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)

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.

Keywords: Glycine max; LAMP; LFD; allergen detection; food allergy; lateral flow dipstick; loop-mediated isothermal amplification; multicopy gene; rapid test; soybean.

Figure 1

Localization of individual LAMP primers, including FITC and biotin-labeled LoopF and FIP primers, respectively, leading to a double-labeled LAMP product that is detected using a lateral flow dipstick (LFD). Illustrations of LAMP product formation are detailed elsewhere [18,19,20]. FITC- and biotin-labeled LAMP products, loaded by gold particles via an FITC-specific gold-labeled antibody, are captured on the test line by the biotin label. Free gold-labeled antibodies are captured at the control line.

Figure 2

Optimization of the ratio of labeled biotin-FIP and FITC-LoopF primers in fluorescence-based real-time detection of the atpA gene. FIP/biotin-FIP and LoopF/FITC-LoopF primer ratios: blue [1] = 0:1, 0:1; magenta [2] = 0:1, 1:1; green [3] = 1:1, 1:1; red [4] = 1:1; 1:3; black = reaction without DNA template (NTC).

Figure 3

Duplicate detection of LAMP DNA products of the optimized atpA (left) and ORF160b (right) LAMP assays using (a) the lateral flow dipstick (LFD); (b) real-time fluorescence based on SYBR Green I; (c) agarose gel electrophoresis (AGE); (d) visual inspection via turbidity formation. (b: red curves: 1:10³ diluted DNA; blue curves: 1:10⁵ diluted DNA; black curves: NTC).

Figure 4

Specificity of (a) the atpA and (b) the ORF160b LAMP-LFD assays for the detection of allergenic soybean. Cross-reactivity testing with undiluted DNA. Numbers according to Table 1 (PC: positive control tofu).

Figure 5

Screening of different soybean cultivars using (a) the atpA and (b) the ORF160b LAMP-LFD assays. Duplicate analysis of 1:10⁵ diluted DNA (equivalent to 10 mg soybean per kg food) of the 11 soybean cultivars C-1 to C-11 according to Table 2 (PC: positive control yellow soybean (Schoenenberger^®® Hensel^®®); NTC: no template controls).