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Damara, Dandy Satria
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Biochemistry, Genetics & Molecular Biology Chemical Engineering, Chemistry & Bioengineering Environmental Science Materials Science & Nanotechnology Physics

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Development of a Gelatin-Based Genomic Reference Material for Halal Authentication Using Real-Time PCR Rahma, Anisa Aula; Meilani, Nanda Diva; Sulistiawati; Ainaputri, Aliza Salsabila; Damara, Dandy Satria; Malau, Jekmal
Science and Technology Indonesia Vol. 10 No. 1 (2025): January
Publisher : Research Center of Inorganic Materials and Coordination Complexes, FMIPA Universitas Sriwijaya

Show Abstract | Download Original | Original Source | Check in Google Scholar | DOI: 10.26554/sti.2025.10.1.27-42

Abstract

Indonesia, home to over 270 million people, has the largest Muslim population globally, with approximately 87.18% adhering to Islam, driving significant demand for halal products, particularly in the food and pharmaceutical sectors. Gelatin, commonly used in medicinal capsules, often originates from porcine sources, necessitating precise halal authentication methods. This study presents the development of a novel genomic DNA-based Reference Material (RM) for gelatin, specifically for porcine DNA detection, employing Real-Time Polymerase Chain Reaction (qPCR) techniques. The methodology encompassed in-silico primer design, sample extraction optimization, DNA quality and quantity analysis, linearity assessment, limit of detection (LoD) and quantification (LoQ) determination, and RM characterization. Results indicated that the designed primers could reliably and efficiently detect porcine DNA, with optimal annealing at 58°C and primer concentration at 500 nM, achieving a PCR efficiency of 96.74%. The LoD and LoQ for pork meat samples were determined to be 0.02 pg/uL and 0.004 pg/uL, respectively, while the LoD for porcine gelatin was 0.27 ng/uL. The RMs exhibited robust homogeneity (Sig. 0.052), significant intergroup differences (Sig. 0.000), and low variation (CV 0.96%). Short-term storage at -80°C and -20°C preserved Ct value stability and consistency. Conclusively, this study successfully developed a novel gelatin-based genomic DNA RM for halal authentication, offering a scientifically validated tool that strengthens the halal assurance system, addressing Indonesian consumers’ demand for porcine-free products. These findings hold substantial implications for regulatory authorities, especially in Indonesia, and could inform the development of standardized qPCR RMs for porcine DNA detection in halal compliance testing.
Advancements in Real-Time PCR Technologies: A Comprehensive Review of Probe-Based and Non-Probe-Based Assays for Molecular Diagnostics Malau, Jekmal; Zahro, Aurora Fatimatuz; Zahra, Aliya Azkia; Kasasiah, Ahsanal; Meilani, Nanda Diva; Damara, Dandy Satria; Lestari, Agatha Nabilla; Saryono; Wahyono, Daniel Joko
Science and Technology Indonesia Vol. 10 No. 3 (2025): July
Publisher : Research Center of Inorganic Materials and Coordination Complexes, FMIPA Universitas Sriwijaya

Show Abstract | Download Original | Original Source | Check in Google Scholar | DOI: 10.26554/sti.2025.10.3.660-677

Abstract

The decision between probe-based and non-probe-based qPCR assays is crucial, influenced by diagnostic goals and sample characteristics. This review provides an in-depth evaluation of these two assay types, analyzing their principles, strengths, drawbacks, and applications. A thorough review of the literature, primarily sourced from PubMed, was undertaken to explore prominent assay systems, including TaqMan, KASP, rhAmp, HRM, and SYBR Green. Probe-based qPCR assays, exemplified by TaqMan and rhAmp, are distinguished by their high specificity, aptitude for multiplex analysis, and reduced risk of false positives, making them highly suitable for SNP genotyping and pathogen detection. However, their elevated costs and intricate design requirements remain significant challenges. Conversely, non-probe-based assays, such as SYBR Green and HRM, present cost-effective alternatives with straightforward designs. HRM, in particular, is effective in identifying genetic variations like SNPs with remarkable sensitivity. Nonetheless, these methods are susceptible to non-specific amplifications, requiring careful optimization to maintain reliability. The selection of a suitable qPCR assay depends on various factors, including precision, affordability, and multiplexing capabilities, with applications spanning infectious disease detection and genetic disorder analysis. This review emphasizes the indispensable role of qPCR in molecular diagnostics while showcasing recent technological advances that aim to mitigate existing constraints and enhance diagnostic precision and accessibility.
Co-Authors Ahsanal Kasasiah Ainaputri, Aliza Salsabila Daniel Joko Wahyono Jekmal Malau Lestari, Agatha Nabilla Meilani, Nanda Diva Rahma, Anisa Aula Saryono Sulistiawati Zahra, Aliya Azkia Zahro, Aurora Fatimatuz