Development and Application of qPCR and RPA Genus- and Species-Specific Detection of <i>Phytophthora sojae</i> and <i>P. sansomeana</i> Root Rot Pathogens of Soybean

Rojas, J. Alejandro; Miles, Timothy D.; Coffey, Michael D.; Martin, Frank N.; Chilvers, Martin I.

doi:10.1094/pdis-09-16-1225-re

Cited by 55 publications

(32 citation statements)

References 40 publications

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“…Non-isothermal molecular-based assays (e.g., polymerase chain reaction (PCR) and quantitative PCR (qPCR)) have been developed to identify Phytophthora species using several nuclear and mitochondrial loci (e.g., ypt1 gene and atp9-nad9 spacer region) [1,[11][12][13][14][15][16][17][18][19][20] (Figure 1). Figure 1 details the loci commonly used for Phytophthora species identification in both isothermal and non-isothermal assays; citations for the primers used are included in the figure legend ( Figure 1).…”

Section: Introductionmentioning

confidence: 99%

“…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%

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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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“…Indeed, several RPA assays for diagnosing plant diseases have been developed (Rojas et al . ; Strayer‐Scherer et al . ).…”

Section: Introductionmentioning

confidence: 99%

“…RPA technology has high sensitivity, requires minimal sample preparation and enables rapid amplification (<20 min) at a low reaction temperature (37-42°C) without initial heating for DNA denaturation (Lobato and O'Sullivan 2017). Indeed, several RPA assays for diagnosing plant diseases have been developed (Rojas et al 2017;Strayer-Scherer et al 2019). Moreover, RPA assays can be combined with lateral flow (LF) dipsticks to achieve visual detection of pathogens (Poulton and Webster 2018).…”

Section: Introductionmentioning

confidence: 99%

Rapid and equipment‐free detection of Phytophthora capsici using lateral flow strip‐based recombinase polymerase amplification assay

Yu¹,

Shen²,

Dai

et al. 2019

Lett Appl Microbiol

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Phytophthora capsici is an important oomycete pathogen that causes devastating diseases in various crops. Methods for the rapid detection of P. capsici are important for disease control and eradication programmes. Here, we developed an assay based on lateral flow strip‐based recombinase polymerase amplification (LF‐RPA) for the rapid and equipment‐free detection of P. capsici. The specific primers and a probe were designed using the sequence of Ypt1, and the optimal assay conditions were 40°C for 20 min. The specificity of the assay was verified using closely related oomycetes and fungal species, and its detection limit was 10 pg of genomic DNA. In combination with a simple DNA extraction method, the LF‐RPA assay enabled detection of P. capsici in diseased pepper samples without specialized equipment within 30 min. Consequently, the LF‐RPA assay developed in this study enables rapid and equipment‐free detection of P. capsici and has potential for further development as a diagnostic kit for application in the field or in resource‐limited laboratories. Significance and Impact of the Study We developed a novel assay based on lateral flow strip‐based recombinase polymerase amplification (LF‐RPA) for the rapid and equipment‐free detection of Phytophthora capsici. In combination with a simple DNA extraction method, the LF‐RPA assay detected P. capsici in field samples without specialized equipment within 30 min. The assay has potential for further development as a diagnostic kit for application in the field or in resource‐limited laboratories.

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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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Development and Application of qPCR and RPA Genus- and Species-Specific Detection of Phytophthora sojae and P. sansomeana Root Rot Pathogens of Soybean

Cited by 55 publications

References 40 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

Rapid and equipment‐free detection of Phytophthora capsici using lateral flow strip‐based recombinase polymerase amplification assay

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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