APLIKASI HIGH RESOLUTION MELTING ANALYSIS (HRMA) DALAM REAL-TIME PCR UNTUK DETEKSI CEMARAN DAGING TIKUS DALAM BAKSO MENGGUNAKAN PRIMER BERTARGET GEN CYTOCHROME C OXIDASE I

R. ARINDRA HANURAGA, Prof. Dr. Sudjadi, M.S., Apt.

2014 | Skripsi | FARMASI

Keamanan sumber daging dalam bakso harus semakin dijaga kualitasnya. Hal ini dikarenakan banyaknya isu dan kasus pencampuran sumber daging dalam bakso sapi dengan daging tikus. Kasus ini sudah termasuk pelanggaran hukum, juga berpotensi menularkan penyakit yang mempunyai vektor tikus, seperti pes dan leptospirosis. Oleh karena itu deteksi yang akurat perlu dilakukan untuk merespon permasalahan tersebut. Pada penelitian ini ditawarkan metode Real-Time PCR yang dikombinasi dengan High Resolution Melting Analysis (HRMA). Dicari sepasang primer yang mengamplifikasi sekuen gen cytochrome c oxidase I (COI) pada DNA sapi (Bos taurus) dan tikus (Rattus norvegicus). Sampel yang digunakan adalah daging murni tikus dan sapi, serta model bakso campuran tikus dan sapi rasio 0%, 5%, 10%, 15%, 25%, 50%, 75%, 100%. Preparasi sampel dilakukan dengan ekstraksi DNA dari daging atau bakso, kemudian diuji kualitas dan kuantitasnya. Amplifikasi sekuen menggunakan Real-Time PCR, yang mana dilakukan optimasi suhu annealing, kemudian dilanjutkan dengan HRMA yang melihat Tm spesifik pada melt-peak dari produk PCR-nya. Suhu annealing optimal sebesar 53,1oC. Raw data melt-curve kemudian diolah menggunakan HRMA menjadi normalized melt-curve, melt-peak, dan melt-difference. Pada melt-curve didapatkan daerah melting produk PCR tikus dan sapi sekitar 81-82oC dan 83-84,5oC. Melt-peak memperjelas hasil dengan puncak Tm 81,7oC dan 83,6oC. Kurva Melt-difference terhadap sampel bakso 0% dapat melihat rasio dari campuran daging tikus dalam sampel bakso sapi.

Security source of meat in the meatball should be maintained, because there still found many issues and cases of mixing meat sources in beef meatball with rat meat. This cases were against the law and also have the potential to transmit disease by rat vectors, such as plague and leptospirosis. Therefore, accurate detection needs to be done to respond to these problems. In this study, Real-Time PCR method was used and combined with High Resolution Melting Analysis (HRMA). A pair of primers was searched for amplifying the cytochrome c oxidase I (COI) gene sequences in mitochondrial DNA of a cow (Bos taurus) and rat (Rattus norvegicus). The sample consist of pure rat meat and beef, as well as models of rat and beef meatball mixtures with ratio of 0%, 5%, 10%, 15%, 25%, 50%, 75%, 100%. Sample preparation was done by extracting DNA from meat or meatballs, then tested its quality and quantity. The amplification of sequences was done with Real-Time PCR. The annealing temperature was optimized first, then followed by HRMA which observe the melt-specific peak (Tm) of its PCR products. The optimal annealing temperature was found 53,1oC. The melt-curve raw data were collected and then processed by HRMA to become normalized melt-curve, melt-peak, and melt-difference. The melt-curve showed the PCR product melting-regions of rat and cow around 81-82oC and 83-84,5oC. Melt-peak clarify the results by Tm 81,7oC and 83,6oC. The melt-difference curve of samples that compared to ratio 0% meatballs could saw the ratio of the meat mixture of rat meat in beef meatball samples.

Kata Kunci : Real-Time PCR, HRMA, cemaran bakso, Bos taurus, Rattus norvegicus