scholarly journals LAMP-LFD Based on Isothermal Amplification of Multicopy Gene ORF160b: Applicability for Highly Sensitive Low-Tech Screening of Allergenic Soybean (Glycine max) in Food

Foods ◽  
2020 ◽  
Vol 9 (12) ◽  
pp. 1741
Author(s):  
Stefanie M. Allgöwer ◽  
Chris A. Hartmann ◽  
Clarissa Lipinski ◽  
Vera Mahler ◽  
Stefanie Randow ◽  
...  
Keyword(s):  
Glycine Max ◽  
Lateral Flow ◽  
Isothermal Amplification ◽  
Detection Methods ◽  
Current Standard ◽  
Allergen Detection ◽  
Multicopy Gene ◽  
The Matrix ◽  
Soybean Allergy ◽  
Food Matrices

Soybean (Glycine max) allergy can be life threatening. A lack of causative immunotherapy of soybean allergy makes soybean avoidance indispensable. Detection methods are essential to verify allergen labeling and unintentional allergen cross contact during food manufacture. Here, we aimed at evaluating our previously described primers for loop-mediated isothermal amplification (LAMP) of multicopy gene ORF160b, combined with a lateral flow dipstick (LFD)-like detection, for their performance of soybean detection in complex food matrices. The results were compared with those obtained using quantitative real-time Polymerase Chain Reaction (qPCR) as the current standard of DNA-based allergen detection, and antibody-based commercial lateral flow device (LFD) as the current reference of protein-based rapid allergen detection. LAMP-LFD allowed unequivocal and reproducible detection of 10 mg/kg soybean incurred in three representative matrices (boiled sausage, chocolate, instant tomato soup), while clear visibility of positive test lines of two commercial LFD tests was between 10 and 102 mg/kg and depending on the matrix. Sensitivity of soybean detection in incurred food matrices, commercial retail samples, as well as various processed soybean products was comparable between LAMP-LFD and qPCR. The DNA-based LAMP-LFD proved to be a simple and low-technology soybean detection tool, showing sensitivity and specificity that is comparable or superior to the investigated commercial protein-based LFD.

scholarly journals The Development of Highly Specific and Sensitive Primers for the Detection of Potentially Allergenic Soybean (Glycine max) Using Loop-Mediated Isothermal Amplification Combined with Lateral Flow Dipstick (LAMP-LFD)

Foods ◽  
2020 ◽  
Vol 9 (4) ◽  
pp. 423 ◽  
Author(s):  
Stefanie M. Allgöwer ◽  
Chris A. Hartmann ◽  
Thomas Holzhauser
Keyword(s):  
Glycine Max ◽  
Detection Method ◽  
Lateral Flow ◽  
Isothermal Amplification ◽  
Loop Mediated Isothermal Amplification ◽  
Lateral Flow Dipstick ◽  
Soybean Allergy ◽  
Systematic Development ◽  
Multicopy Genes ◽  
Selection Of

The soybean (Glycine max) has been recognized as a frequent elicitor of food allergy worldwide. A lack of causative immunotherapy of soybean allergy makes soybean avoidance essential. Therefore, sensitive and specific methods for soybean detection are needed to allow for soybean verification in foods. Loop-mediated isothermal amplification (LAMP) represents a rapid and simple DNA-based detection method principally suitable for field-like applications or on-site analytical screening for allergens during the manufacturing of foods. This work describes the systematic development and selection of suitable LAMP primers based on soybean multicopy genes. The chemistry applied allows for a versatile detection of amplified DNA, using either gel electrophoresis, fluorescence recording, or a simple Lateral Flow Dipstick (LFD). LAMP based on the ORF160b gene was highly specific for the soybean and may allow for a detection level equivalent to approximately 10 mg soy per kg food. Various soybean cultivars were detectable at a comparable level of sensitivity. LAMP combined with LFD-like detection facilitates a simple, highly specific and sensitive detection of the soybean without the need for expensive analytical equipment. In contrast to the majority of antibody-based methods for soybean detection, all identified primer sequences and optimized protocols are disclosed and broadly available to the community.

scholarly journals The Potential Use of Isothermal Amplification Assays for In-Field Diagnostics of Plant Pathogens

Plants ◽  
2021 ◽  
Vol 10 (11) ◽  
pp. 2424
Author(s):  
Aleksandr V. Ivanov ◽  
Irina V. Safenkova ◽  
Anatoly V. Zherdev ◽  
Boris B. Dzantiev
Keyword(s):  
Molecular Diagnostics ◽  
Fungal Pathogens ◽  
Plant Pathogens ◽  
Isothermal Amplification ◽  
Detection Methods ◽  
Nucleic Acid Detection ◽  
Advantages And Disadvantages ◽  
Fungal Phytopathogens ◽  
Highly Sensitive Detection ◽  
And Control

Rapid, sensitive, and timely diagnostics are essential for protecting plants from pathogens. Commonly, PCR techniques are used in laboratories for highly sensitive detection of DNA/RNA from viral, viroid, bacterial, and fungal pathogens of plants. However, using PCR-based methods for in-field diagnostics is a challenge and sometimes nearly impossible. With the advent of isothermal amplification methods, which provide amplification of nucleic acids at a certain temperature and do not require thermocyclic equipment, going beyond the laboratory has become a reality for molecular diagnostics. The amplification stage ceases to be limited by time and instruments. Challenges to solve involve finding suitable approaches for rapid and user-friendly plant preparation and detection of amplicons after amplification. Here, we summarize approaches for in-field diagnostics of phytopathogens based on different types of isothermal amplification and discuss their advantages and disadvantages. In this review, we consider a combination of isothermal amplification methods with extraction and detection methods compatible with in-field phytodiagnostics. Molecular diagnostics in out-of-lab conditions are of particular importance for protecting against viral, bacterial, and fungal phytopathogens in order to quickly prevent and control the spread of disease. We believe that the development of rapid, sensitive, and equipment-free nucleic acid detection methods is the future of phytodiagnostics, and its benefits are already visible.

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