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. 2020 Mar 12;9(3):332.
doi: 10.3390/foods9030332.

Development of a DNA-Based Detection Method for Cocos Nucifera Using TaqMan™ Real-Time PCR

Jasmin Wrage  1   2 , Oxana Kleyner  1 , Sascha Rohn  2 , Jürgen Kuballa  1
Affiliations

Development of a DNA-Based Detection Method for Cocos Nucifera Using TaqMan™ Real-Time PCR

Jasmin Wrage et al. Foods. .

Abstract

So far, only a few cases of immunoglobulin E (IgE)-mediated coconut allergies have been described in the literature. Due to a growing consumption of coconut-containing foods in occidental countries, the number of coconut allergies may also increase. As there is no causative immunotherapy in clinical routine, appropriate food labelling is particularly important, also with regard to cross-contamination, to prevent serious health consequences. The purpose of this study was to develop a DNA-based detection method for coconut (Cocos nucifera). Initially, three sets of coconut-specific primers were designed and tested. A TaqMan™ probe was then developed to identify and quantify coconut by real-time PCR assay. With 27 other plant and animal species, the specificity of the primer/probe system was tested and cross reactivity was excluded. In a dilution series, a limit of detection of 1 pg/µL was determined. Thus, the developed real-time PCR assay is a suitable method to detect coconut in food.

Keywords: Cocos nucifera; TaqMan™ probe; coconut allergy; primer design; real-time PCR.

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Conflict of interest statement

The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

Figures

Figure 1
Figure 1
Gel electrophoresis of DNA extracted from different palm species to test the specificity of different sets of primer. Lane 1: Chilean wine palm (Jubaea chilensis); lane 2: oil palm (Elaeis guineensis); lane 3: coconut palm (Cocos nucifera), lane 4: jelly palm (Butia capitata); lane 5: non-template sample with water; lane 6: 100 bp DNA ladder. (a) PCR amplification with the primer set cocosPRK. (b) PCR amplification using the primer set cocosITS109. (c) PCR amplification performed with the primer set cocosITS197.
Figure 2
Figure 2
Gel electrophoresis of DNA extracted from coconut, coconut chips, and coconut blossom sugar. Lane 1–2: coconut chips; lane 3–4: coconut; lane 5: 100 bp DNA ladder; lane 6–7: coconut blossom sugar; lane 8: non-template sample with water.
Figure 3
Figure 3
Specificity test of the primer/probe system for coconut identification. No amplification was detected with species other than coconut.
Figure 4
Figure 4
Sensitivity test of the real-time PCR assay with 10-fold diluted DNA of coconut in a concentration range from 100 ng/µL to 0.1 pg/µL. The reactions were performed in duplicates. (a) The amplification plot shows Ct values for samples from 100 ng/µL to 1 pg/µL. (b) Standard curve of the coconut TagMan real-time PCR system by plotting Ct values against the log10 input DNA concentration.
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