Development of a recombinase polymerase based isothermal amplification combined with lateral flow assay (HLB-RPA-LFA) for rapid detection of "Candidatus Liberibacter asiaticus"

Ghosh, Dilip K.; Kokane, Sunil; Kokane, Amol; Warghane, Ashish; Motghare, Manali; Bhose, Sumit; Sharma, Ashwani; Reddy, M. Krishna

doi:10.1371/journal.pone.0208530

Cited by 69 publications

(51 citation statements)

References 40 publications

(44 reference statements)

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“…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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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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“…Regarding maceration, several reagents have been applied to prepare plant crude macerates that can be directly utilized in RPA reaction. Those reagents include TE buffer (Ahmed et al 2018), NaOH (Karakkat et al 2018;Qian et al 2018), standard ELISA grinding buffer (Miles et al 2015), and commercial buffers GEB (Kumar et al 2018), GEB 2 (Miles et al 2015;Li et al 2017), GEB 4 (Zhang et al 2014) and AMP1 (Kalyebi et al 2015;Ghosh et al 2018) from Agdia, Inc. In contrast, our investigation on standard ELISA grinding buffer revealed that no component of the buffer was essential for obtaining RPA amplification.…”

Section: Discussionmentioning

confidence: 82%

Development and evaluation of a recombinase polymerase amplification assay for rapid detection of strawberry red stele pathogen

et al. 2020

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Phytophthora fragariae causes drastic damage in strawberry crops. P. fragariae infects strawberry roots and causes red stele root rot. Although P. fragariae is a quarantine organism, its spread in Finland continues as more and more fields contract the disease. The spread can be halted through developing rapid and reliable detection assays. We have developed a rapid recombinase polymerase amplification (RPA) assay for P. fragariae targeting the Phytophthora mitochondrial DNA intergenic atp9-nad9 marker. The assay is DNA-extraction free and capable of detecting as low as 10 fg of P. fragariae genomic DNA. We found the assay reliable for diagnosing field plants when samples are adequately collected. We also applied the RPA assay to the detection of the pathogen in the soil through coupling the assay with baiting with the host plant. The results suggest that if only a small number of samples are analysed, the baiting results will not be reliable.

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“…Crude macerates have been prepared with different buffers. Ready-to-use buffers from Agdia, Inc. such as GEB , GEB 2 (Miles et al, 2015;Li et al, 2017), GEB 4 (Zhang et al, 2014) and AMP1 (Kalyebi et al, 2015;Ghosh et al, 2018) have been utilized. Similarly, NaOH (Karakkat et al, 2018;Qian et al, 2018), standard ELISA grinding buffer (Miles et al, 2015), and TE buffer (Ahmed et al 2018) have also been used for maceration.…”

Section: Introductionmentioning

confidence: 99%

RPA-PCR couple: an approach to expedite plant diagnostics and overcome PCR inhibitors

Munawar

Martin

Toljamo

et al. 2020

Preprint

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Plant diseases are often diagnosed by the method of DNA extraction followed by PCR. DNA extraction from plant tissue can be a recalcitrant and lengthy process, and sometimes ends up with inhibitors that reduce PCR amplification efficiency. Here we present a unique approach, 'RPA-PCR couple', to exclude the DNA extraction step from the standard plant diagnostic process. The process crudely macerates plant tissue in water for a few minutes, and then transfers the macerate supernatant to a Recombinase Polymerase Amplification (RPA) reaction. Following an incubation of 20 minutes at 39 0 C, the RPA reaction can be directly utilized in PCR amplification. In RPA-PCR couple, the RPA reaction is run at slower reaction kinetics to promotes amplification of long amplicons and the slower reaction kinetics are achieved by lowering RPA components concentrations. In this proof of concept study, we targeted Phytophthora intergenic mitochondrial spacer between atp9 and nad9 genes and the two common Phytophthora pathogens of strawberry: P. fragariae and P. cactorum. We presented coupling of RPA with real time TaqMan and SYBR Green PCR assays, and conventional PCR amplification aimed at Sanger sequencing. We found the RPA-PCR couple specific and capable of detecting as low as 10 fg of Phytophthora genomic DNA. Moreover, comparing RPA-PCR couple with the routine method of DNA extraction followed by PCR generated comparable results for the field samples. The idea of RPA-PCR couple to exclude DNA extraction may have vast application in different fields such as clinical diagnostics, food inspection and soil sciences.An alternative DNA amplification method, Recombinase Polymerase Amplification (RPA), has been developed that addresses many of these limitations of DNA extraction (Piepenburg et al., 2006). RPA is an isothermal amplification where primers of 30-35 nucleotides length bind with recombinase enzyme, and these nucleoprotein complexes scan DNA to pair up with homologous region. This results in D-loop structure formation, where recombinase dissociates from primers and polymerase extend the primers. Bidirectional extension of primers by polymerase with strand displacement activity leads to exponential amplification of the target nucleic acid. Commercially available formulations for RPA assays can be obtained from TwistDx (United Kingdom), the patent holder of the technology, and Agdia (France). TwistDx, besides supplying reagents for RPA technology, aims at supporting the deployment of RPA technology in various fields including life sciences, diagnostics, microfluidics/lab-on-chip applications, agriculture, and defence.

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Development of a recombinase polymerase based isothermal amplification combined with lateral flow assay (HLB-RPA-LFA) for rapid detection of "Candidatus Liberibacter asiaticus"

Cited by 69 publications

References 40 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

Development and evaluation of a recombinase polymerase amplification assay for rapid detection of strawberry red stele pathogen

RPA-PCR couple: an approach to expedite plant diagnostics and overcome PCR inhibitors

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