Rapid identification and quantitation of single plant seed allergen using paper-based microfluidics

Abstract

Nucleic acid amplification is a sensitive and powerful tool for allergen detection. However, it is limited due to the relatively cumbersome methods required to extract nucleic acids from single plant seed allergen (e.g. peanut and soybean). In view of this, an approach of extracting nucleic acid with untreated glass-fiber paper (paper-based microfluidics) was applied for nucleic acid capture and purification from plant seed allergen and commercial products. After cut by hollow cylindrical cutter, a certain size the paper chip it used to absorb DNA. And this paper-based microfluidics with DNA was directly applied for amplification by loop-mediated isothermal amplification (LAMP). To evaluate the adsorption performance of paper chip to DNA, CTAB and SDS method were used as comparisons. From amplification results, the established technique has good specificity, high repeatability (C.V. values are 4.41% and 6.17% for peanut and soybean) and favorable sensitivity (7.39 ng/μL or peanut and 6.6 ng/μL for soybean), and successfully used for commercial products (2 kinds of candy and 2 kinds of cakes containing peanut, and 2 kinds of drinks, candy and 2 kinds of biscuits containing soybean). This speed and flexible detection method makes it suit for applications in point-of-care (POC) detection at different scenario, such as custom house and import port.

Fig 1. Process of LAMP method for nucleic acid detection extracted by paper-based microfluidics.

Fig 2. Adsorption of DNA and H₂O on paper-based microfluidics.

Notes: A, fluorescence of paper chip; B and C standard line of different concentration of DNA.

Fig 3. Specificity of LAMP detection with paper-based microfluidics extraction.

Notes: A1 & A2, LAMP amplification of peanut and soybean; B1 & B2, corresponding ladder electrophoresis bands of peanut and soybean.

Fig 4. LAMP detection and electrophoresis verification based on the extraction DNA by paper-based microfluidics.

Notes: A1 & A2, LAMP amplification curves of peanut and soybean; B1 & B2, corresponding ladder electrophoresis bands of peanut and soybean.

Fig 5. LAMP detection of different concentrations of DNA on paper-based microfluidics.

Notes: A1 & A2, amplification curves of peanut and soybean DNA with different concentrations; B1 & B2, linear relationship between peanut and soybean DNA concentrations and Ct value.

Fig 6. Sensitivity of LAMP detection with paper-based microfluidics extraction.

Notes: A1, B1 & C1, the fluorescence amplification results, melting curve results, and agarose gel electrophoresis results of different peanut diluted samples; A2, B2 & C2, the fluorescence amplification results, melting curve results, and agarose gel electrophoresis results of different soybean diluted samples.

Fig 7. Repeatability of LAMP detection with paper-based microfluidics extraction.

Notes: A1 & A2, the amplification results of peanut and soybean with 5 repeats; B1 & B2, Ct value of peanut and soybean with 5 repeats.

Fig 8. Detection of peanut and soybean allergens in different areas and varieties by LAMP with paper-based microfluidics extraction.

Notes: 8A1 & 8C1, amplification curves appeared in peanut samples from different regions and varieties; 8B1 & 8D1, Tm values of peanut from different varieties and different places; 8A2 & 8C2, amplification curves appeared in soybean samples from different regions and varieties; 8B2 & 8D2, Tm values of soybean from different varieties and different places.

Fig 9. Detection of peanut and soybean allergens in commercial products.

Notes: A1 & A2, LAMP amplification curves of different products containing peanut and soybean; B1 & B2, corresponding ladder electrophoresis bands o of different products containing peanut and soybean.