scholarly journals Development of a quantitative loop-mediated isothermal amplification assay for the field detection ofErysiphe necator

PeerJ ◽  
2018 ◽  
Vol 6 ◽  
pp. e4639 ◽  
Author(s):  
Lindsey D. Thiessen ◽  
Tara M. Neill ◽  
Walter F. Mahaffee
Keyword(s):  
Powdery Mildew ◽  
Pathogen Detection ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Lamp Assay ◽  
Isothermal Amplification ◽  
Management Tool ◽  
Willamette Valley ◽  
Loop Mediated Isothermal Amplification ◽  
Grape Powdery Mildew

Plant pathogen detection systems have been useful tools to monitor inoculum presence and initiate management schedules. More recently, a loop-mediated isothermal amplification (LAMP) assay was successfully designed for field use in the grape powdery mildew pathosystem; however, false negatives or false positives were prevalent in grower-conducted assays due to the difficulty in perceiving the magnesium pyrophosphate precipitate at low DNA concentrations. A quantitative LAMP (qLAMP) assay using a fluorescence resonance energy transfer-based probe was assessed by grape growers in the Willamette Valley of Oregon. Custom impaction spore samplers were placed at a research vineyard and six commercial vineyard locations, and were tested bi-weekly by the lab and by growers. Grower-conducted qLAMP assays used a beta-version of the Smart-DART handheld LAMP reaction devices (Diagenetix, Inc., Honolulu, HI, USA), connected to Android 4.4 enabled, Bluetooth-capable Nexus 7 tablets for output. Quantification by a quantitative PCR assay was assumed correct to compare the lab and grower qLAMP assay quantification. Growers were able to conduct and interpret qLAMP results; however, theErysiphe necatorinoculum quantification was unreliable using the beta-Smart-DART devices. The qLAMP assay developed was sensitive to one spore in early testing of the assay, but decreased to >20 spores by the end of the trial. The qLAMP assay is not likely a suitable management tool for grape powdery mildew due to losses in sensitivity and decreasing costs and portability for other, more reliable molecular tools.

scholarly journals Evaluation of Loop-Mediated Isothermal Amplification for Rapid Detection of Erwinia amylovora on Pear and Apple Fruit Flowers

Plant Disease ◽  
2011 ◽  
Vol 95 (4) ◽  
pp. 423-430 ◽  
Author(s):  
Todd N. Temple ◽  
Kenneth B. Johnson
Keyword(s):  
Rapid Detection ◽  
Pathogen Detection ◽  
Fire Blight ◽  
Management Practices ◽  
Erwinia Amylovora ◽  
Lamp Assay ◽  
Apple Fruit ◽  
Isothermal Amplification ◽  
Loop Mediated Isothermal Amplification ◽  
Forecasting Models

Fire blight of pear and apple is frequently an inoculum-limited disease but weather-based forecasting models commonly assume that the pathogen is omnipresent. To improve fire blight risk assessment during flowering, we developed a rapid pathogen detection protocol that uses loop-mediated isothermal amplification (LAMP) to detect DNA of epiphytic Erwinia amylovora on samples of pear and apple flowers. LAMP detected a single flower colonized epiphytically by E. amylovora in a sample of 100 flower clusters (approximately 600 flowers). Samples of 100 flower clusters from orchards (approximately one sample per hectare) were washed and subjected to LAMP, which was completed in 2 h. In three experimental orchards inoculated with E. amylovora, positive LAMP reactions were attained from nine of nine 100-flower cluster samples; pathogen populations in the floral washes averaged 5.2 × 103 CFU per flower as determined by dilution plating. Samples of pear and apple flowers obtained from 60 commercial orchards located in Oregon, Washington, California, and Utah resulted in detection of E. amylovora by LAMP assay from 34 sites, 20 of which developed fire blight. Of samples at early bloom, 10% were positive for epiphytic E. amylovora compared with 28% at petal fall; pathogen density in washes of positive samples averaged 3.2 × 102 CFU per flower. In another 26 orchards, all floral washes were negative for E. amylovora by LAMP and by dilution plating; a light severity of fire blight was observed in 8 of these orchards. Overall, positive detection of epiphytic E. amylovora in commercial orchards by LAMP-based scouting generally occurred at later stages of bloom after heat (risk) units had begun to accumulate, an indication that weather-based forecasting models may be an adequate measure of fire blight risk for many orchardists. Nonetheless, several orchardists communicated that information from the LAMP-based rapid detection protocol resulted in modification of their fire blight management practices.

scholarly journals The qPCR and loop mediated isothermal amplification for rapid detection of Ustilago tritici

2019 ◽  
Author(s):  
Hanwen Yan ◽  
Jian Zhang ◽  
Dongfang Ma ◽  
Junliang Yin
Keyword(s):  
Detection Limit ◽  
Pathogen Detection ◽  
Puccinia Striiformis ◽  
Lamp Assay ◽  
Alternaria Solani ◽  
Bipolaris Sorokiniana ◽  
Isothermal Amplification ◽  
Loose Smut ◽  
Loop Mediated Isothermal Amplification ◽  
Ustilago Tritici

Wheat loose smut caused by Ustilago tritici a seed-borne disease, is difficult to control due to the expansion of wheat planting area and difficulty of pathogen detection. In this study, real-time fluorescence quantitative PCR (qPCR) and loop-mediated isothermal amplification (LAMP) assays were used to rapidly amplify the DNA of U. tritici. Five pairs primers for qPCR and two series primers for LAMP were designed. Firstly, the specify of primers were carried out by using the DNAs of U. tritici, Fusarium graminearum, Blumeria graminis, Rhizoctonia cerealis, Puccinia striiformis, Bipolaris sorokiniana, and Alternaria solani as templates. Then the amplification systems are optimized. Finally, the sensitivity of qPCR and LAMP assays were quantified. The results show that using the primers pairs Y430F/R, Y307F/R, Y755F/R and Y139F/R for qPCR, primers L-139 and L-988 for LAMP assay could be used for U. tritici detection. In the sensitivity test, the detection limit of qPCR assay is 10 pg μL-1 of genomic DNA, the detection limit of LAMP assay is 100 fg μL -1 . We successfully performed qPCR and LAMP assays on two wheat loose smut wheat samples, and confirmed sequenced U. tritici infection by subsequently sequencing. This paper established two methods for U. tritici detection, which could be used for wheat loose smut diagnose in lab and field.

scholarly journals qPCR and loop mediated isothermal amplification for rapid detection of Ustilago tritici

PeerJ ◽  
2019 ◽  
Vol 7 ◽  
pp. e7766 ◽  
Author(s):  
Hanwen Yan ◽  
Jian Zhang ◽  
Dongfang Ma ◽  
Junliang Yin
Keyword(s):  
Detection Limit ◽  
Genomic Dna ◽  
Pathogen Detection ◽  
Puccinia Striiformis ◽  
Lamp Assay ◽  
Bipolaris Sorokiniana ◽  
Isothermal Amplification ◽  
Loose Smut ◽  
Loop Mediated Isothermal Amplification ◽  
Ustilago Tritici

Loose smut of wheat caused by the basidiomycete fungus Ustilago tritici, a seed-borne disease, is difficult to control because of the expanse of wheat planting area and difficulty in pathogen detection. In this study, real-time fluorescence quantitative PCR (qPCR) and loop-mediated isothermal amplification (LAMP) assays are used to rapidly amplify the DNA of U. tritici. Five pairs of primers for qPCR and two series primers for LAMP were designed. Primarily, the specificity of the primer was assessed by using genomic DNA of U. tritici, Fusarium graminearum, Blumeria graminis, Rhizoctonia cerealis, Puccinia striiformis, Bipolaris sorokiniana, and Alternaria solani as templates. Further, the amplification systems were optimized. Finally, the sensitivity of qPCR and LAMP assays were evaluated. The results showed that the primer Y-430 F/R, Y-307 F/R, Y-755 F/R, and Y-139 F/R for qPCR and primers L-139 and L-988 for LAMP could be used for U. tritici detection. In the sensitivity test, the detection limit of qPCR assay was identified as 10 pg μL−1 of genomic DNA, the detection limit for LAMP assay was 100 fg μL−1. We successfully performed qPCR and LAMP assays on wheat loose smut wheat samples. This paper establishes two methods for U. tritici detection, which can be used for diagnosis of wheat loose smut in the laboratory and in the field.

Erwinia amylovora loop-mediated isothermal amplification (LAMP) assay for rapid pathogen detection and on-site diagnosis of fire blight

2013 ◽  
Vol 92 (3) ◽  
pp. 332-339 ◽  
Author(s):  
Andreas Bühlmann ◽  
Joël F. Pothier ◽  
Fabio Rezzonico ◽  
Theo H.M. Smits ◽  
Michael Andreou ◽  
...  
Keyword(s):  
Pathogen Detection ◽  
Fire Blight ◽  
Erwinia Amylovora ◽  
Lamp Assay ◽  
Isothermal Amplification ◽  
Loop Mediated Isothermal Amplification

scholarly journals The qPCR and loop mediated isothermal amplification for rapid detection of Ustilago tritici

2019 ◽  
Author(s):  
Hanwen Yan ◽  
Jian Zhang ◽  
Dongfang Ma ◽  
Junliang Yin
Keyword(s):  
Detection Limit ◽  
Pathogen Detection ◽  
Puccinia Striiformis ◽  
Lamp Assay ◽  
Alternaria Solani ◽  
Bipolaris Sorokiniana ◽  
Isothermal Amplification ◽  
Loose Smut ◽  
Loop Mediated Isothermal Amplification ◽  
Ustilago Tritici

Wheat loose smut caused by Ustilago tritici a seed-borne disease, is difficult to control due to the expansion of wheat planting area and difficulty of pathogen detection. In this study, real-time fluorescence quantitative PCR (qPCR) and loop-mediated isothermal amplification (LAMP) assays were used to rapidly amplify the DNA of U. tritici. Five pairs primers for qPCR and two series primers for LAMP were designed. Firstly, the specify of primers were carried out by using the DNAs of U. tritici, Fusarium graminearum, Blumeria graminis, Rhizoctonia cerealis, Puccinia striiformis, Bipolaris sorokiniana, and Alternaria solani as templates. Then the amplification systems are optimized. Finally, the sensitivity of qPCR and LAMP assays were quantified. The results show that using the primers pairs Y430F/R, Y307F/R, Y755F/R and Y139F/R for qPCR, primers L-139 and L-988 for LAMP assay could be used for U. tritici detection. In the sensitivity test, the detection limit of qPCR assay is 10 pg μL-1 of genomic DNA, the detection limit of LAMP assay is 100 fg μL -1 . We successfully performed qPCR and LAMP assays on two wheat loose smut wheat samples, and confirmed sequenced U. tritici infection by subsequently sequencing. This paper established two methods for U. tritici detection, which could be used for wheat loose smut diagnose in lab and field.

scholarly journals Development of a loop-mediated isothermal amplification assay for the detection of Tilletia controversa based on genome comparison

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Somayyeh Sedaghatjoo ◽  
Monika K. Forster ◽  
Ludwig Niessen ◽  
Petr Karlovsky ◽  
Berta Killermann ◽  
...  
Keyword(s):  
Test Performance ◽  
Genomic Dna ◽  
Lamp Assay ◽  
Fungal Species ◽  
Isothermal Amplification ◽  
Genome Comparison ◽  
Performance Study ◽  
Loop Mediated Isothermal Amplification ◽  
Tilletia Controversa ◽  
Genomic Regions

AbstractTilletia controversa causing dwarf bunt of wheat is a quarantine pathogen in several countries. Therefore, its specific detection is of great phytosanitary importance. Genomic regions routinely used for phylogenetic inferences lack suitable polymorphisms for the development of species-specific markers. We therefore compared 21 genomes of six Tilletia species to identify DNA regions that were unique and conserved in all T. controversa isolates and had no or limited homology to other Tilletia species. A loop-mediated isothermal amplification (LAMP) assay for T. controversa was developed based on one of these DNA regions. The specificity of the assay was verified using 223 fungal samples comprising 43 fungal species including 11 Tilletia species, in particular 39 specimens of T. controversa, 92 of T. caries and 40 of T. laevis, respectively. The assay specifically amplified genomic DNA of T. controversa from pure cultures and teliospores. Only Tilletia trabutii generated false positive signals. The detection limit of the LAMP assay was 5 pg of genomic DNA per reaction. A test performance study that included five laboratories in Germany resulted in 100% sensitivity and 97.7% specificity of the assay. Genomic regions, specific to common bunt (Tilletia caries and Tilletia laevis together) are also provided.

scholarly journals Development and validation of a one-step reverse transcription loop-mediated isothermal amplification (RT-LAMP) for rapid detection of ZIKV in patient samples from Brazil

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Severino Jefferson Ribeiro da Silva ◽  
Keith Pardee ◽  
Udeni B. R. Balasuriya ◽  
Lindomar Pena
Keyword(s):  
Reverse Transcription ◽  
Rapid Detection ◽  
Quantitative Polymerase Chain Reaction ◽  
Rna Extraction ◽  
Lamp Assay ◽  
Isothermal Amplification ◽  
Serum Samples ◽  
Low Resource ◽  
Loop Mediated Isothermal Amplification ◽  
One Step

AbstractWe have previously developed and validated a one-step assay based on reverse transcription loop-mediated isothermal amplification (RT-LAMP) for rapid detection of the Zika virus (ZIKV) from mosquito samples. Patient diagnosis of ZIKV is currently carried out in centralized laboratories using the reverse transcription-quantitative polymerase chain reaction (RT-qPCR), which, while the gold standard molecular method, has several drawbacks for use in remote and low-resource settings, such as high cost and the need of specialized equipment. Point-of-care (POC) diagnostic platforms have the potential to overcome these limitations, especially in low-resource countries where ZIKV is endemic. With this in mind, here we optimized and validated our RT-LAMP assay for rapid detection of ZIKV from patient samples. We found that the assay detected ZIKV from diverse sample types (serum, urine, saliva, and semen) in as little as 20 min, without RNA extraction. The RT-LAMP assay was highly specific and up to 100 times more sensitive than RT-qPCR. We then validated the assay using 100 patient serum samples collected from suspected cases of arbovirus infection in the state of Pernambuco, which was at the epicenter of the last Zika epidemic. Analysis of the results, in comparison to RT-qPCR, found that the ZIKV RT-LAMP assay provided sensitivity of 100%, specificity of 93.75%, and an overall accuracy of 95.00%. Taken together, the RT-LAMP assay provides a straightforward and inexpensive alternative for the diagnosis of ZIKV from patients and has the potential to increase diagnostic capacity in ZIKV-affected areas, particularly in low and middle-income countries.

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