Optimization of loop-mediated isothermal amplification (LAMP) assay for robust visualization in SARS-CoV-2 and emerging variants diagnosis

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.

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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

Scientific Reports ◽

10.1038/s41598-021-83371-1 ◽

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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Reverse transcription-loop mediated isothermal amplification (RT-LAMP) assay for detection of AhR receptor responsive xenobiotics

Toxicology Mechanisms and Methods ◽

10.1080/15376516.2021.1884923 ◽

2021 ◽

pp. 1-13

Author(s):

Deeksha Sharma ◽

Payal Rani ◽

Suneel Kumar Onteru ◽

Partha Roy ◽

Rakesh Kumar Tyagi ◽

...

Keyword(s):

Reverse Transcription ◽

Lamp Assay ◽

Isothermal Amplification ◽

Loop Mediated Isothermal Amplification

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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 ◽

10.1007/s13205-020-02602-w ◽

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.

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

Parasites & Vectors ◽

10.1186/s13071-020-04506-3 ◽

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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Loop-mediated isothermal amplification (LAMP) assay for detection of sesame phyllody phytoplasmas in Vietnam

Folia Microbiologica ◽

10.1007/s12223-020-00842-0 ◽

2021 ◽

Author(s):

Nguyen Bao Quoc ◽

Nguyen Thi Thanh Xuan ◽

Nguyen Mai Nghiep ◽

Nguyen Doan Nguyen Phuong ◽

Ton Bao Linh ◽

...

Keyword(s):

Lamp Assay ◽

Isothermal Amplification ◽

Loop Mediated Isothermal Amplification

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A loop-mediated isothermal amplification (LAMP) assay to identify isotype 1 β-tubulin locus SNPs in synthetic double-stranded Haemonchus contortus DNA

Journal of Parasitic Diseases ◽

10.1007/s12639-021-01414-w ◽

2021 ◽

Author(s):

Livio M. Costa-Junior ◽

Umer N. Chaudhry ◽

Philip J. Skuce ◽

Seamus Stack ◽

Neil D. Sargison

Keyword(s):

Genetic Susceptibility ◽

Management Practices ◽

Anthelmintic Resistance ◽

Lamp Assay ◽

Gastrointestinal Nematode ◽

Isothermal Amplification ◽

Loop Mediated Isothermal Amplification ◽

Snp Typing ◽

Field Samples ◽

Β Tubulin

AbstractDevelopment of sustainable gastrointestinal nematode (GIN) control strategies depends on the ability to identify the frequencies of drug-susceptible and resistant genotypes in GIN populations arising from management practices undertaken on individual farms. Resistance to BZ drugs in GINs has been shown to be conferred by the presence of defined SNPs in the isotype 1 β-tubulin locus. Loop-mediated isothermal amplification (LAMP) assays are amenable to use on a range of DNA templates and are potentially adaptable to use in practical, cost-effective, pen-side diagnostic platforms that are needed to detect anthelmintic resistance in the field. In this study, we designed primers and examined LAMP assays to detect each of the three major isotype 1 β-tubulin SNPs conferring genetic susceptibility to BZ drugs. We used artificial pools of synthetic DNA, containing different proportions of susceptible and resistant SNPs to determine reproducibility of the assays. We demonstrated the detection of each of the isotype 1 β-tubulin SNPs conferring susceptibility to BZ drugs using the optimal LAMP assay. Isotype 1 β-tubulin SNP typing was effective in detecting BZ susceptibility, but the accuracy was reduced in samples with less than 60 % susceptible DNA. Our results show the potential for LAMP SNP typing to detect genetic susceptibility or resistance to anthelmintic drugs in livestock GINs, and some of the limitations in our approach that will need to be overcome in order to evaluate this assay using field samples.

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