Development of a Recombinase Polymerase Amplification Assay for Rapid Detection of the Mycobacterium avium subsp. paratuberculosis

Hansen, Søren Gustenhoff; Schafer, James A.; Fechner, Kim; Czerny, Claus Peter; Wahed, Ahmed Abd El

doi:10.1371/journal.pone.0168733

Cited by 25 publications

(25 citation statements)

References 22 publications

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“…Both tests were able to detect ten imp copies per reaction, regardless if the target was present in pure form or mixed with unrelated DNA from healthy plants. This copy number is in the range to those reported for other bacterial pathogens, like Mycobacterium avium , where the detection limit was 50 target molecules (Hansen et al., ). RPA detection limit is also similar to an isothermal LAMP assay developed for the 16SrX group fruit tree phytoplasmas (De Jonghe, De Roo, & Maes, ) or for a generic TaqMan real‐time PCR procedure based on 16S rDNA primers and probe (Christensen et al., ).…”

Section: Discussionmentioning

confidence: 51%

Rapid detection of “Candidatus Phytoplasma mali” by recombinase polymerase amplification assays

Valasevich

Schneider

2017

Journal of Phytopathology

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Isothermal recombinase polymerase amplification (RPA) assays for the specific detection of "Candidatus Phytoplasma mali (Ca. P. mali)," the causal agent of apple proliferation, were developed. The assays amplify a fragment of the imp gene and amplimers were detected either by fluorescence in real-time mode (TwistAmp ® exo assay) using a fluorophore-labelled probe or by direct visualization employing a lateral flow device (TwistAmp ® nfo assay/Milenia ® HybriDetect). The RPA assays specifically amplified DNA from "Ca. P. mali" strains, and cross-reactivity with other phytoplasmas or plant DNA was not observed. The limit of detection was determined with a cloned imp standard, and positive results were obtained down to 10 copies with both RPA assay formats. In comparison with a TaqMan real-time PCR assay based on the same target gene, the RPA assays were equally sensitive, but results were obtained faster.Simplified nucleic acid extraction procedures from plant tissue with Tris-and CTABbased buffers revealed that crude Tris-DNA extracts were a suitable source for RPA tests while larger concentrations of CTAB were inhibitory. This is the first report of RPA-based assays for the detection of "Ca. P. mali". The assays are suitable for highthroughput screening of plant material and point-of-care diagnostic and can be potentially combined with a simplified DNA extraction procedure.

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Section: Discussionmentioning

confidence: 51%

Rapid detection of “Candidatus Phytoplasma mali” by recombinase polymerase amplification assays

Valasevich

Schneider

2017

Journal of Phytopathology

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“…Substances present in the sample matrix can interfere with the enzymatic nucleic acid amplification. Previous studies [ [41] , [42] , [43] ] have reported that the presence of background DNA or specific concentration of ions in the sample could have a negative influence on the RPA sensitivity. In our study, the lower limit of detection obtained for YMMV could be explained by the higher levels of polysaccharides contained in extracts obtained from D. alata compared to D. rotundata leaves [ 18 ].…”

Section: Discussionmentioning

confidence: 99%

Rapid detection of potyviruses from crude plant extracts

Silva

Oyekanmi

Nkere

et al. 2018

Analytical Biochemistry

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Potyviruses (genus Potyvirus; family Potyviridae) are widely distributed and represent one of the most economically important genera of plant viruses. Therefore, their accurate detection is a key factor in developing efficient control strategies. However, this can sometimes be problematic particularly in plant species containing high amounts of polysaccharides and polyphenols such as yam (Dioscorea spp.). Here, we report the development of a reliable, rapid and cost-effective detection method for the two most important potyviruses infecting yam based on reverse transcription-recombinase polymerase amplification (RT-RPA).The developed method, named ‘Direct RT-RPA’, detects each target virus directly from plant leaf extracts prepared with a simple and inexpensive extraction method avoiding laborious extraction of high-quality RNA. Direct RT-RPA enables the detection of virus-positive samples in under 30 min at a single low operation temperature (37 °C) without the need for any expensive instrumentation.The Direct RT-RPA tests constitute robust, accurate, sensitive and quick methods for detection of potyviruses from recalcitrant plant species. The minimal sample preparation requirements and the possibility of storing RPA reagents without cold chain storage, allow Direct RT-RPA to be adopted in minimally equipped laboratories and with potential use in plant clinic laboratories and seed certification facilities worldwide.

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“…At present, RPA assays have been established and reported for the rapid detection of several pathogens [ 1 11 18 ]. Moreover, a real-time RPA assay for detecting M. paratuberculosis DNA has also been developed [ 7 ]. However, an RPA-LFD assay for M. paratuberculosis detection has not been established.…”

Section: Introductionmentioning

confidence: 99%

Rapid visual detection of Mycobacterium avium subsp. paratuberculosis by recombinase polymerase amplification combined with a lateral flow dipstick

et al. 2018

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Paratuberculosis (Johne's disease) is a chronic debilitating disease of domestic and wild ruminants. However, widespread point-of-care testing is infrequent due to the lack of a robust method. The isothermal recombinase polymerase amplification (RPA) technique has applied for rapid diagnosis. Herein, RPA combined with a lateral flow dipstick (LFD) assay was developed to estimate DNA from Mycobacterium avium subsp. paratuberculosis. First, analytical specificity and sensitivity of the RPA-nfo primer and probe sets were assessed. The assay successfully detected M. paratuberculosis DNA in 30 min at 39℃ with a detection limit of up to eight copies per reaction, which was equivalent to that of the real-time quantitative polymerase chain reaction (qPCR) assay. The assay was specific, as it did not amplify genomes from five other Mycobacterium spp. or five pathogenic enteric bacteria. Six hundred-twelve clinical samples (320 fecal and 292 serum) were assessed by RPA-LFD, qPCR, and enzyme-linked immunosorbent assay, respectively. The RPA-LFD assay yielded 100% sensitivity, 97.63% specificity, and 98.44% concordance rate with the qPCR results. This is the first report utilizing an RPA-LFD assay to visualize and rapidly detect M. paratuberculosis. Our results show this assay should be a useful method for the diagnosis of paratuberculosis in resource-constrained settings.

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Development of a Recombinase Polymerase Amplification Assay for Rapid Detection of the Mycobacterium avium subsp. paratuberculosis

Cited by 25 publications

References 22 publications

Rapid detection of “Candidatus Phytoplasma mali” by recombinase polymerase amplification assays

Rapid detection of “Candidatus Phytoplasma mali” by recombinase polymerase amplification assays

Rapid detection of potyviruses from crude plant extracts

Rapid visual detection of Mycobacterium avium subsp. paratuberculosis by recombinase polymerase amplification combined with a lateral flow dipstick

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