Rapid and sensitive detection of Vibrio cholerae by loop-mediated isothermal amplification targeted to the gene of outer membrane protein ompW

Srisuk, Chutima; Chaivisuthangkura, Parin; Rukpratanporn, Sombat; Longyant, Siwaporn; Sridulyakul, Pattarin; Sithigorngul, Paisarn

doi:10.1111/j.1472-765x.2009.02749.x

Cited by 41 publications

(35 citation statements)

References 24 publications

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“…In the case of V. parahaemolyticus, the detection limit of dLAMP-LFD in pure culture was 4.1 CFU per reaction, which was 100 times more sensitive than that of dPCR. Additionally, the detection limit of dLAMP-LFD in a pure culture was also equivalent to results in previous reports of LAMP detection in Pseudomonas putida (4.8 CFU per reaction: Mao et al 2012) and V. cholerae (8 CFU per reaction: Srisuk et al 2010) and in the triplex-LAMP detection of V. harveyi, V. anguillarum, and V. alginolyticus (5 CFU per reaction: Yu et al 2013).…”

Section: Discussionsupporting

confidence: 86%

“…This technique uses the strand displacement activity of Bst DNA polymerase and primers that are specific to six regions on the target DNA under conditions with constant temperature (Notomi et al 2000). The LAMP technique has been used for the identification of various bacteria causing diseases in aquatic animals (Yamazaki et al 2008a;Han and Ge 2010;Srisuk et al 2010;Yongjum et al 2010;Prompamorn et al 2011;Surasilp et al 2011;Mao et al 2012). Typically, LAMP amplicons are monitored by agarose gel electrophoresis.…”

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confidence: 99%

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Development of Duplex Loop‐Mediated Isothermal Amplification (dLAMP) Combined with Lateral Flow Dipstick (LFD) for the Rapid and Specific Detection of Vibrio vulnificus and V. parahaemolyticus

et al. 2016

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Loop-mediated isothermal amplification (LAMP) is a rapid, specific, and effective DNA amplification assay. In this study, a duplex LAMP (dLAMP) assay combined with chromatographic lateral flow dipstick (LFD) was developed for the simultaneous identification of Vibrio vulnificus and V. parahaemolyticus. Each primer set that comprised F3, B3, FIP, and BIP recognized the V. vulnificus rpoS gene and the V. parahaemolyticus tlh gene. Digoxigenilated and biotinylated LAMP amplicons of V. vulnificus and V. parahaemolyticus, respectively, were hybridized with corresponded fluorescein isothiocyanate (FITC)-labeled probes for LFD detection. The optimum conditions were 90 min at 63°C. The dLAMP-LFD had high specificity to V. vulnificus and V. parahaemolyticus and showed no cross-amplification with 59 isolates of other bacteria. The detection limit of dLAMP-LFD with a pure culture of V. vulnificus was 2.2 × 10 4 CFU/mL, corresponding to 41 CFU per reaction, which was equal to that of duplex PCR (dPCR). In the case of V. parahaemolyticus, the detection limit of dLAMP-LFD with a pure culture was 2.2 × 10 3 CFU/mL, corresponding to 4.1 CFU per reaction, 100 times more sensitive than that of dPCR. In the spiked shrimp samples, the detection limit of dLAMP-LFD for V. vulnificus was 2.2 × 10 5 CFU/g, corresponding to 410 CFU per reaction, 10 times more sensitive than that of dPCR. In the case of V. parahaemolyticus, the detection limit of dLAMP-LFD in spiked shrimp samples was 2.2 × 10 4 CFU/g, corresponding to 41 CFU per reaction, 100 times more sensitive than that of dPCR. These results demonstrate that the simple dLAMP-LFD assay had high specificity and high sensitivity for the concurrent detection of V. vulnificus and V. parahaemolyticus.

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Section: Discussionsupporting

confidence: 86%

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confidence: 99%

Development of Duplex Loop‐Mediated Isothermal Amplification (dLAMP) Combined with Lateral Flow Dipstick (LFD) for the Rapid and Specific Detection of Vibrio vulnificus and V. parahaemolyticus

et al. 2016

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“…5–7 times higher than that of LAMP assay for Vibrio harveyi in added shellfish cultures (17·2 CFU per reaction; Cao et al. 2010) and V. cholerae in spiked shrimp sample (20 CFU per reaction; Srisuk et al. 2010).…”

Section: Discussionmentioning

confidence: 87%

The development of loop-mediated isothermal amplification combined with lateral flow dipstick for detection of Vibrio parahaemolyticus

Prompamorn

Sithigorngul

Rukpratanporn

et al. 2011

Letters in Applied Microbiology

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Aims: The current study was aimed to develop a loop‐mediated isothermal amplification (LAMP) combined with amplicon detection by chromatographic lateral flow dipstick (LFD) assay for rapid and specific detection of Vibrio parahaemolyticus. Methods and Results: Biotinylated LAMP amplicons were produced by a set of four designed primers that recognized specifically the V. parahaemolyticus thermolabile haemolysin (tlh) gene followed by hybridization with an FITC‐labelled probe and LFD detection. The optimized time and temperature conditions for the LAMP assay were 90 min at 65°C. The LAMP–LFD method accurately identified 28 isolates of V. parahaemolyticus but did not detect 24 non‐parahaemolyticus Vibrio isolates and 35 non‐Vibrio bacterial isolates. The sensitivity of LAMP–LFD for V. parahaemolyticus detection in pure cultures was 120 CFU ml−1. In the case of spiked shrimp samples without enrichment, the detection limit for V. parahaemolyticus was 1·8 × 103 CFU g−1 or equivalent to 3 CFU per reaction while that of conventional PCR was 30 CFU per reaction. Conclusions: The established LAMP–LFD assay targeting tlh gene was specific, rapid and sensitive for identification of V. parahaemolyticus. Significance and Impact of the Study: The developed LAMP–LFD assay provided a valuable tool for detection of V. parahaemolyticus and can be used effectively for identification of V. parahaemolyticus in contaminated food sample.

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“…OmpW is an outer membrane protein that serves as a receptor for Escherichia coli colicin S4 (4). The ompW gene has served as a species-specific gene of V. cholerae in many detection studies (5,6). This gene was found to have an 11-bp deletion in the PT6 strains, but the deletion was not detected in any PT1 strain (3).…”

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Outer Membrane Protein OmpW Is the Receptor for Typing Phage VP5 in the Vibrio cholerae O1 El Tor Biotype

Zhang

Liu

et al. 2014

J Virol

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Phage typing is used for the subtyping of clones of epidemic bacteria. In this study, we identified the outer membrane protein OmpW as the receptor for phage VP5, one of the typing phages for the Vibrio cholerae O1 El Tor biotype. A characteristic 11-bp deletion in ompW was observed in all epidemic strains resistant to VP5, suggesting that this mutation event can be used as a tracing marker in cholera surveillance. Seven cholera pandemics have been recorded historically. The first six pandemics were putatively caused by the classical biotype of Vibrio cholerae serogroup O1, whereas the ongoing seventh pandemic is caused by the O1 El Tor biotype (1). Subtyping of V. cholerae strains is useful in epidemiological and microbiological studies. A phage-biotyping scheme, in which five typing phages (VP1 to VP5) were included, was established for El Tor strain subtyping in the 1970s in China (2). During surveillance of V. cholerae strains, almost all El Tor strains from epidemics were found to be sensitive to all five of these typing phages and are designated phage type 1 (PT1) strains. However, during the 1998 cholera epidemic in Sichuan Province, some patient strains were resistant to phage VP5. These strains belong to the PT6 group and coexisted with PT1 strains. PT6 strains became predominant in 1999 and 2000 but disappeared after 2001 (3), which makes them unique among the epidemic strains.Phages infect susceptible bacterial strains, beginning with binding to receptors on the surfaces of bacteria. OmpW is an outer membrane protein that serves as a receptor for Escherichia coli colicin S4 (4). The ompW gene has served as a species-specific gene of V. cholerae in many detection studies (5, 6). This gene was found to have an 11-bp deletion in the PT6 strains, but the deletion was not detected in any PT1 strain (3). Therefore, a role for OmpW in VP5 infection was suspected.In this study, ompW genes from 44 strains isolated from 1998 to 2001, including 22 PT6 and 11 PT1 strains from Sichuan and 11 PT1 strains from other provinces, were sequenced. The same 11-bp deletion was in all PT6 strains (Fig. 1). The deletion corresponds to nucleotides (nt) 298 to 308 of ompW and causes a shift in the reading frame. A new stop codon is generated at 350 nt, ahead of the original stop codon (Fig. 1). All the PT1 strains had intact and identical ompW sequences.To assess the possible role of the ompW gene in VP5 infection, we constructed an ompW deletion mutant, designated N16961-dompW, with suicide plasmid pWM91 (7) from the VP5-sensitive strain N16961. The N16961-dompW mutant lost sensitivity to VP5 ( Fig. 2A). This sensitivity was restored when it was complemented with plasmid pBR322-ompW (designated strain N16961-dompW-R) containing the intact ompW gene cloned from N16961 but not when it obtained pBR322-ompW(del) containing ompW with an 11-bp deletion cloned from the PT6 strain VC631. VC631 became sensitive to VP5 when it obtained plasmid pBR322-ompW but remained resistant when it obtained pBR322-ompW(del) ( Fig. 2A), indicating t...

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Rapid and sensitive detection of Vibrio cholerae by loop-mediated isothermal amplification targeted to the gene of outer membrane protein ompW

Cited by 41 publications

References 24 publications

Development of Duplex Loop‐Mediated Isothermal Amplification (dLAMP) Combined with Lateral Flow Dipstick (LFD) for the Rapid and Specific Detection of Vibrio vulnificus and V. parahaemolyticus

Development of Duplex Loop‐Mediated Isothermal Amplification (dLAMP) Combined with Lateral Flow Dipstick (LFD) for the Rapid and Specific Detection of Vibrio vulnificus and V. parahaemolyticus

The development of loop-mediated isothermal amplification combined with lateral flow dipstick for detection of Vibrio parahaemolyticus

Outer Membrane Protein OmpW Is the Receptor for Typing Phage VP5 in the Vibrio cholerae O1 El Tor Biotype

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