Penanda DNA: Uji Halal pada Makanan Olahan Daging Menggunakan Primer Multiplex PCR (Polymerase Chain Reaction)

Abstract

Multiplex-PCR (mPCR) is a technique that is gaining popularity due to its ability to detect multiple species in a single tube, resulting in faster results with more efficient and cost-effective resources than the conventional PCR method. This study aimed to design mPCR genetic markers for the detection of bovine/rat/chicken/pork organisms in processed foods. The procedure was divided into two stages: in silico and in vitro. The primers were designed based on mitochondrial cytochrome genes obtained from the NCBI GeneBank. Following alignment, the selected DNA fragments were used as the target sequence for the primer design. The sequences of all primer pairs were aligned on the Oli2go primary pooler to determine whether there was a possibility of cross reaction. The results of this study indicated that primers designed for the COX1 gene in rats and bovine, the KEF22 r01 gene in pork, and the ND6 gene in chicken produced amplicons with sizes of 622 bp, 552 bp, 588 bp, and 272 bp, respectively. However, the amplicons generated from pork, bovine and mouse marker primers were difficult to distinguish when electrophoresed in the same 2% agarose gel. Meanwhile, the amplicons of the primer markers of chickens can be distinguished when electrophoresed with pork/cows/rat. The study concludes that the designed primer pairs can be used together in mPCR if the difference in amplicon size is greater than 200 bp.Key words: Multiplex PCR; DNA markers; Halal meat test.ABSTRAKMultiplex-PCR merupakan metode yang saat ini sedang populer untuk digunakan karena dapat melakukan pendeteksian lebih dari satu spesies dalam satu tabung, sehingga hasil yang diperoleh lebih cepat, efisien, dan murah dibanding metode PCR biasa. Penelitian ini bertujuan untuk merancang penanda genetik multipleks-PCR untuk deteksi adanya organisme sapi/tikus/ayam/babi pada makanan olahan. Metode yang digunakan terdiri dari dua tahap, yaitu secara in silico dan in vitro. Primer dirancang dengan menggunakan gen sitokrom mitokondria yang didapat dari GeneBank NCBI. Setelah dilakukan alignment, fragmen DNA yang terpilih dijadikan sebagai target sekuen untuk desain primer. Semua sikuen pasangan primer di-alignment pada Oli2go primer pooler untuk melihat kemungkinan adanya cross reaction. Hasil dari penelitian ini yaitu primer tikus dan sapi dirancang dengan menggunakan gen COX1, primer babi menggunakan gen KEF22_r01, dan primer ayam menggunakan gen ND6 menghasilkan amplikon dengan ukuran berturut-turut 622 pb, 552 pb, 588 pb dan 272 pb. Namun, amplikon yang dihasilkan dari primer penanda babi, sapi dan tikus sulit dibedakan saat dielektroforesis dalam sumur gel agaros 2% yang sama. Sedangkan amplikon dari pasangan primer penanda ayam dapat terbedakan saat dielektroforesis baik bersama babi/sapi/tikus. Kesimpulan dari penelitian ini adalah hasil rancangan primer penanda telah dapat digunakan secara bersama jika perbedaan ukuran amplikon >200 pb.Kata kunci: Multiplex PCR; Penanda DNA; Uji halal daging.

Adulteration Test of Chicken DNA (Gallus gallus) by the Multiplex PCR Method Using a Specific Primer for Mitochondrial DNA CO1

“…DNA bands that are formed in the mix 1 and mix 2 columns have different product sizes according to their respective target lengths. After the visualization process using the electrophoresis method, the target DNA bands will separate specifically which can be seen based on the thickness of the target DNA, and the thickness of the target DNA formed is based on the weight and amount of the targeted DNA [22].…”

Successful Primer Picking and Pooling for the Design of Multiplex PCR Primers Specific to Pork, Beef, Chicken, and Rat DNA

“…The results of research by Dzikrina et al (2022) have successfully used m-PCR and multiplex primers that produce amplicons with the size of chicken primer 272 bp, beef primer 522 bp, rat primer 622 bp, and pork primer 588 bp. However, the visualization of m-PCR products by electrophoresis on a 1.5-2.0% agarose gel showed that the amplicon from the pig, Beef, and rat primers could not be clearly distinguished.…”

“…Beef and pork primers were designed using the mitochondrial genome sequences of cattle (Bos taurus) and pigs (Sus scrofa) from the NCBI Genebank site (https://www.ncbi.nlm.nih.gov/) with the "complete genome" category. In contrast, rat and chicken primers used the sequences from Dzikrina et al 2022. After the Beef and pork primers were successfully redesigned, the sequence homology test was carried out with BLAST on the database contained on the NCBI website.…”