scholarly journals A loop‐mediated isothermal amplification (LAMP) assay based on unique markers derived from genotyping by sequencing data for rapid in planta diagnosis of Panama disease caused by Tropical Race 4 in banana

2019 ◽  
Vol 68 (9) ◽  
pp. 1682-1693 ◽  
Author(s):  
N. Ordóñez ◽  
M. Salacinas ◽  
O. Mendes ◽  
M. F. Seidl ◽  
H. J. G. Meijer ◽  
...  
Keyword(s):  
Lamp Assay ◽  
Genotyping By Sequencing ◽  
Isothermal Amplification ◽  
In Planta ◽  
Sequencing Data ◽  
Panama Disease ◽  
Loop Mediated Isothermal Amplification ◽  
Unique Markers ◽  
Race 4

scholarly journals Novel Multiplex and Loop-Mediated Isothermal Amplification (LAMP) Assays for Rapid Species and Mating-Type Identification of Oculimacula acuformis and O. yallundae (Causal Agents of Cereal Eyespot), and Application for Detection of Ascospore Dispersal and in planta Use

2020 ◽  
Author(s):  
Kevin M. King ◽  
Gavin J. Eyres ◽  
Jon West ◽  
Clara Siraf ◽  
Pavel Matusinsky ◽  
...  
Keyword(s):  
Multiplex Pcr ◽  
Mating Type ◽  
Field Experiments ◽  
Lamp Assay ◽  
Isothermal Amplification ◽  
In Planta ◽  
Loop Mediated Isothermal Amplification ◽  
Multiplex Pcr Assay ◽  
Species Specific ◽  
Oculimacula Acuformis

Eyespot, caused by the related fungal pathogens Oculimacula acuformis (OA) and O. yallundae (OY), is an important cereal stem-base disease in temperate parts of the world. Both species are dispersed mainly by splash-dispersed conidia but are also known to undergo sexual reproduction yielding apothecia containing ascospores. Field diagnosis of eyespot can be challenging with other pathogens causing similar symptoms, which complicates eyespot management strategies. Differences between OA and OY (e.g. host pathogenicity and fungicide sensitivity) require that both be targeted for effective disease management. Here, we develop and apply two molecular methods for species-specific and mating-type (MAT1-1 or MAT1-2) discrimination of OA and OY isolates. First, a multiplex PCR-based diagnostic assay targeting the MAT idiomorph region was developed allowing simultaneous determination of both species and mating type. This multiplex-PCR assay was successfully applied to type a global collection of isolates. Second, the development of loop-mediated isothermal amplification (LAMP) assays targeting beta-tubulin sequences is described, which allow fast (<9 min) species-specific discrimination of global OA and OY isolates. The LAMP assay can detect very small amounts of target DNA (1 pg) and was successfully applied in planta. In addition, mating-type specific LAMP assays were also developed for rapid (<12 min) genotyping of OA and OY isolates. Finally, the multiplex PCR-based diagnostic was applied, in conjunction with spore trapping in field experiments, to provide evidence of the wind dispersal of ascospores from a diseased crop. The results indicate an important role of the sexual cycle in the dispersal of eyespot.

scholarly journals Loop-Mediated Isothermal Amplification and Polymerase Chain Reaction Methods for Specific and Rapid Detection of Rhodococcus fascians

Plant Disease ◽  
2013 ◽  
Vol 97 (4) ◽  
pp. 517-529 ◽  
Author(s):  
M. Serdani ◽  
M. Curtis ◽  
M. L. Miller ◽  
J. Kraus ◽  
M. L. Putnam
Keyword(s):  
Polymerase Chain Reaction ◽  
Lamp Assay ◽  
Isothermal Amplification ◽  
In Planta ◽  
Bacterial Strains ◽  
Chain Reaction ◽  
Loop Mediated Isothermal Amplification ◽  
Rhodococcus Fascians ◽  
Polymerase Chain

Rhodococcus fascians is a phytopathogenic actinobacterium which causes leafy galls and other plant distortions that result in economically significant losses to nurseries producing ornamental plants. Traditional assays for detection and identification are time-consuming and laborious. We developed a rapid polymerase chain reaction (PCR) diagnostic assay based on two primer pairs, p450 and fas, which target the fasA and fasD genes, respectively, that are essential for pathogenicity. We also developed a faster, more convenient, loop-mediated isothermal amplification (LAMP) assay targeting the fasR gene, which regulates expression of virulence genes. Both assays were evaluated for sensitivity and specificity in vitro and in planta. The p450 and fas primers amplified DNA only from pure cultures of pathogenic reference isolates of R. fascians. Nonpathogenic isolates and 51 other plant-associated bacteria were not amplified. The PCR primers correctly detected pathogenic R. fascians from 73 of 75 (97%) bacterial strains isolated from naturally infected plants. The PCR assay correctly discriminated between pathogenic R. fascians and other bacteria in 132 of 139 (95%) naturally infected plants, and in 34 of 34 (100%) artificially inoculated plants. The fas primers were slightly more accurate than the p450 primers. The LAMP assay accurately detected pathogenic R. fascians in 26 of 28 (93%) naturally infected plants and did not react with 23 asymptomatic plants. The LAMP primers also amplified product for DNA extracts of 40 of 41 bacterial strains isolated from plants with leafy galls. The detection limit of both the PCR and LAMP assays was approximately 103 CFU/30-μl reaction. These new tools allow fast, reliable, and accurate detection of R. fascians in vitro and in planta. The LAMP assay in particular is a significant advancement in rapid R. fascians diagnostics, and enables those with limited laboratory facilities to confirm the presence of this pathogen in infected plants.

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.

scholarly journals Development of a loop-mediated isothermal amplification (LAMP) assay for the identification of the invasive wood borer Aromia bungii (Coleoptera: Cerambycidae) from frass

3 Biotech ◽  
2021 ◽  
Vol 11 (2) ◽  
Author(s):  
Domenico Rizzo ◽  
Nicola Luchi ◽  
Daniele Da Lio ◽  
Linda Bartolini ◽  
Francesco Nugnes ◽  
...  
Keyword(s):  
Lamp Assay ◽  
Isothermal Amplification ◽  
Molecular Diagnostic ◽  
Diagnostic Tools ◽  
Loop Mediated Isothermal Amplification ◽  
Larval Stages ◽  
Longhorn Beetle ◽  
Rapid Monitoring ◽  
Major Pest ◽  
Wood Borer

AbstractThe red-necked longhorn beetle Aromia bungii (Faldermann, 1835) (Coleoptera: Cerambycidae) is native to east Asia, where it is a major pest of cultivated and ornamental species of the genus Prunus. Morphological or molecular discrimination of adults or larval specimens is required to identify this invasive wood borer. However, recovering larval stages of the pest from trunks and branches causes extensive damage to plants and is timewasting. An alternative approach consists in applying non-invasive molecular diagnostic tools to biological traces (i.e., fecal pellets, frass). In this way, infestations in host plants can be detected without destructive methods. This paper presents a protocol based on both real-time and visual loop-mediated isothermal amplification (LAMP), using DNA of A. bungii extracted from fecal particles in larval frass. Laboratory validations demonstrated the robustness of the protocols adopted and their reliability was confirmed performing an inter-lab blind panel. The LAMP assay and the qPCR SYBR Green method using the F3/B3 LAMP external primers were equally sensitive, and both were more sensitive than the conventional PCR (sensitivity > 103 to the same starting matrix). The visual LAMP protocol, due to the relatively easy performance of the method, could be a useful tool to apply in rapid monitoring of A. bungii and in the management of its outbreaks.

scholarly journals Validation of loop-mediated isothermal amplification for the detection of Loa loa infection in Chrysops spp in experimental and natural field conditions

2021 ◽  
Vol 14 (1) ◽  
Author(s):  
Glory Ngongeh Amambo ◽  
Raphael Awah Abong ◽  
Fanny Fri Fombad ◽  
Abdel Jelil Njouendou ◽  
Franck Nietcho ◽  
...  
Keyword(s):  
Lamp Assay ◽  
Isothermal Amplification ◽  
Field Conditions ◽  
Infection Rates ◽  
Loa Loa ◽  
Significant Drop ◽  
Natural Field ◽  
North West ◽  
Loop Mediated Isothermal Amplification ◽  
The North

Abstract Background The mass drug administration of ivermectin for onchocerciasis control has contributed to a significant drop in Loa loa microfilaria loads in humans that has, in turn, led to reduction of infection levels in Chrysops vectors. Accurate parasite detection is essential for assessing loiasis transmission as it provides a potential alternative or indirect strategy for addressing the problem of co-endemic loiasis and lymphatic filariasis through the Onchocerciasis Elimination Programme and it further reflects the true magnitude of the loiasis problem as excess human mortality has been reported to be associated with the disease. Although microscopy is the gold standard for detecting the infection, the sensitivity of this method is compromised when the intensity of infection is low. The loop-mediated isothermal amplification (LAMP) assay of parasite DNA is an alternative method for detecting infection which offers operational simplicity, rapidity and versatility of visual readout options. The aim of this study was to validate the Loa loa LAMP assay for the detection of infected Chrysops spp. under experimental and natural field conditions. Methods Two sets of 18 flies were fed on volunteers with either a low (< 10 mf/ml) or high (> 30,000mf/ml) microfilarial load. The fed flies were maintained under laboratory conditions for 14 days and then analysed using LAMP for the detection of L. loa infection. In addition, a total of 9270 flies were collected from the north-west, east, and south-west regions (SW 1 and 2) of Cameroon using sweep nets and subjected to microscopy (7841 flies) and LAMP (1291 flies plus 138 nulliparous flies) analyses. Results The LAMP assay successfully detected parasites in Chrysops fed on volunteers with both low and high microfilariaemic loads. Field validation and surveillance studies revealed LAMP-based infection rates ranging from 0.5 to 31.6%, with the lowest levels in SW 2 and the highest infection rates in SW 1. The LAMP assay detected significantly higher infection rates than microscopy in four of the five study sites. Conclusion This study demonstrated the potential of LAMP as a simple surveillance tool. It was found to be more sensitive than microscopy for the detection of experimental and natural L. loa infections in Chrysops vectors.

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