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Effectiveness Binding Buffer in Porcine DNA Extraction from Meat Products Using Spin-Column Filter Paper RT-PCR 1Department of Food Science and Biotechnology, Faculty Of Agricultural Technology, Universitas Brawijaya, Malang, Indonesia Abstract Real-time PCR (RT-PCR) is recommended by the National Standardization Agency of Indonesia (BSN) for halal authentication due to its high sensitivity and specificity, with DNA quality serving as a critical determinant of success. One widely used approach is spin column based DNA extraction, which involves lysis, binding, washing, and elution steps, where the DNA binding stage plays a key role in determining concentration and purity. This study aimed to evaluate the effectiveness of binding buffer in DNA extraction using a spin column filter paper system for detecting porcine DNA in fresh and processed meat products. DNA extraction was performed using grade 3 filter paper (three layers) as the binding matrix combined with chaotropic salt-based binding buffers (guanidine hydrochloride and guanidinium thiocyanate). The optimized buffer concentration of 5.5 M yielded DNA with a concentration of 10 nanogram/microliter and a purity of 1.84, while application to processed meat samples produced DNA concentrations ranging from 20-43 nanogram/microliter with purity values of 1.89-1.96. In comparison, commercial kits yielded lower DNA concentrations (10.45-20.84 nanogram/microliter) with purity values of 1.7-2.0. Validation using RT-PCR confirmed successful detection of porcine DNA with Ct values between 13 and 21, highlighting the sensitivity of this method. In conclusion, the spin column filter paper method with a 5.5 M binding buffer proved more effective than commercial kits in producing high-quality DNA and provides a reliable tool for porcine DNA detection in halal authentication. Keywords: DNA_extraction- Filter_paper- Porcine_DNA- RT-PCR- spin_column- Topic: Food science and biotechnology |
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