THE UTILIZATION OF GENETIC MARKERS AND MULTIPLEX-PCR FOR DETECTION THE HALALNESS OF PROCESSED MEAT PRODUCTS
Diah Kusumawaty (a*), Nurul Faridah (a), Didik Priyandoko (a), Hanina Dzikrina (a), Azzania Febriani (b), Any Aryani (a)

(a) Program Studi Biologi, Fakultas Pendidikan Matematika dan Ilmu Pengetahuan Alam
Universitas Pendidikan Indonesia, Jl. Dr. Setiabudhi No.229, Kel.Isola, Kec. Sukasari, Kota Bandung, Jawa Barat 40154
* diah.kusumawaty[at]upi.edu
(b) Program Studi Biologi, SITH ITB, Jl. Ganesha 10 Bandung, Indonesia

Abstract

The increase in consumption of processed meat products has led to an increase in cases of mixing processed meat products with non-halal meat such as pork and rats. The use of DNA markers and Multiplex-PCR continues to be developed as a method of species detection in processed meat products. The main objective of this study was to design multiplex primer sequences of pigs, rats, cattle, and chickens that produce amplicons that can be distinguished by agarose gel electrophoresis for the detection of halal processed meat products. The primer design was performed using mitochondrial genome data and the NCBI-Primary BLAST site to obtain specific pig and bovine primer sequences. In silico Single and Multiplex-PCR simulations were performed using a Primer Pooler. In vitro validation was carried out through Single-PCR and multiplex-PCR annealing temperature optimization using samples of chicken, beef, pork, rat and processed meat products such as meatballs, sausages and nuggets. The results of the primary design found specific pig and bovine primer sequences with the ND5 target gene and the amplicon size of pigs and cattle was 860 bp and 1187 bp. The results of the in silico simulation showed that multiplex primers were found in the mitochondrial genome and amplified the target sequence to produce an amplicon with the longest size of 1202 bp. The validation results using the DNA amplification method with PCR showed that the halal marker gene multiplex primers succeeded in specifically amplifying the target sequence at the optimal annealing temperature of 60oC and the results of agarose gel electrophoresis visualization showed the amplicons for cow (1187 bp), pig (860 bp), rat (622 bp), and chicken (272 bp) which could be differentiated using 1.5% agarose electrophoresis. The multiplex primer amplification using samples of processed meat products can detect the species used in processed meat products so that the halalness can be determined.

Keywords: DNA markers-Halal detection- Multiplex-PCR- Primer design- Processed Meat Products

Topic: Biology