Fast real‐time detection of Potato spindle tuber viroid by RT‐LAMP

Lenarčič, Rok; Morisset, Dany; Mehle, Nataša; Ravnikar, Maja

doi:10.1111/ppa.12017

Cited by 50 publications

(41 citation statements)

References 33 publications

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“…Oligonucleotides (Thermo Scientific, Schwerte, Germany) were HPLC purified. PCR primers were designed as previously described (22), and LAMP primers were designed according to Lenari et al (23). The isolated RNA was reverse transcribed into ssDNA using reverse primers and Maxima Reverse Transcriptase (Thermo Scientific).…”

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confidence: 99%

Shining a Light on LAMP Assays — A Comparison of LAMP Visualization Methods Including the Novel use of Berberine

et al. 2015

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The need for simple and effective assays for detecting nucleic acids by isothermal amplification reactions has led to a great variety of end point and real-time monitoring methods. Here we tested direct and indirect methods to visualize the amplification of potato spindle tuber viroid (PSTVd) by loop-mediated isothermal amplification (LAMP) and compared features important for one-pot in-field applications. We compared the performance of magnesium pyrophosphate, hydroxynaphthol blue (HNB), calcein, SYBR Green I, EvaGreen, and berberine. All assays could be used to distinguish between positive and negative samples in visible or UV light. Precipitation of magnesium-pyrophosphate resulted in a turbid reaction solution. The use of HNB resulted in a color change from violet to blue, whereas calcein induced a change from orange to yellow-green. We also investigated berberine as a nucleic acid-specific dye that emits a fluorescence signal under UV light after a positive LAMP reaction. It has a comparable sensitivity to SYBR Green I and EvaGreen. Based on our results, an optimal detection method can be chosen easily for isothermal real-time or end point screening applications.

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confidence: 99%

Shining a Light on LAMP Assays — A Comparison of LAMP Visualization Methods Including the Novel use of Berberine

et al. 2015

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“…LAMP is a highly specific and rapid technique, and it also circumvents the sensitivity of PCR and qPCR to inhibitors in plant extracts (Francois et al 2011); furthermore, its isothermal nature provides the potential for it to be deployed in the field (Kogovšek et al 2015;Tomlinson et al 2010a). LAMP has shown a comparable or better performance to other detection methods and a wide applicability for the detection of plant pathogenic bacteria (Lenarčič et al 2014), viroids (Lenarčič et al 2013), fungi (Tomlinson et al 2010b) and phytoplasmas (Bekele et al 2011;Dickinson 2015;Hodgetts et al 2011;Kogovšek et al 2015;Tomlinson et al 2010a).…”

Section: Introductionmentioning

confidence: 99%

Rapid loop-mediated isothermal amplification assays for grapevine yellows phytoplasmas on crude leaf-vein homogenate has the same performance as qPCR

et al. 2016

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A fluorescence-based real-time loop-mediated isothermal amplification (LAMP) assay for 'Candidatus Phytoplasama solani' (Bois noir phytoplasma; BNp) detection was developed and optimised for rapid laboratory and on-site BNp detection. This assay is highly specific, rapid and as sensitive as qPCR. It was validated according to European and Mediterranean Plant Protection Organisation recommendations. In addition, 286 grapevine leaf samples from the 2015 growing season were tested with this new real-time LAMP assay and an assay previously developed for detection of Flavescence dorée phytoplasma (FDp). These LAMP assays for detection of both BNp and FDp used without any DNA extraction step, which is a required step for qPCR analysis, were comparably effective to qPCR, and positive results were obtained in less than 35 min.

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“…Boonham et al (2004) found that the real time RT-PCR was 1000 times more sensitive than the chemiluminescent hybridization detection of PSTVd and it could detect up to 1 ×10 -6 dilutions of infected RNA. Later, the same method was used by Lenarcic et al (2012) who reported that the sensitivity was found up to 10 PSTVd RNA copies. Similarly, the detection limit of the realtime RT-PCR assay developed by Botermans et al (2013) was 2.5× 10 -5 dilutions (serial dilutions of infected tomato leaf sap in healthy tomato leaf sap).…”

Section: Discussionmentioning

confidence: 85%

Duplex realtime RT-PCR assay for the detection of Potato spindle tuber viroid (PSTVd) along with ef 1-α gene of potato

et al. 2015

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SYBR green based realtime RT-PCR assay coupled with melt curve analysis was developed for the detection of Potato spindle tuber viroid (PSTVd) along with and without internal control from potato. The amplification of the specific targets was identified by their melting points viz., 85.93±0.22, 85.62±0.34 and 82.07 ±0.23 for primer pairs PSTVd-1F/PSTVd-1R, PSTVd-NFP/PSTVd-NRP and PSTVd-QFP1/PSTVd-QRP1, respectively. The realtime RT-PCR assay was 1×10 4 times more sensitive than RT-PCR assay and the assay could detect up to 20 copies of the target using serially diluted plasmid and up to 0.025 fg of total RNA from infected tissues diluted in healthy plant RNA. Duplex realtime RT-PCR assay was also standardized to detect PSTVd along with elongation factor 1-α (ef-1α) gene, a stable housekeeping gene of potato. Duplex realtime RT-PCR assay showed two melt peaks indicating the successful amplification both the PSTVd RNA and internal control RNA. The developed assays could consistently detect PSTVd and proved to be highly sensitive and rapid for the detection of PSTVd in post entry quarantine testing.

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Fast real‐time detection of Potato spindle tuber viroid by RT‐LAMP

Cited by 50 publications

References 33 publications

Shining a Light on LAMP Assays — A Comparison of LAMP Visualization Methods Including the Novel use of Berberine

Shining a Light on LAMP Assays — A Comparison of LAMP Visualization Methods Including the Novel use of Berberine

Rapid loop-mediated isothermal amplification assays for grapevine yellows phytoplasmas on crude leaf-vein homogenate has the same performance as qPCR

Duplex realtime RT-PCR assay for the detection of Potato spindle tuber viroid (PSTVd) along with ef 1-α gene of potato

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