UJI KINERJA METODE REAL TIME-POLYMERASE CHAIN REACTION (RT-PCR) PROBE TaqMan SPESIFIK GEN CYT B UNTUK MENDETEKSI CEMARAN DAGING ANJING DALAM BAKSO

MUTHIA RAHAYU IRESHA, Dr. Deni Pranowo, S.Si., M.Si.; Tri Joko Raharjo, S.Si., M.Si., Ph.D.

2017 | Skripsi | S1 KIMIA

Penelitian mengenai uji kinerja metode Real Time-PCR probe TaqMan telah dilakukan untuk mendeteksi kontaminasi daging anjing dalam produk bakso. Penelitian bertujuan untuk mengembangkan metode identifikasi DNA anjing pada bakso berbasis uji DNA sehingga dapat digunakan sebagai metode uji untuk mendeteksi kehalalan pangan. Metode RT-PCR probe TaqMan menggunakan primer spesifik gen sitokrom b yaitu primer forward (5- TGG ACA AAG CAA CCC TAA CA-3) dan primer reverse (5-CCG GTT TCG TGT AGA AAT AGG A-3), serta menggunakan probe TaqMan spesifik gen sitokrom b (5-/56- FAM/TCA TCC TCC/ZEN/CTT TCA TCA TCG CAG C/3IABkFQ/-3). Tahapan penelitian terdiri atas uji kinerja metode RT-PCR probe TaqMan pada bakso (uji spesifitas, uji presisi, uji sensitivitas, dan uji batas deteksi), serta uji deteksi kontaminasi daging anjing pada produk bakso kemasan. Isolasi DNA pada bakso menggunakan metode fenol KIAA. Amplifikasi fragmen DNA dilakukan dengan kondisi RT-PCR pra-denaturasi (95 �°C selama 5 menit), denaturasi (95 �°C selama 30 detik), annealing (52 �°C selama 30 detik), dan ekstensi (72 �°C selama 1 menit). Hasil uji metode RT-PCR probe TaqMan diketahui spesifik terhadap DNA anjing dalam sampel bakso. Metode ini juga menghasilkan presisi tinggi dengan nilai RSD sebesar 4,3%. Metode ini dapat mengamplifikasi DNA anjing hingga konsentrasi DNA anjing 5,0 pg dengan batas deteksi DNA anjing hingga konsentrasi terendah daging anjing 1%. Aplikasi metode terhadap 10 sampel bakso kemasan diketahui bahwa tidak ditemukan adanya cemaran daging anjing pada produk bakso.

The research on the performance test of TaqMan probe Real Time-PCR method has been done to detect dog meat contamination in meatball products. The aims is to develop a method of DNA identification of dogs in meatball products based on DNA test so that it can be used as a test method to detect halal food. The TaqMan probe RT-PCR method use the canine-specific primers that is primer forward (5-TGG ACA AAG CAA CCC TAA CA-3) and primer reverse (5-CCG GTT TCG TGT AGA AAT AGG A-3), and also use a canine-specific cytochrome b gene TaqMan probe (5-/56-FAM/TCA TCC TCC/ZEN/CTT TCA TCG CAG C/3IABkFQ/-3). The stage of research consist of the performance test in TaqMan probe RT-PCR method on meatballs (specificity test, precision test, sensitivity test, and limit detection test) and dog meat contamination test on meatball products. Isolation of DNA in meatballs use phenol CIAA method. Amplification of DNA fragments was performed with pre-denaturation RT-PCR conditions (95 �°C for 5 minute), denaturation (95 �°C for 30 seconds), annealing (52 �°C for 30 seconds), and extension (72 �°C for 1 minute ). TaqMan probe RT-PCR method result is known specific to dog DNA on the meatballs samples. This method also produces high precision with RSD value about 4.3%. This method can amplify dog DNA until the concentration is 5.0 pg with the lowest concentration of dog meat limit detection test is 1%. The application of the method on 10 samples of meatballs product is obtained contamination of dog meat was not found.

Kata Kunci : Analisis halal, Bakso, DNA anjing, Real Time-PCR probe TaqMan, Sitokrom b.