scholarly journals Development and evaluation of a loop-mediated isothermal amplification (LAMP) assay for the detection of Tomato brown rugose fruit virus (ToBRFV)

2020 ◽  
Author(s):  
Alian Sarkes ◽  
Heting Fu ◽  
David Feindel ◽  
Michael W. Harding ◽  
Jie Feng
Keyword(s):  
Colour Change ◽  
Diagnostic System ◽  
Lamp Assay ◽  
The United States ◽  
Visual Observation ◽  
Isothermal Amplification ◽  
Experimental Conditions ◽  
Rna Molecules ◽  
Loop Mediated Isothermal Amplification ◽  
Target Rna

AbstractTomato brown rugose fruit virus (ToBRFV) is a member of Tobamovirus infecting tomato and pepper. Within North America, both the United States and Mexico consider ToBRFV to be a regulated pest. In Canada, the presence of ToBRFV has been reported, but an efficient diagnostic system has not yet been established. Here, we describe the development and assessment of a loop-mediated isothermal amplification (LAMP)-based assay to detect ToBRFV. The LAMP test was efficient and robust, and results could be obtained within 35 min with an available RNA sample. Amplification was possible when either water bath or oven were used to maintain the temperature at isothermal conditions (65°C), and results could be read by visual observation of colour change. Detection limit of the LAMP was eight target RNA molecules. Under the experimental conditions tested, LAMP was as sensitive as qPCR and 100 times more sensitive than the currently used rt-PCR. We recommend this sensitive, efficient LAMP protocol to be used for routine lab testing of ToBRFV.

scholarly journals Loop-Mediated Isothermal Amplification for the Diagnostic Detection of Meloidogyne chitwoodi and M. fallax

Plant Disease ◽  
2019 ◽  
Vol 103 (1) ◽  
pp. 12-18 ◽  
Author(s):  
Lei Zhang ◽  
Cynthia Gleason
Keyword(s):  
Pacific Northwest ◽  
Intergenic Spacer ◽  
Lamp Assay ◽  
The United States ◽  
Isothermal Amplification ◽  
Root Knot Nematode ◽  
Meloidogyne Chitwoodi ◽  
Loop Mediated Isothermal Amplification ◽  
The Pacific ◽  
Potato Nematodes

Meloidogyne chitwoodi is a root-knot nematode that parasitizes a broad range of plants. In the Pacific Northwest (PNW) of the United States, M. chitwoodi is a major potato pest. The nematodes infect roots and tubers; blemishes caused by the nematodes on the tubers significantly affect potato marketability. M. chitwoodi is a quarantine pathogen by many regulatory agencies, limiting potato trade opportunities when it is present. A loop-mediated isothermal amplification (LAMP) assay was developed to amplify the intergenic spacer (IGS2)-18S region of the ribosomal rDNA of M. chitwoodi. Using the LAMP assay, we could detect the presence of M. chitwoodi from infected Washington State soil samples. The LAMP primers showed specificity for DNA from M. chitwoodi and the closely related species M. fallax. There was no cross reaction of the LAMP primers with DNA from tropical nematodes M. incognita, M. arenaria, and M. javanica, or the Northern root-knot nematode M. hapla. The LAMP assays can be completed within 45 min, and they were 100 times more sensitive in nematode detection than conventional PCR. The LAMP assay will facilitate detection of potato nematodes M. chitwoodi and M. fallax. Knowledge of potato nematodes, particularly M. chitwoodi in PNW soils, will aid management decisions.

scholarly journals Development of Loop-Mediated Isothermal Amplification Assay for Rapid Detection of Cucurbit Leaf Crumple Virus

2020 ◽  
Vol 21 (5) ◽  
pp. 1756 ◽  
Author(s):  
Sumyya Waliullah ◽  
Kai-Shu Ling ◽  
Elizabeth J. Cieniewicz ◽  
Jonathan E. Oliver ◽  
Pingsheng Ji ◽  
...  
Keyword(s):  
Lamp Assay ◽  
Visual Observation ◽  
Cross Reactivity ◽  
Isothermal Amplification ◽  
Infected Leaf ◽  
Loop Mediated Isothermal Amplification ◽  
Efficient Detection ◽  
Field Samples ◽  
Optimum Reaction ◽  
Cucurbit Leaf Crumple Virus

A loop-mediated isothermal amplification (LAMP) assay was developed for simple, rapid and efficient detection of Cucurbit leaf crumple virus (CuLCrV), one of the most important begomoviruses that infects cucurbits worldwide. A set of six specific primers targeting a total 240 nt sequence regions in the DNA A of CuLCrV were designed and synthesized for detection of CuLCrV from infected leaf tissues using real-time LAMP amplification with the Genie® III system, which was further confirmed by gel electrophoresis and SYBR™ Green I DNA staining for visual observation. The optimum reaction temperature and time were determined, and no cross-reactivity was seen with other begomoviruses. The LAMP assay could amplify CuLCrV from a mixed virus assay. The sensitivity assay demonstrated that the LAMP reaction was more sensitive than conventional PCR, but less sensitive than qPCR. However, it was simpler and faster than the other assays evaluated. The LAMP assay also amplified CuLCrV-infected symptomatic and asymptomatic samples more efficiently than PCR. Successful LAMP amplification was observed in mixed virus-infected field samples. This simple, rapid, and sensitive method has the capacity to detect CuLCrV in samples collected in the field and is therefore suitable for early detection of the disease to reduce the risk of epidemics.

scholarly journals Detecting Potentially Virulent Vibrio vulnificus Strains in Raw Oysters by Quantitative Loop-Mediated Isothermal Amplification

2011 ◽  
Vol 77 (8) ◽  
pp. 2589-2595 ◽  
Author(s):  
Feifei Han ◽  
Fei Wang ◽  
Beilei Ge
Keyword(s):  
Pure Culture ◽  
Vibrio Vulnificus ◽  
Lamp Assay ◽  
The United States ◽  
Isothermal Amplification ◽  
Quantitative Detection ◽  
Strain Atcc ◽  
Content Type ◽  
Loop Mediated Isothermal Amplification ◽  
Negative Results

ABSTRACTVibrio vulnificusis a leading cause of seafood-related deaths in the United States. Sequence variations in the virulence-correlated gene (vcg) have been used to distinguish between clinical and environmentalV. vulnificusstrains, with a strong association between clinical ones and the C sequence variant (vcgC). In this study,vcgCwas selected as the target to design a loop-mediated isothermal amplification (LAMP) assay for the rapid, sensitive, specific, and quantitative detection of potentially virulentV. vulnificusstrains in raw oysters. No false-positive or false-negative results were generated among the 125 bacterial strains used to evaluate assay specificity. The detection limit was 5.4 CFU per reaction for a virulentV. vulnificusstrain (ATCC 33815) in pure culture, 100-fold more sensitive than that of PCR. In spiked raw oysters, the assay was capable of detecting 2.5 × 103CFU/g ofV. vulnificusATCC 33815, while showing negative results for a nonvirulentV. vulnificusstrain (515-4c2) spiked at 107CFU/g. After 6 h of enrichment, the LAMP assay could detect 1 CFU/g of the virulentV. vulnificusstrain ATCC 33815. Standard curves generated in pure culture and spiked oysters suggested a good linear relationship between cell numbers of the virulentV. vulnificusstrain and turbidity signals. In conclusion, the LAMP assay developed in this study could quantitatively detect potentially virulentV. vulnificusin raw oysters with high speed, specificity, and sensitivity, which may facilitate better control ofV. vulnificusrisks associated with raw oyster consumption.

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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