No abstract

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.Foods 2020, 9, 423 2 of 19 soybeans may remain indiscernible or hidden due to mislabeling or the cross contact of allergen-free food products with allergenic foods, e.g., during the food manufacturing process [7]. As part of the Allergen Bureau of Australia & New Zealand, the VITAL (Voluntary Incidental Trace Allergen Labeling) expert panel established reference doses for allergenic foods on the basis of individual clinical threshold data. Accordingly, 1 mg soy protein, equivalent to 2.5 mg soy, was modeled as the eliciting dose (ED) for an allergic reaction in 5% of the soy allergic population (ED05) [8]. Thus, 95% of soy allergic subjects are thought to be safe at a level of 2.5 mg soy. Recently, the soy protein reference dose was lowered to 0.5 mg, however, without peer-reviewed publication. Accordingly, for the detection of 1.25−2.5 mg soy in a serving size of 100 g, a method sensitivity from less than 12.5 to 25 mg soy per kg food is required to verify the absence or presence of soy at a level that likely improves food safety for the majority of soy allergic subjects. The availability of specific and sensitive methods for the detection of allergenic foods is essential for the verification of compliance with labeling requirements and the risk management of cross contact with allergenic foods, such as those based on the implementation of VITAL reference doses.Currently, the two most prominent analytical techniques for soybean detection in food are protein-based enzyme-linked immunosorbent assays (ELISA) [9,10] and nucleic acid (DNA)-based quantitative polymerase chain reaction (qPCR) assays [11,12]. Both types of methods ...

In this prospective and monocentric study, we investigated the performance of a commercialized real-time polymerase chain reaction (RT-PCR) test system for the specific detection of DNA from Candida albicans, C. dubliniensis, C. glabrata, C. krusei, C. lusitaniae, C. parapsilosis, and C. tropicalis in human milk samples of patients suspicious of mammary candidiasis. For this purpose, 43 breast-feeding women with characteristic symptoms of mammary candidiasis and 40 asymptomatic controls were enrolled. By culture, Candida spp. were detected in 8.8 % (4/46) and 9.3 % (4/43) of patient and control samples, respectively. Candida albicans (2/46), C. parapsilosis (1/46), and C. guilliermondii (1/46) were present in patient samples, and C. lusitaniae (3/43) and C. guilliermondii (1/43) were present in the controls. After RT-PCR was applied, Candida spp. were found to be present in 67.4 % (31/46) and 79.1 % (34/43) of patient and control samples investigated, respectively. PCR detection of C. albicans and C. parapsilosis revealed only a low sensitivity and specificity of 67.4 % and 41.9 %, respectively. Our data do not support the use of Candida RT-PCR for sensitive and specific diagnosis of mammary candidiasis.

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.