Development of a rapid and sensitive recombinase polymerase amplification‐lateral flow assay for detection of<i>Burkholderia mallei</i>

Saxena, Apoorva; Pal, Vijai; Tripathi, Nagesh K.; Goel, Ajay

doi:10.1111/tbed.13126

Cited by 20 publications

(14 citation statements)

References 23 publications

(43 reference statements)

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“…The RPA-LF method has been adapted to detect many pathogens which requires minimal equipment and a quick turnaround time of 30 min. It has been applied for detection of pathogens in various samples, such as blood ( Srisrattakarn et al, 2020 ), plasma ( Qi et al, 2018 ), foodstuffs ( Du et al, 2018a ; Du et al, 2018b ), stool ( Crannell et al, 2014a ) and environmental samples such as plant ( Londono, Harmon & Polston, 2016 ), soil and water ( Saxena et al, 2019 ). To the best of our knowledge, this is the first report of the application of the RPA-LF method for direct detection of E .…”

Section: Discussionmentioning

confidence: 99%

Rapid detection ofEnterococcusand vancomycin resistance using recombinase polymerase amplification

Panpru

Srisrattakarn

Panthasri

et al. 2021

PeerJ

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Vancomycin-resistant enterococci (VRE), especially Enterococcus faecium, have been a global concern, often causing serious healthcare-associated infections. We established a rapid approach for detecting E. faecium and vancomycin-resistance genes (vanA and vanB) in clinical samples using isothermal recombinase polymerase amplification (RPA) combined with a lateral-flow (LF) strip. Specific RPA primer sets and probes for ddl (to identify the presence of E. faecium) vanA and vanB genes were designed. The RPA reaction was performed under isothermal condition at 37 °C within 20 min and read using the LF strip within a further 5 min. A total of 141 positive blood-cultures and 136 stool/rectal swab samples were tested using RPA-LF method compared to the conventional PCR method. The RPA-LF method exhibited 100% sensitivity in both blood-culture (60 E. faecium; 35 vanA type and two vanB type) and stool/rectal-swab samples (63 E. faecium and 36 vanA type) without cross-reaction (100% specificity). The lower detection limit of the RPA-LF was approximately 10 times better than that of the conventional PCR method. The RPA-LF method is an alternative rapid method with excellent sensitivity and specificity for detecting E. faecium, vanA, and vanB, and it has the potential to be used as a point-of-care device for VRE therapy and prevention.

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Section: Discussionmentioning

confidence: 99%

Rapid detection ofEnterococcusand vancomycin resistance using recombinase polymerase amplification

Panpru

Srisrattakarn

Panthasri

et al. 2021

PeerJ

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show abstract

“…Rapid and accurate on-site detection is essential because of the fast onset and outbreak of the pathogen [41]. The RPA-LFD combined method is a promising solution because it is fast, simple and can target specific gene sequences [34]. Selection of the detection target was important for specificity.…”

Section: Discussionmentioning

confidence: 99%

“…Chemical labeling enables end-point reading of the amplification product as a colored signal from gold nanoparticles (AuNPs) by the naked eye on lateral flow dipsticks (LFDs) [32]. The RPA-LFD method has been applied for the detection of many pathogens, including goose parvovirus [33], Burkholderia mallei [34], Trichinella spp. [35], Mycoplasma bovis [36], Candidatus Liberibacter asiaticus [37], Pasteurella multocida [38] and Phytophthora soja [39].…”

Section: Introductionmentioning

confidence: 99%

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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“…Rapid and accurate on-site detection is essential because of the fast onset and outbreak of the pathogen [38]. The RPA-LFD combined method should be a promising solution for the rapidness, simplicity and the possibility to target specific gene sequences [34].…”

Section: Discussionmentioning

confidence: 99%

“…Chemical labeling enables end-point reading of the amplification product as colored signals from gold nanoparticles (AuNP) with the naked eye on lateral flow dipsticks (LFD) [32]. The RPA-LFD method has been applied to the detection of many pathogenic bacteria including goose parvovirus [33], Burkholderia mallei [34], Trichinella spp. [35], Mycoplasma bovis [36] and Candidatus Liberibacter asiaticus [37].…”

Section: Introductionmentioning

confidence: 99%

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

Yu¹,

Zhao

Wu³

et al. 2019

Preprint

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Background: Vibrio alginolyticus is an important pathogen that has to be closely monitored and controlled in the mariculture industry for its strong pathogenicity, quick onset after infection and high mortality rate to aquatic animals. Fast, simple and specific methods are demanded for its on-site detection to effectively control its outbreaks and prevent economic losses. 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 for 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 the isothermal recombinase polymerase amplification (RPA) and lateral flow dipsticks (LFD). The method targeted a virulence gene toxR that was reported to have a good coverage for the V. alginolyticus pathogenic strains. To ensure the specificity, the primer-probe set of the RPA system was carefully designed to recognize regions in gene toxR that were diverged in different Vibrio species but conserved in V. alginolyticus pathogenic strains. The primer-probe set was determined after a systematic screening on amplification performance, primer-dimer formation and false positive signals.The RPA-LFD method was confirmed to have a high specificity to V. alginolyticus pathogenic strains without any cross reaction with other Vibrio species, and was able to detect as low as 1 colony forming unit per microliter of the bacterium without DNA extraction. The method finishes detection within 30 min under the temperature between 35oC and 45oC, and the visual signal on the dipstick was directly read with naked eye. In an application simulation, randomly infected shrimp homogenate samples were 100% accurately detected. Conclusions: The RPA-LFD method developed in this study is fast, simple, highly specific and independent of complicated equipment. It is well applicable to the on-site detection of V. alginolyticus pathogenic strains for the mariculture industry.

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Development of a rapid and sensitive recombinase polymerase amplification‐lateral flow assay for detection ofBurkholderia mallei

Cited by 20 publications

References 23 publications

Rapid detection ofEnterococcusand vancomycin resistance using recombinase polymerase amplification

Rapid detection ofEnterococcusand vancomycin resistance using recombinase polymerase amplification

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

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