Rapid and Sensitive Detection of <i>Vibrio alginolyticus</i> by Loop‐Mediated Isothermal Amplification Combined with a Lateral Flow Dipstick Targeted to the <i>rpoX</i> Gene

Plaon, Saranya; Longyant, Siwaporn; Sithigorngul, Paisarn; Chaivisuthangkura, Parin

doi:10.1080/08997659.2015.1037468

Cited by 17 publications

(14 citation statements)

References 27 publications

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“…The detection limit of multiplex PCR is 10 CFU or 5 × 10 2 to 5 × 10 1 copies of genomic DNA [45]. The detection limit of LAMP is 2 × 10 4 CFU/ g of spiked shrimp [46]. The quantitative PCR method has a better detection limit than those of multiplex PCR and LAMP, at 0.14-0.4 pg of genomic DNA per reaction [47], which is at the same level as our RPA-LFD method.…”

Section: Discussionmentioning

confidence: 68%

Fast, simple and highly specific molecular detection of Vibrio alginolyticus pathogenic strains using a visualized isothermal amplification method

Yü¹,

Zhao

Chen³

et al. 2020

BMC Vet Res

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Background: Vibrio alginolyticus is an important pathogen that has to be closely monitored and controlled in the mariculture industry because of its strong pathogenicity, quick onset after infection and high mortality rate in aquatic animals. Fast, simple and specific methods are needed for on-site detection to effectively control outbreaks and prevent economic losses. The detection specificity towards the pathogenic strains has to be emphasized to facilitate pointed treatment and prevention. Polymerase chain reaction (PCR)-based molecular approaches have been developed, but their application is limited due to the requirement of complicated thermal cycling machines and trained personnel. Results: A fast, simple and highly specific detection method for V. alginolyticus pathogenic strains was established based on isothermal recombinase polymerase amplification (RPA) and lateral flow dipsticks (LFD). The method targeted the virulence gene toxR, which is reported to have good coverage for V. alginolyticus pathogenic strains. To ensure the specificity of the method, the primer-probe set of the RPA system was carefully designed to recognize regions in the toxR gene that diverge in different Vibrio species but are conserved in V. alginolyticus pathogenic strains. The primer-probe set was determined after a systematic screening of amplification performance, primer-dimer formation and false positive signals. The RPA-LFD method was confirmed to have high specificity for V. alginolyticus pathogenic strains without any cross reaction with other Vibrio species or other pathogenic bacteria and was able to detect as little as 1 colony forming unit (CFU) per reaction without DNA purification, or 170 fg of genomic DNA, or 6.25 × 10 3 CFU/25 g in spiked shrimp without any enrichment. The method finishes detection within 30 min at temperatures between 35°C and 45°C, and the visual signal on the dipstick can be directly read by the naked eye. In an application simulation, randomly spiked shrimp homogenate samples were 100% accurately detected.

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Section: Discussionmentioning

confidence: 68%

Fast, simple and highly specific molecular detection of Vibrio alginolyticus pathogenic strains using a visualized isothermal amplification method

Yü¹,

Zhao

Chen³

et al. 2020

BMC Vet Res

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“…Vibrio alginolyticus is a serious pathogen and causes great losses to mariculture worldwide (Balebona et al, 1998;George et al, 2005;Li, Yu, Qin et al, 2018a;Liu et al, 2001). test, LAMP and so on Liu et al, 2004;Plaon et al, 2015). However, tedious and time-consuming operation of these methods limits their applications in the aquaculture farms.…”

Section: Discussionmentioning

confidence: 99%

Selection and characterization of ssDNA aptamers specifically recognizing pathogenic Vibrio alginolyticus

Liu

et al. 2019

Journal of Fish Diseases

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Vibrio alginolyticus (V. alginolyticus) is a major opportunistic pathogen to both marine animals and humans, which has also caused heavy economic losses to mariculture. The aim of this study was to develop highly specific aptamers for V. alginolyticus. Single‐stranded DNA (ssDNA) aptamers with high binding affinity to viable V. alginolyticus were generated by Systematic Evolution of Ligands by Exponential Enrichment (SELEX) and identified by flow cytometric analysis in this study. The selected aptamers showed high specificity for V. alginolyticus and low apparent binding for other bacteria. The aptamers formed distinct stem‐loop structures, which could form the basis of aptamers’ specific binding to the target V. alginolyticus. Aptamer VA2 and VA8 showed particularly high binding affinity constant (Kd) of 14.31 ± 4.26 and 90.00 ± 13.51 nM, respectively. The aptamers produced no cytotoxic effects in vitro and in vivo. ssDNA aptamers were successfully selected against the viable bacteria pathogen V. alginolyticus by SELEX. The aptamers selected in this study could be not only applied as specific chemical molecular probes for studying V. alginolyticus pathogenesis to Trachinotus ovatus, but also developing rapid convenient diagnosis assay for V. alginolyticus infection, even when applied to the complex sample matrix, such as food and environment samples.

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“…Lateral flow dipstick (LFD) was developed as well for simultaneous detection of pathogenic Leptospira spp. (Nurul Najian et al 2016), measles virus (Xu et al 2016), animal babesiosis, caused by Babesia bovis and Babesia bigemina , foot-and-mouth diseases (Waters et al 2014), Japanese encephalitis (Deng et al 2015), P. vivax, and P. falciparum (Yongkiettrakul et al 2014;Kongkasuriyachai et al 2017), Jembrana disease virus (Kusumawati et al 2015), canine parvovirus (Sun et al 2014), Vibrio alginolyticus (Plaon et al 2015), Macrobrachium rosenbergii nodavirus and shrimp yellow head virus (Khunthong et al 2013).…”

Section: Lamp End-point Detection Methodsmentioning

confidence: 99%

Loop-mediated isothermal amplification (LAMP): a versatile technique for detection of micro-organisms

et al. 2018

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Rapid and Sensitive Detection of Vibrio alginolyticus by Loop‐Mediated Isothermal Amplification Combined with a Lateral Flow Dipstick Targeted to the rpoX Gene

Cited by 17 publications

References 27 publications

Fast, simple and highly specific molecular detection of Vibrio alginolyticus pathogenic strains using a visualized isothermal amplification method

Fast, simple and highly specific molecular detection of Vibrio alginolyticus pathogenic strains using a visualized isothermal amplification method

Selection and characterization of ssDNA aptamers specifically recognizing pathogenic Vibrio alginolyticus

Loop-mediated isothermal amplification (LAMP): a versatile technique for detection of micro-organisms

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