Sains Malaysiana 49(8)(2020): 1959-1967

http://dx.doi.org/10.17576/jsm-2020-4908-18

Analisis Tindak Balas Berantai Polimerase (PCR) Simpleks dan Multipleks ke atas Produk Surimi Terawat Terma bagi Pengesanan DNA Lembu dan Babi

(Simplex and Multiplex Polymerase Chain Reaction (PCR) Analysis of Heat Treated Surimi Product for Bovine and Porcine DNA Detection)

SAFIYYAH SHAHIMI, NUR SYAFIQA AKMAL AHMAD GHAZALI, AISHAH ELIAS, NURUL AQILAH MOHD ZAINI & SAHILAH ABD MUTALIB*

Department of Food Science, Faculty of Science and Technology, Universiti Kebangsaan Malaysia, 43600 UKM Bangi, Selangor Darul Ehsan, Malaysia

Received: 31 October 2019/Accepted: 8 April 2020

ABSTRAK

Kajian ini dilakukan bagi mengesan DNA babi dan lembu daripada gelatin yang ditambah ke dalam surimi dengan menggunakan teknik tindak balas berantai polimerase (PCR) khusus-spesies simpleks dan multipleks. Bebola ikan berasaskan surimi telah dihasilkan dengan penambahan 5% (w/w) gelatin lembu dan 5% (w/w) gelatin babi serta dimasak menggunakan kaedah rebusan, pemanggangan, penggorengan dan autoklaf. Sebanyak tiga primer digunakan dalam analisis PCR iaitu sitokrom oksidase II (COII) untuk penentuan DNA lembu; dan dua primer untuk menentukan DNA babi, DNA mitokondria tRNA-ATP8 dan Elemen Nuklear Berselang Pendek (SINE). Analisis PCR simpleks menggunakan tiga primer khusus-spesies berjaya mengesan DNA lembu dan babi dalam kesemua sampel dengan penghasilan amplikon 165 bp (COII), 212 bp (tRNA-ATP8) dan 161 bp (SINE). Pengoptimuman PCR multipleks dengan menggunakan primer tRNA-ATP8 dan SINE telah dilakukan dengan menggunakan program kitar; penyahaslian awal pada 95 °C selama dua min, diikuti 30 kitaran penyahaslian pada 94 °C selama 1 min, penyepuhan pada 55 °C selama 1 min, pemanjangan pada 72 °C selama dua min dan pemanjangan akhir pada 72 °C selama sepuluh min telah berjaya mengamplifikasi dua gen sasaran bagi pengesanan DNA babi. Faktor seperti suhu (121 °C) dan tekanan (15 psi) terhadap rawatan terma didapati memberikan kesan kepada kualiti DNA di dalam sampel yang diautoklaf dan ini ditunjukkan dengan penghasilan amplikon yang pudar pada gel agarose. Oleh itu, kajian analisis PCR multipleks menggunakan primer babi khusus-spesies adalah kaedah mudah dan cepat untuk menentukan kehadiran DNA babi yang terkandung di dalam produk makanan terproses seperti surimi.

Kata kunci: DNA babi; gelatin babi; khusus-spesies; PCR multipleks; surimi

ABSTRACT

This research was carried out to detect the presence of pig and bovine DNA from gelatine added into surimi using species-specific simplex and multiplex polymerase chain reaction (PCR). Surimi-based fish balls were made with the addition of 5% (w/w) bovine gelatin and 5% (w/w) porcine gelatin and cooked using boiling, roasting, pan frying, and autoclaving methods. Three species-specific primers used in PCR analysis were cytochrome oxidase II (COII) for determination of bovine DNA; and two primers for determination of porcine DNA, mitochondrial DNA tRNA-ATP8 and Short Interspersed Nuclear Elements (SINE). Simplex PCR analysis using three species specific primers has been successfully detected bovine and porcine DNA in all samples by producing 165 bp (COII), 212 bp (tRNA-ATP8) and 161 bp (SINE) amplicons, respectively. Optimization of multiplex PCR using tRNA-ATP8 and SINE primers was done with step cycle of initial denaturation at 95 °C for two min, followed by 30 cycles of denaturation at 94 °C for one min, annealing at 55 °C for one min, polymerization at 72 °C for two min and a final extension at 72 °C for ten min was found to successfully amplify two target genes for detection of pig DNA. Several factors such as temperature (121 °C) and pressure (15 psi) during the heat treatment were found to affect the quality of DNA in the autoclaved samples, which shown by the faded amplification products in agarose gel. Thus, multiplex PCR analysis using porcine species-specific primer are reliable and useful in order to determine the presence of porcine DNA in processed food products such as surimi.

Keywords: Multiplex PCR; porcine DNA; porcine gelatin; species-specific; surimi

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*Corresponding author; email: sahilah@ukm.edu.my