scholarly journals Development of Rapid Isothermal Amplification Assays for Detection of Phytophthora spp. in Plant Tissue

2015 ◽  
Vol 105 (2) ◽  
pp. 265-278 ◽  
Author(s):  
Timothy D. Miles ◽  
Frank N. Martin ◽  
Michael D. Coffey
Keyword(s):  
Real Time ◽  
Internal Control ◽  
Plant Tissue ◽  
Pathogen Detection ◽  
Phytophthora Ramorum ◽  
Isothermal Amplification ◽  
Sample Collection ◽  
Phytophthora Spp ◽  
Wide Range ◽  
Tissue Maceration

Several isothermal amplification techniques recently have been developed that are tolerant of inhibitors present in many plant extracts, which can reduce the need for obtaining purified DNA for running diagnostic assays. One such commercially available technique that has similarities with real-time polymerase chain reaction (PCR) for designing primers and a labeled probe is recombinase polymerase amplification (RPA). This technology was used to develop two simple and rapid approaches for detection of Phytophthora spp.: one genus-specific assay multiplexed with a plant internal control and the other species-specific assays for Phytophthora ramorum and P. kernoviae. All assays were tested for sensitivity (ranging from 3 ng to 1 fg of DNA) and specificity using DNA extracted from more than 136 Phytophthora taxa, 21 Pythium spp., 1 Phytopythium sp., and a wide range of plant species. The lower limit of linear detection using purified DNA was 200 to 300 fg of DNA in all pathogen RPA assays. Six different extraction buffers were tested for use during plant tissue maceration and the assays were validated in the field by collecting 222 symptomatic plant samples from over 50 different hosts. Only 56 samples were culture positive for Phytophthora spp. whereas 91 were positive using the Phytophthora genus-specific RPA test and a TaqMan real-time PCR assay. A technique for the generation of sequencing templates from positive RPA amplifications to confirm species identification was also developed. These RPA assays have added benefits over traditional technologies because they are rapid (results can be obtained in as little as 15 min), do not require DNA extraction or extensive training to complete, use less expensive portable equipment than PCR-based assays, and are significantly more specific than current immunologically based methods. This should provide a rapid, field-deployable capability for pathogen detection that will facilitate point-of-sample collection processing, thereby reducing the time necessary for accurate diagnostics and making management decisions.

scholarly journals Evaluation of Molecular Markers for Phytophthora ramorum Detection and Identification: Testing for Specificity Using a Standardized Library of Isolates

2009 ◽  
Vol 99 (4) ◽  
pp. 390-403 ◽  
Author(s):  
F. N. Martin ◽  
M. D. Coffey ◽  
K. Zeller ◽  
R. C. Hamelin ◽  
P. Tooley ◽  
...  
Keyword(s):  
Molecular Markers ◽  
Real Time ◽  
Pathogen Detection ◽  
Species Group ◽  
False Positives ◽  
Phytophthora Ramorum ◽  
Sscp Analysis ◽  
Oxidase Gene ◽  
Phytophthora Spp ◽  
Field Samples

Given the importance of Phytophthora ramorum from a regulatory standpoint, it is imperative that molecular markers for pathogen detection are fully tested to evaluate their specificity in detection of the pathogen. In an effort to evaluate 11 reported diagnostic techniques, we assembled a standardized DNA library using accessions from the World Phytophthora Genetic Resource Collection for 315 isolates representing 60 described Phytophthora spp. as well as 11 taxonomically unclassified isolates. These were sent blind to collaborators in seven laboratories to evaluate published diagnostic procedures using conventional (based on internal transcribed spacer [ITS] and cytochrome oxidase gene [cox]1 and 2 spacer regions) and real-time polymerase chain reaction (based on ITS and cox1 and 2 spacer regions as well as β-tubulin and elicitin genes). Single-strand conformation polymorphism (SSCP) analysis using an automated sequencer for data collection was also evaluated for identification of all species tested. In general, the procedures worked well, with varying levels of specificity observed among the different techniques. With few exceptions, all assays correctly identified all isolates of P. ramorum and low levels of false positives were observed for the mitochondrial cox spacer markers and most of the real-time assays based on nuclear markers (diagnostic specificity between 96.9 and 100%). The highest level of false positives was obtained with the conventional nested ITS procedure; however, this technique is not stand-alone and is used in conjunction with two other assays for diagnostic purposes. The results indicated that using multiple assays improved the accuracy of the results compared with looking at a single assay alone, in particular when the markers represented different genetic loci. The SSCP procedure accurately identified P. ramorum and was helpful in classification of a number of isolates to a species level. With one exception, all procedures accurately identified P. ramorum in blind evaluations of 60 field samples that included examples of plant infection by 11 other Phytophthora spp. The SSCP analysis identified eight of these species, with three identified to a species group.

scholarly journals Development of a high-throughput real-time PCR system for detection of enzootic pathogens in pigs

2019 ◽  
Vol 32 (1) ◽  
pp. 51-64 ◽  
Author(s):  
Nicole B. Goecke ◽  
Charlotte K. Hjulsager ◽  
Jesper S. Krog ◽  
Kerstin Skovgaard ◽  
Lars E. Larsen
Keyword(s):  
Real Time ◽  
High Throughput ◽  
Real Time Pcr ◽  
Pathogen Detection ◽  
Detection System ◽  
Working Hours ◽  
Negative Influence ◽  
Optimal Choice ◽  
National Veterinary Institute ◽  
Wide Range

Respiratory and intestinal diseases in pigs can have significant negative influence on productivity and animal welfare. A wide range of real-time PCR (rtPCR) assays are used in our laboratory (National Veterinary Institute, Technical University of Denmark) for pathogen detection, and PCR analyses are performed on traditional rtPCR platforms in which a limited number of samples can be analyzed per day given limitations in equipment and personnel. To mitigate these restrictions, rtPCR assays have been optimized for the high-throughput rtPCR BioMark platform (Fluidigm). Using this platform, we developed a high-throughput detection system that can be used for simultaneous examination of 48 samples with detection specificity for 18 selected respiratory and enteric viral and bacterial pathogens of high importance to Danish pig production. The rtPCR assays were validated and optimized to run under the same reaction conditions using a BioMark 48.48 dynamic array (DA) integrated fluidic circuit chip, and the sensitivity and specificity were assessed by testing known positive samples. Performance of the 48.48DA was similar to traditional rtPCR analysis, and the specificity of the 48.48DA was high. Application of the high-throughput platform has resulted in a significant reduction in cost and working hours and has provided production herds with a new innovative service with the potential to facilitate the optimal choice of disease control strategies such as vaccination and treatment.

scholarly journals Development of an Assay for Rapid Detection and Quantification of Verticillium dahliae in Soil

2012 ◽  
Vol 102 (3) ◽  
pp. 331-343 ◽  
Author(s):  
Guillaume J. Bilodeau ◽  
Steven T. Koike ◽  
Pedro Uribe ◽  
Frank N. Martin
Keyword(s):  
Real Time ◽  
Verticillium Dahliae ◽  
Internal Control ◽  
Real Time Pcr ◽  
Pcr Amplification ◽  
Intergenic Spacer ◽  
Single Copy ◽  
Taqman Assay ◽  
Wide Range ◽  
Pathogen Populations

Verticillium dahliae is responsible for Verticillium wilt on a wide range of hosts, including strawberry, on which low soil inoculum densities can cause significant crop loss. Determination of inoculum density is currently done by soil plating but this can take 6 to 8 weeks to complete and delay the grower's ability to make planting decisions. To provide a faster means for estimating pathogen populations in the soil, a multiplexed TaqMan real-time polymerase chain reaction (PCR) assay based on the ribosomal DNA (rDNA) intergenic spacer (IGS) was developed for V. dahliae. The assay was specific for V. dahliae and included an internal control for evaluation of inhibition due to the presence of PCR inhibitors in DNA extracted from soil samples. An excellent correlation was observed in regression analysis (R2 = 0.96) between real-time PCR results and inoculum densities determined by soil plating in a range of field soils with pathogen densities as low as 1 to 2 microsclerotia/g of soil. Variation in copy number of the rDNA was also evaluated among isolates by SYBR Green real-time PCR amplification of the V. dahliae-specific amplicon compared with amplification of several single-copy genes and was estimated to range from ≈24 to 73 copies per haploid genome, which translated into possible differences in results among isolates of ≈1.8 cycle thresholds. Analysis of the variation in results of V. dahliae quantification among extractions of the same soil sample indicated that assaying four replicate DNA extractions for each field sample would provide accurate results. A TaqMan assay also was developed to help identify colonies of V. tricorpus on soil plates.

scholarly journals Comparison of Real-Time PCR, Reverse Transcriptase Real-Time PCR, Loop-Mediated Isothermal Amplification, and the FDA Conventional Microbiological Method for the Detection of Salmonella spp. in Produce

2011 ◽  
Vol 77 (18) ◽  
pp. 6495-6501 ◽  
Author(s):  
Guodong Zhang ◽  
Eric W. Brown ◽  
Narjol González-Escalona
Keyword(s):  
Reverse Transcriptase ◽  
Real Time ◽  
Internal Control ◽  
Real Time Pcr ◽  
Culture Method ◽  
Isothermal Amplification ◽  
Molecular Methods ◽  
Salmonella Spp ◽  
Content Type ◽  
Loop Mediated Isothermal Amplification

ABSTRACTContamination of foods, especially produce, withSalmonellaspp. is a major concern for public health. Several methods are available for the detection ofSalmonellain produce, but their relative efficiency for detectingSalmonellain commonly consumed vegetables, often associated with outbreaks of food poisoning, needs to be confirmed. In this study, the effectiveness of three molecular methods for detection ofSalmonellain six produce matrices was evaluated and compared to the FDA microbiological detection method. Samples of cilantro (coriander leaves), lettuce, parsley, spinach, tomato, and jalapeno pepper were inoculated withSalmonellaserovars at two different levels (105and <101CFU/25 g of produce). The inoculated produce was assayed by the FDASalmonellaculture method (Bacteriological Analytical Manual) and by three molecular methods: quantitative real-time PCR (qPCR), quantitative reverse transcriptase real-time PCR (RT-qPCR), and loop-mediated isothermal amplification (LAMP). Comparable results were obtained by these four methods, which all detected as little as 2 CFU ofSalmonellacells/25 g of produce. All control samples (not inoculated) were negative by the four methods. RT-qPCR detects only liveSalmonellacells, obviating the danger of false-positive results from nonviable cells. False negatives (inhibition of either qPCR or RT-qPCR) were avoided by the use of either a DNA or an RNA amplification internal control (IAC). Compared to the conventional culture method, the qPCR, RT-qPCR, and LAMP assays allowed faster and equally accurate detection ofSalmonellaspp. in six high-risk produce commodities.

scholarly journals Real-time loop-mediated isothermal amplification: an early-warning tool for quarantine plant pathogen detection

AMB Express ◽  
2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Chiara Aglietti ◽  
Nicola Luchi ◽  
Alessia Lucia Pepori ◽  
Paola Bartolini ◽  
Francesco Pecori ◽  
...  
Keyword(s):  
Real Time ◽  
Early Warning ◽  
Plant Pathogen ◽  
Pathogen Detection ◽  
Isothermal Amplification ◽  
Loop Mediated Isothermal Amplification ◽  
Time Loop ◽  
Plant Pathogen Detection

scholarly journals Faster, Simpler, More-Specific Methods for Improved Molecular Detection of Phytophthora ramorum in the Field

2007 ◽  
Vol 73 (12) ◽  
pp. 4040-4047 ◽  
Author(s):  
J. A. Tomlinson ◽  
I. Barker ◽  
N. Boonham
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Color Change ◽  
Lamp Assay ◽  
Phytophthora Ramorum ◽  
Specific Detection ◽  
Pcr Assay ◽  
Lamp Product ◽  
Wide Range ◽  
Pcr Method

ABSTRACT Phytophthora ramorum is the causal agent of sudden oak death. The pathogen also affects a wide range of tree, shrub, and herbaceous species in natural and landscaped environments as well as plants in the nursery industry. A TaqMan real-time PCR method for the detection of this pathogen in the field has been described previously; this paper describes the development of a number of assays based on this method which have various advantages for use in the field. A scorpion real-time PCR assay that is twice as fast as TaqMan was developed, allowing the detection of P. ramorum in less than 30 min. Also designed was a loop-mediated isothermal amplification (LAMP) assay, which allowed sensitive and specific detection of P. ramorum in 45 min using only a heated block. A positive reaction was identified by the detection of the LAMP product by color change visible to the naked eye.

Loop-Mediated Isothermal Amplification for Detection of Endogenous Sad1 Gene in Cotton: An Internal Control for Rapid Onsite GMO Testing

2018 ◽  
Vol 101 (5) ◽  
pp. 1657-1660 ◽  
Author(s):  
Monika Singh ◽  
Rajesh K Bhoge ◽  
Gurinderjit Randhawa
Keyword(s):  
Real Time ◽  
Internal Control ◽  
Acyl Carrier Protein ◽  
Genetically Modified ◽  
Isothermal Amplification ◽  
Bt Cotton ◽  
Gm Crop ◽  
Loop Mediated Isothermal Amplification ◽  
Control Objective ◽  
Time Amplification

Abstract Background: Confirming the integrity of seed samples in powdered form is important prior to conducting a genetically modified organism (GMO) test. Rapid onsite methods may provide a technological solution to check for genetically modified (GM) events at ports of entry. In India, Bt cotton is the commercialized GM crop with four approved GM events; however, 59 GM events have been approved globally. GMO screening is required to test for authorized GM events. The identity and amplifiability of test samples could be ensured first by employing endogenous genes as an internal control. Objective: A rapid onsite detection method was developed for an endogenous reference gene, stearoyl acyl carrier protein desaturase (Sad1) of cotton, employing visual and real-time loop-mediated isothermal amplification (LAMP). Methods: The assays were performed at a constant temperature of 63°C for 30 min for visual LAMP and 62ºC for 40 min for real-time LAMP. Positive amplification was visualized as a change in color from orange to green on addition of SYBR® Green or detected as real-time amplification curves. Results: Specificity of LAMP assays was confirmed using a set of 10 samples. LOD for visual LAMP was up to 0.1%, detecting 40 target copies, and for real-time LAMP up to 0.05%, detecting 20 target copies. Conclusions: The developed methods could be utilized to confirm the integrity of seed powder prior to conducting a GMO test for specific GM events of cotton. Highlights: LAMP assays for the endogenous Sad1 gene of cotton have been developed to be used as an internal control for onsite GMO testing in cotton.

scholarly journals Clinical assessment and validation of a rapid and sensitive SARS-CoV-2 test using reverse-transcription loop-mediated isothermal amplification

2020 ◽  
Author(s):  
Melis N Anahtar ◽  
Graham EG McGrath ◽  
Brian A Rabe ◽  
Nathan A Tanner ◽  
Benjamin A White ◽  
...  
Keyword(s):  
Reverse Transcription ◽  
Color Change ◽  
Limit Of Detection ◽  
Isothermal Amplification ◽  
Clinical Samples ◽  
Sample Collection ◽  
Clinical Workflow ◽  
Assay Sensitivity ◽  
Loop Mediated Isothermal Amplification ◽  
Wide Range

Amid the enduring COVID-19 pandemic, there is an urgent need for expanded access to rapid and sensitive SARS-CoV-2 testing worldwide. Here we present a simple clinical workflow that uses a sensitive and highly specific colorimetric reverse-transcription loop-mediated isothermal amplification (RT-LAMP) to detect SARS-CoV-2 and takes forty minutes from sample collection to result. This test requires no specialized equipment and costs a few dollars per sample. Nasopharyngeal samples collected in saline were added either directly (unprocessed) to RT-LAMP reactions or first inactivated by a combined chemical and heat treatment step to inhibit RNases and lyse virions and human cells. The specimens were then amplified with two SARS-CoV-2-specific primer sets and an internal specimen control; the resulting color change was visually interpreted. While direct addition of unprocessed specimens to RT-LAMP reactions could reliably detect samples with abundant SARS-CoV-2, the assay sensitivity markedly increased after the addition of an inactivation step. In 62 clinical samples with a wide range of SARS-CoV-2 nucleic acid concentrations, the assay had 87.5% sensitivity and 100% specificity with a limit of detection at least 25 copies/μL, making it an ideal test to rule in infection. To increase sensitivity, samples that tested negative for SARS-CoV-2 by direct sample addition could be reflexed to a purification step, to increase the effective per-reaction sample input volume. In 40 purified samples, the assay yielded a 90% sensitivity and 100% specificity, with a limit of detection comparable to commercially available real-time PCR-based diagnostics that have received Emergency Use Authorization (EUA) from the FDA. This test for SARS-CoV-2 can be performed in a range of settings for a fraction of the price of other available tests, with limited equipment, and without relying on over-burdened supply chains to increase overall testing capacity.

scholarly journals Detection and Quantification of Phytophthora ramorum from California Forests Using a Real-Time Polymerase Chain Reaction Assay

2004 ◽  
Vol 94 (10) ◽  
pp. 1075-1083 ◽  
Author(s):  
Katherine J. Hayden ◽  
David Rizzo ◽  
Justin Tse ◽  
Matteo Garbelotto
Keyword(s):  
Polymerase Chain Reaction ◽  
Real Time ◽  
Pathogen Detection ◽  
Phytophthora Species ◽  
Phytophthora Ramorum ◽  
Plant Diseases ◽  
Chain Reaction ◽  
Nested Polymerase Chain Reaction ◽  
New Host ◽  
Polymerase Chain

The timely and accurate detection of pathogens is a critical aid in the study of the epidemiology and biology of plant diseases. In the case of regulated organisms, the availability of a sensitive and reliable assay is essential when trying to achieve early detection of the pathogen. We developed and tested a real-time, nested polymerase chain reaction (PCR) assay for the detection of Phytophthora ramorum, causal agent of sudden oak death. This technique then was implemented as part of a widespread environmental screen throughout California. The method here described is sensitive, detecting less than 12 fg of pathogen DNA, and is specific for P. ramorum when tested across 21 Phytophthora spp. Hundreds of symptomatic samples from 33 sites in 14 California counties were assayed, resulting in the discovery of 10 new host species and 23 infested areas, including 4 new counties. With the exception of a single host, PCR-based discovery of new hosts and infested areas always was confirmed by traditional pathogen isolations and inoculation studies. Nonetheless, molecular diagnostics were key in early pathogen detection, and steered the direction of further research on this newly discovered and generalist Phytophthora species.

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