Development of multiplex PCR method for simultaneous detection of five tropical fruit species combined with capillary electrophoresis

다국어 초록 (Multilingual Abstract)

Development of multiplex PCR method for simultaneous detection of five tropical fruit species combined with capillary electrophoresis by Jae-Eun Cha Master of Food Science and Biotechnology Graduate School of Biotechnology, Kyung Hee University. In this research, a multiplex polymerase chain reaction (PCR) assay was developed to detect five tropical fruit species with potential allergenic properties. Novel species-specific primer set with high specificity and sensitivity were designed and also can be applied in processed foods. The primer set targets the chloroplast genes matK and ITS. The obtained amplicon sizes were 114 bp for mango, 132 bp for pineapple, 158 bp for avocado, 183 bp for banana, and 242 bp for coconut. The multiplex PCR assay’s specificity was validated with 27 different plant species including five tropical fruit species, demonstrating no cross-reactivity and confirming detection sensitivity at approximately 1 pg/μL. Its applicability was further verified using 32 commercially available food products. Particularly, juice products containing two distinct fruit types displayed amplification signals for both species, highlighting the method’s ability to detect multiple species simultaneously. Therefore, this study aims to develop a multiplex PCR assay to simultaneously and sensitively detect the presence of five potentially allergenic tropical fruit species, thereby preventing allergic reactions among consumers and enhancing future regulations on food allergy labeling.

목차 (Table of Contents)

• 1. INTRODUCTION 1
• 2. MATERIALS AND METHODS 5
• 2.1. Sample preparation 5
• 2.2. DNA extraction 6
• 2.3. Primer design 8
• 2.4. Single PCR specificity and sensitivity 11
• 2.5. Multiplex PCR specificity and sensitivity 12
• 2.6. Application and verification of the developed multiplex PCR assay 13
• 3. RESULTS AND DISCUSSION 14
• 3.1. Specificity and sensitivity of novel species-specific primers 14
• 3.2. Specificity and sensitivity of multiplex PCR using species-specific primers 19
• 3.3. Application and validation in commercial food products 25
• 4. CONCLUSION 31
• 5. REFERENCES 32