Loop‐mediated isothermal amplification for detection of the tomato and potato late blight pathogen,
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              hytophthora infestans
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Hansen, Zachariah R.; Knaus, Brian J.; Tabima, Javier F.; Press, Caroline M.; Judelson, Howard S.; Grünwald, Niklaus J.; Smart, Christine D.

doi:10.1111/jam.13079

Cited by 62 publications

(29 citation statements)

References 50 publications

(136 reference statements)

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“…Four primers were constructed (F3/B3, FIP/BIP) based on the Ypt1 gene sequence that was able to target six distinct regions of P. infestans target genomic DNA. The Ypt1 gene showed high sensitivity and specificity in the LAMP assay (1.28 × 10 -4 ng μL -1 , i.e., 128 fg) in this study, unlike ITS II (2 pg), which was used to detect P. infestans in a previous study (Hansen et al, 2016) and yielded similar results to our nested PCR assay results. Furthermore, the Ypt1 gene is more sensitive and specific than ITS II, which has the drawback of cross reactivity with other species, such as P. nicotianae (Kroon et al, 2004; Blair et al, 2008, 2012; Hansen et al, 2016).…”

Section: Discussionsupporting

confidence: 83%

“…The internal transcribed spacer (ITS) areas of the nuclear-encoded ribosomal RNA genes (rDNA) are broadly used to recognize and detect Phytophthora species (Cooke et al, 2000; Silvar et al, 2005; Pavón et al, 2008), and P. infestans -specific primers based on the ITS for LAMP detection have also been used in diseased plant tissues, soil, and water to rapidly detect the pathogen (Hansen et al, 2016). However, they are not adequately flexible to separate closely related taxa frequently (Kroon et al, 2004; Schena and Cooke, 2006; Chen et al, 2013; Li et al, 2013).…”

Section: Introductionmentioning

confidence: 99%

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Evaluation of Different PCR-Based Assays and LAMP Method for Rapid Detection of Phytophthora infestans by Targeting the Ypt1 Gene

Khan

Yue

et al. 2017

Front. Microbiol.

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Late blight, caused by the oomycete Phytophthora infestans, is one of the most devastating diseases affecting potato and tomato worldwide. Early diagnosis of the P. infestans pathogen causing late blight should be the top priority for addressing disease epidemics and management. In this study, we performed a loop-mediated isothermal amplification (LAMP) assay, conventional polymerase chain reaction (PCR), nested PCR, and real-time PCR to verify and compare the sensitivity and specificity of the reaction based on the Ypt1 (Ras-related protein) gene of P. infestans. In comparison with the PCR-based assays, the LAMP technique led to higher specificity and sensitivity, using uncomplicated equipment with an equivalent time frame. All 43 P. infestans isolates, yielded positive detection results using LAMP assay showing no cross reaction with other Phytophthora spp., oomycetes or fungal pathogens. The LAMP assay yielded the lowest detectable DNA concentration (1.28 × 10-4 ng μL-1), being 10 times more sensitive than nested PCR (1.28 × 10-3 ng μL-1), 100 times more sensitive than real-time PCR (1.28 × 10-2 ng μL-1) and 103 times more sensitive than the conventional PCR assay (1.28 × 10-1 ng μL-1). In the field experiment, the LAMP assay outperformed the other tests by amplifying only diseased tissues (leaf and stem), and showing no positive reaction in healthy tissues. Overall, the LAMP assay developed in this study provides a specific, sensitive, simple, and effective visual method for detection of the P. infestans pathogen, and is therefore suitable for application in early prediction of the disease to reduce the risk of epidemics.

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Section: Discussionsupporting

confidence: 83%

Section: Introductionmentioning

confidence: 99%

Evaluation of Different PCR-Based Assays and LAMP Method for Rapid Detection of Phytophthora infestans by Targeting the Ypt1 Gene

Khan

Yue

et al. 2017

Front. Microbiol.

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“…Moreover, it can be monitored with the naked eye by adding colorimetric indicators, such as calcein, which produces a green fluorescent signal if the LAMP reaction is positive (Zhang et al, 2014). With these advantages, the LAMP method has been widely used for detection of plant pathogens, such as viruses (Hadersdorfer et al, 2011; Peng et al, 2012), bacteria (Hodgetts et al, 2015), nematodes (Kang et al, 2015), oomycetes (Hansen et al, 2016), and fungi (Shen et al, 2016). In recent years, the LAMP-based assay has grown in popularity for the detection of many plant-pathogenic fungi (Niessen and Vogel, 2010; Denschlag et al, 2012; Niessen, 2015).…”

Section: Introductionmentioning

confidence: 99%

Rapid and Sensitive Detection of Didymella bryoniae by Visual Loop-Mediated Isothermal Amplification Assay

Yao

et al. 2016

Front. Microbiol.

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Didymella bryoniae is a pathogenic fungus that causes gummy stem blight (GSB) in Cucurbitaceae crops (e.g., cantaloupe, muskmelon, cucumber, and watermelon). GSB produces lesions on the stems and leaves, and can also be spread by seeds. Here, we developed a rapid, visual, and sensitive loop-mediated amplification (LAMP) assay for D. bryoniae detection based on sequence-characterized amplified regions (GenBank accession nos GQ872461 and GQ872462) common to the two random amplification of polymorphic DNA group genotypes (RGI and RGII) of D. bryoniae; ideal conditions for detection were optimized for completion in 45 min at 63°C. The sensitivity and specificity of the LAMP assay were further analyzed in comparison with those of a conventional polymerase chain reaction (PCR). The sensitivity of the LAMP assay was 1000-fold higher than that of conventional PCR with a detection limit of 0.1 fg μL-1 of targeted DNA. The LAMP assay could be accomplished in about 45 min, with the results visible to the naked eye. The assay showed high specificity in discriminating all D. bryoniae isolates from seven other fungal pathogens that occur in Cucurbitaceae crops. The LAMP assay also detected D. bryoniae infection in young muskmelon leaves with suspected early symptoms of GSB disease. Hence, the technique has great potential for developing rapid and sensitive visual detection methods for the D. bryoniae pathogen in crops and seeds. This method has potential application in early prediction of disease and reducing the risk of epidemics.

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“…Other isothermal DNA-based diagnostics such as LAMP and TwistDX are now being used as they may be more rapid than PCR, use less-expensive equipment or be less prone to inhibition by chemicals present naturally at times in some of the components of the air spora (Hansen et al 2016;Thiessen et al 2016). These isothermal DNA amplification methods offer the prospect of samples to be analysed using relatively simple equipment and even in the field.…”

Section: Air Sampling For Enhanced Disease Controlmentioning

confidence: 99%

Novel Technologies for the detection of Fusarium head blight disease and airborne inoculum

West

Canning

Perryman

et al. 2017

Trop. plant pathol.

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Many pathogens are dispersed by airborne spores, which can vary in space and time. We can use air sampling integrated with suitable diagnostic methods to give a rapid warning of inoculum presence to improve the timing of control options, such as fungicides. Air sampling can also be used to monitor changes in genetic traits of pathogen populations such as the race structure or frequency of fungicide resistance. Although some image-analysis methods are possible to identify spores, in many cases, species-specific identification can only be achieved by DNA-based methods such as qPCR and LAMP and in some cases by antibody-based methods (lateral flow devices) and biomarker-based methods ('electronic noses' and electro-chemical biosensors). Many of these methods also offer the prospect of rapid on-site detection to direct disease control decisions. Thresholds of spore concentrations that correspond to a disease risk depend on the sampler (spore-trap) location (whether just above the crop canopy, on a UAV or drone, or on a tall building) and also need to be considered with weather-based infection models. Where disease control by spore detection is not possible, some diseases can be detected at early stages using optical sensing methods, especially chlorophyll fluorescence. In the case of Fusarium infections on wheat, it is possible to map locations of severe infections, using optical sensing methods, to segregate harvesting of severely affected areas of fields to avoid toxins entering the food chain. This is most useful where variable crop growth or microclimates within fields generate spatially variable infection, i.e. parts of fields that develop disease, while other areas have escaped infection and do not develop any disease.

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Loop‐mediated isothermal amplification for detection of the tomato and potato late blight pathogen, P hytophthora infestans

Abstract: This is the first report of a LAMP assay for the detection of P. infestans, the causal organism of potato and tomato late blight. These assays have potential for immediate utility in plant disease research and diagnostic laboratories.

Cited by 62 publications

References 50 publications

Evaluation of Different PCR-Based Assays and LAMP Method for Rapid Detection of Phytophthora infestans by Targeting the Ypt1 Gene

Evaluation of Different PCR-Based Assays and LAMP Method for Rapid Detection of Phytophthora infestans by Targeting the Ypt1 Gene

Rapid and Sensitive Detection of Didymella bryoniae by Visual Loop-Mediated Isothermal Amplification Assay

Novel Technologies for the detection of Fusarium head blight disease and airborne inoculum

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