reported (Table 1). Three moribund or newly dead sea cucumbers showing disease signs were collected for bacteriologic assays at each sampling time from these areas. All samples were transported to our lab at 4°C within 24 h. The body weights of the sea cucumbers were ...

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“…However, studies of the distribution, dynamics of this species have rarely been addressed in China (38, 44, 46). When we examined the diversity of vibrios in Shenzhen coastal waters, we found that V. alginolyticus -like species was the dominant Vibrio species.…”

Section: Introductionmentioning

confidence: 99%

Distribution of Vibrio alginolyticus-like species in Shenzhen coastal waters, China

Chen¹,

Li²,

Li³

et al. 2011

Braz. J. Microbiol.

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We investigated the distribution of vibrios in Shenzhen coastal waters in order to obtain valuable information for the aquaculture industry and a health warning system. Quantities of vibrios from surface waters ranged from 0 to 4.40×104 CFUs mL-1 in April (spring), while from 0 to 2.57×103 CFUs mL-1 in September (autumn); the abundance of V. alginolyticus-like species from surface water ranged from 0 to 6.72×103 CFUs mL-1 in April (spring) and from 0 to 1.28×103 CFUs mL-1 in September (autumn); higher counts were observed in spring. The V. alginolyticus-like species was dominant in Shenzhen coastal waters, with the highest abundance in the clean region (stations YMK001 and GDN064) in April, suggesting that Vibrio spp. were naturally occurring bacteria in marine environments. The correlation between the abundance of vibrios (including V. alginolyticus-like species) and environmental factors varied in different regions and different seasons. There were no vibrios detected when the salinity was less than 11.15‰ in the Zhujiang River estuary, which indicated that salinity played a key role in the distribution of vibrios and V. alginolyticus-like species.

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Development of a SYBR Green I real-time PCR for quantitative detection of Vibrio alginolyticus in seawater and seafood

Cited by 59 publications

References 25 publications

Real-time PCR detection and quantification of fish probioticPhaeobacterstrain 27-4 and fish pathogenicVibrioin microalgae, rotifer,Artemiaand first feeding turbot (Psetta maxima) larvae

Real-time PCR detection and quantification of fish probioticPhaeobacterstrain 27-4 and fish pathogenicVibrioin microalgae, rotifer,Artemiaand first feeding turbot (Psetta maxima) larvae

Phenotypic and genetic characterization of bacteria isolated from diseased cultured sea cucumber Apostichopus japonicus in northeastern China

Distribution of Vibrio alginolyticus-like species in Shenzhen coastal waters, China

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