Specific and Sensitive Detection of <i>Phytophthora nicotianae</i> by Nested <scp>PCR</scp> and Loop‐mediated Isothermal Amplification Assays

Li, Benjin; Liu, Peiqing; Xie, Shuanglun; Yin, Rongmei; Weng, Qiyong; Chen, Qinghe

doi:10.1111/jph.12305

Cited by 24 publications

(13 citation statements)

References 28 publications

(37 reference statements)

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“…Isothermal assays used in plant diagnostics are predominately loop-mediated isothermal amplifications (LAMPs) or recombinase polymerase amplifications (RPA) [19][20][21][22][23][24][25][26][27][28][29][30][31][32]. These assays achieve amplification at stable temperatures (65 • C for LAMP and 39-42 • C for RPA assays) and thus do not need thermocyclers to amplify target DNA.…”

Section: Introductionmentioning

confidence: 99%

“…The majority of the currently available isothermal diagnostic assays for Phytophthora are LAMP assays. Currently, LAMP assays have been reported for Phytophthora kernoviae [21], Phytophthora infestans [22], Phytophthora cinnamomi [23], Phytophthora melonis [24], Phytophthora nicotianae [25], Phytophthora sojae, and Phytophthora ramorum [21,26]. LAMP assays utilize four primers designed to anneal to different regions of the target DNA, as well as a unique DNA polymerase, with strand displacement activity enabling target amplification at a constant temperature (65 • C) [33].…”

Section: Introductionmentioning

confidence: 99%

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Validation of a Preformulated, Field Deployable, Recombinase Polymerase Amplification Assay for Phytophthora Species

McCoy

Miles

Bilodeau

et al. 2020

Plants

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Recombinase polymerase amplification (RPA) assays are valuable molecular diagnostic tools that can detect and identify plant pathogens in the field without time-consuming DNA extractions. Historically, RPA assay reagents were commercially available as a lyophilized pellet in microfuge strip tubes, but have become available in liquid form more recently—both require the addition of primers and probes prior to use, which can be challenging to handle in a field setting. Lyophilization of primers and probes, along with RPA reagents, contained within a single tube limits the risk of contamination, eliminates the need for refrigeration, as the lyophilized reagents are stable at ambient temperatures, and simplifies field use of the assays. This study investigates the potential effect of preformulation on assay performance using a previously validated Phytophthora genus-specific RPA assay, lyophilized with primers and probes included with the RPA reagents. The preformulated lyophilized Phytophthora RPA assay was compared with a quantitative polymerase chain reaction (qPCR) assay and commercially available RPA kits using three qPCR platforms (BioRad CFX96, QuantStudio 6 and Applied Biosystems ViiA7) and one isothermal platform (Axxin T16-ISO RPA), with experiments run in four separate labs. The assay was tested for sensitivity (ranging from 500 to 0.33 pg of DNA) and specificity using purified oomycete DNA, as well as crude extracts of Phytophthora-infected and non-infected plants. The limit of detection (LOD) using purified DNA was 33 pg in the CFX96 and ViiA7 qPCR platforms using the preformulated kits, while the Axxin T16-ISO RPA chamber and the QuantStudio 6 platform could detect down to 3.3 pg with or without added plant extract. The LOD using a crude plant extract for the BioRad CFX96 was 330 pg, whereas the LOD for the ViiA7 system was 33 pg. These trials demonstrate the consistency and uniformity of pathogen detection with preformulated RPA kits for Phytophthora detection when conducted by different labs using different instruments for measuring results.

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Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Validation of a Preformulated, Field Deployable, Recombinase Polymerase Amplification Assay for Phytophthora Species

McCoy

Miles

Bilodeau

et al. 2020

Plants

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“…These include tests for P. capsici Leonian [27], P. cinnamomi [28], P. infestans (Mont.) de Bary [29,30], P. melonis Katsura [31], P. nicotinae [32], P. ramorum Werres, De…”

Section: Introductionmentioning

confidence: 99%

“…[33]. The genetic targets of these tests include the Ras-related protein (ypt1) gene [i.e., [30][31][32][33] and the ITS regions [i.e., 22,27].…”

Section: Introductionmentioning

confidence: 99%

A LAMP at the end of the tunnel: a rapid, field deployable assay for the kauri dieback pathogen,Phytophthora agathidicida

Winkworth¹,

Nelson²,

Bellgard

et al. 2019

Preprint

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The collar rot causing oomycete, Phytophthora agathidicida, threatens the long-term survival of the iconic New Zealand kauri. Currently, testing for this pathogen involves an extended soil bioassay that takes 14-20 days and requires specialised staff, consumables, and infrastructure. Here we describe a loop-mediated isothermal amplification (LAMP) assay for the detection of P. agathidicida that targets a portion of the mitochondrial apocytochrome b coding sequence. This assay has high specificity and sensitivity; it did not cross react with a range of other Phytophthora isolates and detected as little as 1 fg of total P. agathidicida DNA or 116 copies of the target locus. Assay performance was further investigated by testing plant tissue baits from flooded soil samples using both the extended bioassay and LAMP testing of DNA extracted from baits. In these comparisons, P. agathidicida was detected more frequently using the LAMP assay. In addition to greater sensitivity, by removing the need for culturing, the hybrid baiting plus LAMP approach is more cost effective than the bioassay and, importantly, does not require a centralised laboratory facility with specialised staff, consumables, and equipment. Such testing will allow us to address outstanding questions about P. agathidicida. For example, the hybrid approach could enable monitoring of the pathogen beyond areas with visible disease symptoms, allow direct evaluation of rates and patterns of spread, and allow the effectiveness of disease control to be evaluated. The hybrid assay also has the potential to empower local communities. These communities could use this diagnostic tool to evaluate the pathogen status of local kauri stands, providing information around which to base their management and allowing informed engagement with wider initiatives.

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“…Therefore, it hardly meets the need of rapid detection at customs. With the advent of molecular technology, other faster and more cost-effective techniques including isothermal diagnostic assays have become the mainstream in rapid detection of many Phytophthora species (Miles et al 2015) such as P. infestans (Hansen et al 2016;Khan et al 2017;Si Ammour et al 2017), P. sojae (Dai et al 2012; Dai et al 2015;Rojas et al 2017), P. nicotianae (Li et al 2015), P. cinnamomi (Dai et al 2019). A loop-mediated isothermal amplification (LAMP) assay for detecting P. hibernalis was developed by Li et al (2019).…”

Section: Introductionmentioning

confidence: 99%

Peer Review #1 of "A recombinase polymerase amplification-lateral flow dipstick assay for rapid detection of the quarantine citrus pathogen in China, Phytophthora hibernalis (v0.1)"

Ioos¹

2019

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Phytophthora hibernalis, the causal agent of brown rot of citrus fruit, is an important worldwide pathogen and a quarantine pest in China. Current diagnosis of the disease relies on disease symptoms, pathogen isolation, and identification by DNA sequencing. However, symptoms caused by P. hibernalis can be confused with those by other Phytophthora and fungal species. Moreover, pathogen isolation, PCR amplification, and sequencing are timeconsuming. In this study, a rapid assay including 20-min recombinase polymerase amplification targeting the Ypt1 gene and 5-min visualization using lateral flow dipsticks was developed for detecting P. hibernalis. This assay was able to detect 0.2 ng of P. hibernalis genomic DNA in a 50-µL reaction system. It was specific to P. hibernalis without detection of other tested species including P. citrophthora, P. nicotianae, P. palmivora, and P. syringae, four other important citrus pathogens. Using this assay, P. hibernalis was also detected from artificially inoculated orange fruits. Results in this study indicated that this assay has the potential application to detect P. hibernalis at diagnostic laboratories, and plant quarantine departments of customs, especially under time-and resource-limited conditions.

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Specific and Sensitive Detection of Phytophthora nicotianae by Nested PCR and Loop‐mediated Isothermal Amplification Assays

Cited by 24 publications

References 28 publications

Validation of a Preformulated, Field Deployable, Recombinase Polymerase Amplification Assay for Phytophthora Species

Validation of a Preformulated, Field Deployable, Recombinase Polymerase Amplification Assay for Phytophthora Species

A LAMP at the end of the tunnel: a rapid, field deployable assay for the kauri dieback pathogen,Phytophthora agathidicida

Peer Review #1 of "A recombinase polymerase amplification-lateral flow dipstick assay for rapid detection of the quarantine citrus pathogen in China, Phytophthora hibernalis (v0.1)"

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