Development of Kit Formulation for Non-Halal Animal DNA Detection using Multiplex RT-PCR Method Joni Kusnadi*, Johanna Helina Soeprajitno, Rosyid Muhaimin Malik, Ainun Sayyidah Zakiyah
Department of Food Science and Biotechnology, Faculty of Agricultural Technology, Universitas Brawijaya, Jalan Veteran, Malang, Indonesia
*jkusnadi[at]ub.ac.id
Abstract
Halal detection of food products is an important process in ensuring the halalness of food products consumed by the public, especially Muslims. Real Time Polymerase Chain Reaction (PCR) is a DNA-based method recommended for detecting the presence of DNA of non-halal animal species. The formulation of PCR mixture components (DNA polymerase, dNTP, MgCl2, buffer, DNA template), and primers are important factors for the success of the detection process. In this study, the optimization of PCR mix composition and primers for several non-halal animal species detection based on probe and intercalating dye was carried out to enhance detection effectiveness using the Multiplex RT-PCR method. The research stages includes primer and probe design, formulation of PCR mix composition, and validation tests to determine the effectiveness of the formulated mixture. Specific primers and probes for identifying pig, rat, and dog DNA has been sucessfully designed. Using intercalating dye-based primers and probe-based primer, the PCR Mix formula 1 was found to be most effective for identify dog, pig, and rat DNA in the tested samples using singleplex RT-PCR method. The Ct values of intercalating dye-based primers 16.23 (Dog), 13.43 (Pig), 15.56 (Rat) respectively, whereas for probe-based primers, the amplification results using were 13.92 (Dog), 12.73 (Pig), 15.28 (Rat), respectively. Validation tests using several parameters conclude that the PCR Mix formulation is effective for detecting non-halal animal DNA, although it is less optimal when implemented in the multiplex RT-PCR system.
Keywords: halal- kit- multiplex- primer- real time PCR
