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A Loop-mediated Isothermal Amplification (LAMP) Assay for Rapid Identification of Bemisia tabaci
Published on: October 29, 2018
Development and application of a loop-mediated isothermal amplification assay for rapid identification of
Jie Luo1, Rudi F Vogel, Ludwig Niessen
1Technische Universität München, Lehrstuhl für Technische Mikrobiologie, Weihenstephaner Steig 16, Freising, Germany.
Abstract:
Aflatoxins are the most thoroughly studied mycotoxins. They are produced by several members of the genus Aspergillus in section Flavi with Aspergillus flavus, Aspergillus parasiticus, and Aspergillus nomius being frequently isolated from contaminated food sources. In this work, we describe the development and evaluation of loop-mediated isothermal amplification (LAMP) assays for rapid detection of the three species in separate analyses. The acl1-gene of A. flavus and amy1-genes of A. nomius and A. parasiticus were used as target genes. The detection limits were 2.4, 7.6 and 20pg of pure DNA/reaction for A. flavus, A. nomius and A. parasiticus, respectively. For specificity testing, DNA extracted from mycelia of representative strains of 39 Aspergillus species, 23 Penicillium species, 75 Fusarium species and 37 other fungal species was used as a template for the specific LAMP primer sets developed for the three target species. The LAMP assay was combined with a DNA extraction method for the analysis of pure fungal cultures as well as artificially contaminated Brazil nuts, peanuts and green coffee beans. It is suggested that the developed LAMP assay is a promising tool in the prediction of a potential aflatoxin risk in food and food raw materials and may therefore be suitable for high throughput analysis in the food industry.
Insights
This study developed rapid loop-mediated isothermal amplification (LAMP) assays to detect three key Aspergillus species. These assays offer a promising tool for predicting aflatoxin contamination risk in food products.
Area of Science:
- Food Science
- Microbiology
- Molecular Biology
Background:
- Aflatoxins, potent mycotoxins, are produced by Aspergillus species, posing significant food safety risks.
- Aspergillus flavus, Aspergillus parasiticus, and Aspergillus nomius are frequently implicated in food contamination.
- Accurate and rapid detection methods are crucial for managing aflatoxin contamination in food.
Purpose of the Study:
- To develop and evaluate loop-mediated isothermal amplification (LAMP) assays for the rapid and specific detection of Aspergillus flavus, Aspergillus nomius, and Aspergillus parasiticus.
- To establish the detection limits and specificity of the developed LAMP assays.
- To assess the applicability of the LAMP assay for analyzing contaminated food samples.
Main Methods:
- Development of species-specific LAMP assays targeting the acl1-gene (A. flavus) and amy1-genes (A. nomius, A. parasiticus).
- Determination of detection limits using purified fungal DNA.
- Specificity testing against a wide range of fungal species.
- Integration of LAMP with a DNA extraction method for analyzing pure cultures and artificially contaminated food matrices (Brazil nuts, peanuts, green coffee beans).
Main Results:
- Successful development of distinct LAMP assays for each target species.
- Achieved low DNA detection limits: 2.4 pg/reaction (A. flavus), 7.6 pg/reaction (A. nomius), and 20 pg/reaction (A. parasiticus).
- Demonstrated high specificity against numerous other fungal species.
- Validated the assay's performance on pure cultures and artificially contaminated food samples.
Conclusions:
- The developed LAMP assays are sensitive and specific for detecting key aflatoxin-producing Aspergillus species.
- This method provides a rapid and promising tool for predicting aflatoxin risk in various food and food raw materials.
- The high-throughput capability makes the LAMP assay suitable for industrial food safety applications.
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