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 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.

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 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 Application of Loop-Mediated Isothermal Amplification combined with colorimetric and lateral flow dipstick visualization as the potential point-of-care testing for diphtheria

2019 ◽  
Author(s):  
Aleksandra Anna Zasada ◽  
Aldona Wiatrzyk ◽  
Urszula Czajka ◽  
Klaudia Brodzik ◽  
Kamila Formińska ◽  
...  
Keyword(s):  
Detection Limit ◽  
Sensitivity And Specificity ◽  
Point Of Care ◽  
Lamp Assay ◽  
Corynebacterium Diphtheriae ◽  
Lateral Flow ◽  
Isothermal Amplification ◽  
Suspected Case ◽  
Point Of Care Testing ◽  
Loop Mediated Isothermal Amplification

Abstract Background Diphtheria outbreaks occurred in endemic areas and imported and indigenous cases are reported in UE/EEA. Because of the high infectiveness and severity of the disease, early and accurate diagnosis of each suspected case is essential for the treatment and management of the case and close contacts. The aim of the study was to establish simple and rapid testing methods based on Loop-Mediated Isothermal Amplification (LAMP) assay for the detection of Corynebacterium diphtheriae and differentiation between toxigenic and non-toxigenic strains.Methods Corynebacterium diphtheriae and Corynebacterium ulcerans isolates from the National Institute of Public Health-National Institute of Hygiene collection were used for the development of LAMP assay for the diagnosis of diphtheria and nontoxigenic C. diphtheriae infections. Various colorimetric methods for visualization of results were investigated. Sensitivity and specificity of the assay were examined using a collection of DNA samples from various gram-positive and gram-negative bacteria.Results The LAMP assay for tox and dtxR genes was developed. The sensitivity and specificity of the assay were calculated as 100%. The detection limit was estimated as 1.42 pg/µl concentration of DNA template when the reaction was conducted for 60 min. However, the detection limit was lowered 10 times for every 10 minutes of reduction in the time of incubation during the reaction. Positive results were successfully detected colorimetrically using hydroxynaphthol blue, calcein, QuantiFluor, and lateral flow Milenia HybriDetect dipsticks.Conclusion The assay developed in the study might be applied for point-of-care testing of diphtheria and other C. diphtheriae infections. It is highly sensitive, specific, inexpensive, easy to use, and suitable for low-resource settings.

scholarly journals Application of Loop-Mediated Isothermal Amplification combined with colorimetric and lateral flow dipstick visualization as the potential point-of-care testing for Corynebacterium diphtheriae

2020 ◽  
Author(s):  
Aleksandra Anna Zasada ◽  
Aldona Wiatrzyk ◽  
Urszula Czajka ◽  
Klaudia Brodzik ◽  
Kamila Formińska ◽  
...  
Keyword(s):  
Detection Limit ◽  
Sensitivity And Specificity ◽  
Point Of Care ◽  
Lamp Assay ◽  
Corynebacterium Diphtheriae ◽  
Lateral Flow ◽  
Isothermal Amplification ◽  
Suspected Case ◽  
Point Of Care Testing ◽  
Loop Mediated Isothermal Amplification

Abstract Background Diphtheria outbreaks occurred in endemic areas and imported and indigenous cases are reported in UE/EEA. Because of the high infectiveness and severity of the disease, early and accurate diagnosis of each suspected case is essential for the treatment and management of the case and close contacts.The aim of the study was to establish simple and rapid testing methods based on Loop-Mediated Isothermal Amplification (LAMP) assay for the detection of Corynebacterium diphtheriae and differentiation between toxigenic and non-toxigenic strains.Methods Corynebacterium diphtheriae and Corynebacterium ulcerans isolates from the National Institute of Public Health-National Institute of Hygiene collection were used for the development of LAMP assay for the diagnosis of diphtheria and nontoxigenic C. diphtheriae infections. Various colorimetric methods for visualization of results were investigated. Sensitivity and specificity of the assay were examined using a collection of DNA samples from various gram-positive and gram-negative bacteria.Results The LAMP assay for tox and dtxR genes was developed. The sensitivity and specificity of the assay were calculated as 100%. The detection limit was estimated as 1.42 pg/µl concentration of DNA template when the reaction was conducted for 60 min. However, the detection limit was lowered 10 times for every 10 minutes of reduction in the time of incubation during the reaction. Positive results were successfully detected colorimetrically using hydroxynaphthol blue, calcein, QuantiFluor, and lateral flow Milenia HybriDetect dipsticks.Conclusion The assay developed in the study might be applied for point-of-care testing of diphtheria and other C. diphtheriae infections as well as for other infections caused by diphtheria-toxin producing Corynebacterium species. It is highly sensitive, specific, inexpensive, easy to use, and suitable for low-resource settings.

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 Application of Loop-Mediated Isothermal Amplification combined with colorimetric and lateral flow dipstick visualization as the potential point-of-care testing for Corynebacterium diphtheriae

2020 ◽  
Author(s):  
Aleksandra Anna Zasada ◽  
Aldona Wiatrzyk ◽  
Urszula Czajka ◽  
Klaudia Brodzik ◽  
Kamila Formińska ◽  
...  
Keyword(s):  
Detection Limit ◽  
Sensitivity And Specificity ◽  
Point Of Care ◽  
Lamp Assay ◽  
Corynebacterium Diphtheriae ◽  
Lateral Flow ◽  
Isothermal Amplification ◽  
Suspected Case ◽  
Point Of Care Testing ◽  
Loop Mediated Isothermal Amplification

Abstract Background Diphtheria outbreaks occurred in endemic areas and imported and indigenous cases are reported in UE/EEA. Because of the high infectiveness and severity of the disease, early and accurate diagnosis of each suspected case is essential for the treatment and management of the case and close contacts. The aim of the study was to establish simple and rapid testing methods based on Loop-Mediated Isothermal Amplification (LAMP) assay for the detection of Corynebacterium diphtheriae and differentiation between toxigenic and non-toxigenic strains. Methods Corynebacterium diphtheriae and Corynebacterium ulcerans isolates from the National Institute of Public Health-National Institute of Hygiene collection were used for the development of LAMP assay for the diagnosis of diphtheria and nontoxigenic C. diphtheriae infections. Various colorimetric methods for visualization of results were investigated. Sensitivity and specificity of the assay were examined using a collection of DNA samples from various gram-positive and gram-negative bacteria. Results The LAMP assay for tox and dtxR genes was developed. The sensitivity and specificity of the assay were calculated as 100%. The detection limit was estimated as 1.42 pg/µl concentration of DNA template when the reaction was conducted for 60 min. However, the detection limit was lowered 10 times for every 10 minutes of reduction in the time of incubation during the reaction. Positive results were successfully detected colorimetrically using hydroxynaphthol blue, calcein, QuantiFluor, and lateral flow Milenia HybriDetect dipsticks. Conclusion The assay developed in the study might be applied for point-of-care testing of diphtheria and other C. diphtheriae infections as well as for other infections caused by diphtheria-toxin producing Corynebacterium species. It is highly sensitive, specific, inexpensive, easy to use, and suitable for low-resource settings.

scholarly journals Application of Loop-Mediated Isothermal Amplification combined with colorimetric and lateral flow dipstick visualization as the potential point-of-care testing for Corynebacterium diphtheriae

2020 ◽  
Author(s):  
Aleksandra Anna Zasada ◽  
Aldona Wiatrzyk ◽  
Urszula Czajka ◽  
Klaudia Brodzik ◽  
Kamila Formińska ◽  
...  
Keyword(s):  
Detection Limit ◽  
Sensitivity And Specificity ◽  
Point Of Care ◽  
Lamp Assay ◽  
Corynebacterium Diphtheriae ◽  
Lateral Flow ◽  
Isothermal Amplification ◽  
Suspected Case ◽  
Point Of Care Testing ◽  
Loop Mediated Isothermal Amplification

Abstract Background Diphtheria outbreaks occurred in endemic areas and imported and indigenous cases are reported in UE/EEA. Because of the high infectiveness and severity of the disease, early and accurate diagnosis of each suspected case is essential for the treatment and management of the case and close contacts. The aim of the study was to establish simple and rapid testing methods based on Loop-Mediated Isothermal Amplification (LAMP) assay for the detection of Corynebacterium diphtheriae and differentiation between toxigenic and non-toxigenic strains. Methods Corynebacterium diphtheriae and Corynebacterium ulcerans isolates from the National Institute of Public Health-National Institute of Hygiene collection were used for the development of LAMP assay for the diagnosis of diphtheria and nontoxigenic C. diphtheriae infections. Various colorimetric methods for visualization of results were investigated. Sensitivity and specificity of the assay were examined using a collection of DNA samples from various gram-positive and gram-negative bacteria. Results The LAMP assay for tox and dtxR genes was developed. The sensitivity and specificity of the assay were calculated as 100%. The detection limit was estimated as 1.42 pg/µl concentration of DNA template when the reaction was conducted for 60 min. However, the detection limit was lowered 10 times for every 10 minutes of reduction in the time of incubation during the reaction. Positive results were successfully detected colorimetrically using hydroxynaphthol blue, calcein, QuantiFluor, and lateral flow Milenia HybriDetect dipsticks. Conclusion The assay developed in the study might be applied for point-of-care testing of diphtheria and other C. diphtheriae infections as well as for other infections caused by diphtheria-toxin producing Corynebacterium species. It is highly sensitive, specific, inexpensive, easy to use, and suitable for low-resource settings.

scholarly journals Application of Loop-Mediated Isothermal Amplification combined with colorimetric and lateral flow dipstick visualization as the potential point-of-care testing for Corynebacterium diphtheriae

2020 ◽  
Author(s):  
Aleksandra Anna Zasada ◽  
Aldona Wiatrzyk ◽  
Urszula Czajka ◽  
Klaudia Brodzik ◽  
Kamila Formińska ◽  
...  
Keyword(s):  
Detection Limit ◽  
Sensitivity And Specificity ◽  
Point Of Care ◽  
Lamp Assay ◽  
Corynebacterium Diphtheriae ◽  
Lateral Flow ◽  
Isothermal Amplification ◽  
Suspected Case ◽  
Point Of Care Testing ◽  
Loop Mediated Isothermal Amplification

Abstract Background: Diphtheria outbreaks occurred in endemic areas and imported and indigenous cases are reported in UE/EEA. Because of the high infectiveness and severity of the disease, early and accurate diagnosis of each suspected case is essential for the treatment and management of the case and close contacts.The aim of the study was to establish simple and rapid testing methods based on Loop-Mediated Isothermal Amplification (LAMP) assay for the detection of Corynebacterium diphtheriae and differentiation between toxigenic and non-toxigenic strains.Methods: Corynebacterium diphtheriae and Corynebacterium ulcerans isolates from the National Institute of Public Health-National Institute of Hygiene collection were used for the development of LAMP assay for the diagnosis of diphtheria and nontoxigenic C. diphtheriae infections. Various colorimetric methods for visualization of results were investigated. Sensitivity and specificity of the assay were examined using a collection of DNA samples from various gram-positive and gram-negative bacteria.Results: The LAMP assay for tox and dtxR genes was developed. The sensitivity and specificity of the assay were calculated as 100%. The detection limit was estimated as 1.42 pg/µl concentration of DNA template when the reaction was conducted for 60 min. However, the detection limit was lowered 10 times for every 10 minutes of reduction in the time of incubation during the reaction. Positive results were successfully detected colorimetrically using hydroxynaphthol blue, calcein, QuantiFluor, and lateral flow Milenia HybriDetect dipsticks.Conclusion: The assay developed in the study might be applied for point-of-care testing of diphtheria and other C. diphtheriae infections as well as for other infections caused by diphtheria-toxin producing Corynebacterium species. It is highly sensitive, specific, inexpensive, easy to use, and suitable for low-resource settings.

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