Assessment of Commercial DNA Extraction Kits for Porcine Gelatin Detection Using RT-PCR and ddPCR
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Abstract
Gelatin is extensively applied in various industries, including food, beverages, cosmetics, and pharmaceuticals. Although the determination of gelatin species is essential for religious, health, and consumer preference reasons, a standardized analysis method is absent. The challenge in identifying gelatin through DNA-based methods arises from the low DNA content and extensive DNA denaturation in the gelatin matrix. This study assessed the efficacy of two commercial DNA extraction kits, namely the Processed Food DNA Extraction (PF kit) and the DNeasy Mericon Food Kit (DM kit), for extracting DNA from porcine gelatin powder and commercial products derived from gelatin. Additionally, we evaluate the amplification of the extracted porcine DNA using real time polymerase chain reaction (RT-PCR) and droplet digital polymerase chain reaction (ddPCR) techniques. The PF extraction kit demonstrated successful DNA extraction from porcine gelatin powder and commercial samples of porcine gelatin-based candies with a higher concentration (32.24-286.07 ng/uL) and purity (A260/A280 ratio of 1.82-2.33) compared to the DM kit (3.95-7.30 ng/uL and an A260/A280 ratio of 1.29-2.45). RT-PCR and ddPCR analyses yielded positive results for porcine DNA from gelatin powder for both PF and DM kits, albeit with differing Cq values and copy numbers. The choice of DNA extraction kit significantly impacted the amplification results when analyzing commercial samples of porcine gelatin-based candies. Using RT-PCR, all samples yielded negative results with the DM kit, while the PF kit detected one positive result for porcine DNA. Improved outcomes were observed with more sensitive analysis methods such as ddPCR, where the DM kit identified one positive result for porcine DNA while the PF kit detected positive results for all tested candies.
References
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Pacheco Coello, R., J. Pestana Justo, A. Factos Mendoza, and E. Santos Ordoñez (2017). Comparison of Three DnA Extraction Methods for the Detection and Quantification of GMO in Ecuadorian Manufactured Food. Bmc Research Notes, 10(758); 1–7
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Pratiwi, D., N. Fitriani, and A. Rohman (2018). Application of Real-Time Polymerase Chain Reaction for Analysis of Porcine DNA in Gelatine-Containing Capsule Shell for Halal Authentication. International Food Research Journal, 25(4); 1515–1519
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Sultana, S., M. M. Hossain, A. Azlan, M. R. Johan, Z. Z. Chowdhury, and M. E. Ali (2020). TaqMan Probe Based Multiplex Quantitative PCR Assay for Determination of Bovine, Porcine and Fish Dna in Gelatin Admixture, Food Products and Dietary Supplements. Food Chemistry, 325(September); 126756
Sultana, S., M. M. Hossain, N. N. A. Naquiah, and M. E. Ali (2018a). Novel Multiplex PCR-RFLP Assay Discriminates Bovine, Porcine and Fish Gelatin Substitution in Asian Pharmaceuticals Capsule Shells. Food Additives & Contaminants: Part A, 35(9); 1662–1673
Sultana, S., M. M. Hossain, I. S. M. Zaidul, and M. E. Ali (2018b). Multiplex PCR to Discriminate Bovine, Porcine, and Fish Dna in Gelatin and Confectionery Products. LWT-Food Science and Technology, 92(June); 169–176
Uddin, S. M. K., M. M. Hossain, S. Sagadevan, M. Al Amin, and M. R. Johan (2021). Halal and Kosher Gelatin: Applications As Well As Detection Approaches with Challenges and Prospects. Food Bioscience, 44(Part A); 101422
Yap, B. K. and L. H. Gam (2019). Differentiation of Bovine from Porcine Gelatin Capsules Using Gel Electrophoresis Method. Food Chemistry, 274(February); 16–19
Yayla, M. E. A. and C. Ekinci Doğan (2021). Development of a New and Sensitive Method for the Detection of Pork Adulteration in Gelatin and Other Highly Processed Food Products. Food Additives & Contaminants: Part A, 38(6); 881–891
Al-Kahtani, H. A., E. A. Ismail, and M. A. Ahmed (2017). Pork Detection in Binary Meat Mixtures and Some Commercial Food Products Using Conventional and Real-Time Pcr Techniques. Food Chemistry, 219(March); 54–60
Al-Nimry, S., A. A. Dayah, I. Hasan, and R. Daghmash (2021). Cosmetic, Biomedical and Pharmaceutical Applications of Fish Gelatin/Hydrolysates. Marine Drugs, 19(3); 145
Baziwane, D. and Q. He (2003). Gelatin: The Paramount Food Additive. Food Reviews International, 19(4); 423–435
Butler, C., A. Matsumoto, C. Rutherford, and H. K. Lima (2022). Comparison of the Effectiveness of Four Commercial DNA Extraction Kits on Fresh and Frozen Human Milk Samples. Methods and Protocols, 5(4); 63
Demirhan, Y., P. Ulca, and H. Z. Senyuva (2012). Detection of Porcine DNA in Gelatine and Gelatine-Containing Processed Food Products—Halal/kosher Authentication. Meat Science, 90(3); 686–689
Elyasi, H., H. Rahimi, and A. Sepahvend (2020). Gelatin, Halal or Haram. Plant Biotechnology Persa, 2(1); 35–41
Grundy, H., P. Reece, M. Buckley, C. Solazzo, A. Dowle, D. Ashford, A. Charlton, M. Wadsley, and M. J. Collins (2016). A Mass Spectrometry Method for the Determination of the Species of Origin of Gelatine in Foods and Pharmaceutical Products. Food Chemistry, 190(January); 276–284
Ha, L. T. V., L. Vanlerberghe, H. T. Toan, K. Dewettinck, and K. Messens (2015). Comparative Evaluation of Six Extraction Methods for DNA Quantification and PCR Detection in Cocoa and Cocoa-Derived Products. Food Biotechnology, 29(1); 1–19
Hindson, B. J., K. D. Ness, D. A. Masquelier, P. Belgrader, N. J. Heredia, A. J. Makarewicz, I. J. Bright, M. Y. Lucero, A. L. Hiddessen, and T. C. Legler (2011). High-Throughput Droplet Digital PCR System for Absolute Quantitation of DNA Copy Number. Analytical Chemistry, 83(22); 8604–8610
Huggett, J. F., T. Novak, J. A. Garson, C. Green, S. D. Morris-Jones, R. F. Miller, and A. Zumla (2008). Differential Susceptibility of PCR Reactions to Inhibitors: An Important and Unrecognised Phenomenon. BMC Research Notes, 1(70); 1–9
Ismarti, I., D. D. Handoko, K. Triyana, H. M. Salleh, N. A. Fadzillah, and N. F. H. Nordin (2022). Study on Volatile Compounds of Gelatine and The Maillard Reaction Products from Different Species Using SPME-GCMS. Science and Technology Indonesia, 7(2); 132–139
Khayyira, A. S., V. M. Estepane, and A. Malik (2018). Rapid PCR-Based Detection Optimization of Porcine DNA in Gelatin Capsule Shell. International Journal of Applied Pharmaceutics, 10(6); 217–223
Medina, S., R. Perestrelo, P. Silva, J. A. Pereira, and J. S. Câmara (2019). Current Trends and Recent Advances on Food Authenticity Technologies and Chemometric Approaches. Trends in Food Science & Technology, 85; 163–176
Mohamad, N. A., S. Mustafa, A. F. El Sheikha, N. F. Khairil Mokhtar, A. Ismail, and M. E. Ali (2016). Modification of Gelatin–DNA Interaction for Optimised DNA Extraction from Gelatin and Gelatin Capsule. Journal of the Science of Food and Agriculture, 96(7); 2344–2351
Muñoz-Colmenero, M., J. L. Martínez, A. Roca, and E. GarciaVazquez (2016). Detection of Different DNA Animal Species in Commercial Candy Products. Journal of Food Science, 81(3); T801–T809
Nikzad, J., S. Shahhosseini, M. Tabarzad, N. Nafissi-Varcheh, and M. Torshabi (2017). Simultaneous Detection of Bovine and Porcine DNA in Pharmaceutical Gelatin Capsules by Duplex PCR Assay for Halal Authentication. DARU Journal of Pharmaceutical Sciences, 25(3); 1–11
Nuraeni, U., J. Malau, R. T. Astuti, A. Dewantoro, D. Apriori, E. D. Lusiana, and B. Prasetya (2023). Droplet Digital PCR Versus Real-Time PCR for In-House Validation of Porcine Detection and Quantification Protocol: An Artificial Recombinant Plasmid Approach. PLoS One, 18(7); e0287712
Nurilmala, M., N. Darmawan, E. A. W. Putri, A. M. Jacoeb, and T. T. Irawadi (2021). Pangasius Fish Skin and Swim Bladder As Gelatin Sources for Hard Capsule Material. International Journal of Biomaterials, 2021(August); 6658002
Opel, K. L., D. Chung, and B. R. McCord (2010). A Study of PCR Inhibition Mechanisms Using Real Time PCR. Journal of Forensic Sciences, 55(1); 25–33
Pacheco Coello, R., J. Pestana Justo, A. Factos Mendoza, and E. Santos Ordoñez (2017). Comparison of Three DnA Extraction Methods for the Detection and Quantification of GMO in Ecuadorian Manufactured Food. Bmc Research Notes, 10(758); 1–7
Piskata, Z., E. Servusova, V. Babak, M. Nesvadbova, and G. Borilova (2019). The Quality of DNA Isolated from Processed Food and Feed Via Different Extraction Procedures. Molecules, 24(6); 1188
Pratiwi, D., N. Fitriani, and A. Rohman (2018). Application of Real-Time Polymerase Chain Reaction for Analysis of Porcine DNA in Gelatine-Containing Capsule Shell for Halal Authentication. International Food Research Journal, 25(4); 1515–1519
Rohman, A., A. Windarsih, Y. Erwanto, and Z. Zakaria (2020). Review on Analytical Methods for Analysis of Porcine Gelatine in Food and Pharmaceutical Products for Halal Authentication. Trends in Food Science & Technology, 101(July); 122–132
Sani, M. S. A., A. M. Ismail, A. Azid, and M. S. Samsudin (2021). Establishing Forensic Food Models for Authentication and Quantification of Porcine Adulterant in Gelatine and Marshmallow. Food Control, 130(December); 108350
Shabani, H., M. Mehdizadeh, S. M. Mousavi, E. A. Dezfouli, T. Solgi, M. Khodaverdi, M. Rabiei, H. Rastegar, and M. Alebouyeh (2015). Halal Authenticity of Gelatin Using Species-Specific PCR. Food Chemistry, 184(October); 203–206
Shehata, H. R., J. Li, S. Chen, H. Redda, S. Cheng, N. Tabujara, H. Li, K. Warriner, and R. Hanner (2017). Droplet Digital Polymerase Chain Reaction (ddpcr) Assays Integrated with an Internal Control for Quantification of Bovine, Porcine, Chicken and Turkey Species in Food and Feed. PLoS One, 12(8); e0182872
Sultana, S., M. M. Hossain, A. Azlan, M. R. Johan, Z. Z. Chowdhury, and M. E. Ali (2020). TaqMan Probe Based Multiplex Quantitative PCR Assay for Determination of Bovine, Porcine and Fish Dna in Gelatin Admixture, Food Products and Dietary Supplements. Food Chemistry, 325(September); 126756
Sultana, S., M. M. Hossain, N. N. A. Naquiah, and M. E. Ali (2018a). Novel Multiplex PCR-RFLP Assay Discriminates Bovine, Porcine and Fish Gelatin Substitution in Asian Pharmaceuticals Capsule Shells. Food Additives & Contaminants: Part A, 35(9); 1662–1673
Sultana, S., M. M. Hossain, I. S. M. Zaidul, and M. E. Ali (2018b). Multiplex PCR to Discriminate Bovine, Porcine, and Fish Dna in Gelatin and Confectionery Products. LWT-Food Science and Technology, 92(June); 169–176
Uddin, S. M. K., M. M. Hossain, S. Sagadevan, M. Al Amin, and M. R. Johan (2021). Halal and Kosher Gelatin: Applications As Well As Detection Approaches with Challenges and Prospects. Food Bioscience, 44(Part A); 101422
Yap, B. K. and L. H. Gam (2019). Differentiation of Bovine from Porcine Gelatin Capsules Using Gel Electrophoresis Method. Food Chemistry, 274(February); 16–19
Yayla, M. E. A. and C. Ekinci Doğan (2021). Development of a New and Sensitive Method for the Detection of Pork Adulteration in Gelatin and Other Highly Processed Food Products. Food Additives & Contaminants: Part A, 38(6); 881–891
Authors
Gina, S. ., Rahmagiarti, C. ., Ummah, I. M. ., Sumantri, C. ., Suparto, I. H. ., & Darmawan, N. (2024). Assessment of Commercial DNA Extraction Kits for Porcine Gelatin Detection Using RT-PCR and ddPCR. Science and Technology Indonesia, 9(3), 605–612. https://doi.org/10.26554/sti.2024.9.3.605-612
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