Pramitha, Putu Ayu Eka and Dr. Ir. Joni Kusnadi,, M. S and . Dr. Ir. Estri Laras Arumingtyas,, M.Sc., St DESAIN DAN UJI KINERJA PRIMER DAN PROBE SPESIFIK SPESIES ANJING (Canis lupus familiaris) DALAM PRODUK BAKSO DAGING DENGAN METODE REAL-TIME POLYMERASE CHAIN REACTION (qPCR. Sarjana thesis, Universitas Brawijaya.
Abstract
Daging dikenal sebagai sumber protein utama dengan kandungan asam amino esensial penting dibutuhkan oleh tubuh manusia. Pemalsuan daging sapi dengan daging anjing dilakukan oleh pelaku ekonomi akibat tingginya jumlah permintaan akan daging sapi, meningkatnya perdagangan anjing dan nilai harga daging anjing yang lebih rendah dari daging sapi. Oleh karena itu, dibutuhkan metode deteksi menggunakan Real-Time Polymerase Chain Reaction (qPCR) dengan TaqMan probe karena memiliki kemampuan deteksi yang akurat dan dapat diperoleh hasil secara kuantitatif. Tujuan dari penelitian ini adalah memperoleh primer dan probe spesifik yang dirancang dari sekuen gen Cytochrome b dalam mtDNA spesies anjing. Primer dan probe didesain dengan cara manual menggunakan informasi sekuen gen dari database National Center for Biotechnology Information (NCBI). Pemilihan primer dan probe didasarkan pada kriteria primer dan probe yang baik, dimana penentuan kriteria dilakukan secara in-silico menggunakan website Beacon Designer dan Primer3Plus. Kinerja primer dan TaqMan probe diukur berdasarkan tiga parameter, yaitu uji spesifisitas terhadap enam jenis daging hewan berbeda, uji limit of detection dengan serial pengenceran DNA anjing dari 10-10-5 ng/μL dan kadar daging anjing yang dicampur dengan daging sapi, serta uji terhadap produk bakso komersil. Hasil uji metode qPCR berbasis TaqMan Probe diketahui spesifik terhadap DNA spesies anjing mentah maupun daging anjing dalam campuran daging sapi. Primer dan probe yang didesain dapat mendeteksi DNA anjing hingga konsentrasi 10-5 ng/μL dan batas deteksi daging anjing pada campuran daging sapi hingga 0,01%. Metode ini menghasilkan liniearitas 0,99 dan efisiensi PCR 106%. Aplikasi metode terhadap 5 bakso bermerk dan 5 bakso curah, dua sampel menghasilkan nilai Ct yang terdeteksi diatas siklus ke-35. Berdasarkan hasil tersebut, metode amplifikasi DNA anjing dengan primer dan TaqMan Probe yang telah didesain perlu dilakukan validasi kembali seperti penambahan ulangan pengujian terhadap produk bakso komersial.
English Abstract
Meat is known as the main source of protein which contains important essential amino acids needed by the human body. The adulteration of beef with dog meat is carried out by economic actors due to the high demand for beef, the increase in the dog trade and the price of dog meat which is lower than beef. Therefore, a detection method using Real-Time Polymerase Chain Reaction (qPCR) with TaqMan probe is needed because it has accurate detection capabilities and quantitative results can be obtained. The aim of this research was to obtain specific primers and probes designed from the Cytochrome b gene sequence in the mtDNA of the dog species. Primers and probes were designed manually using gene sequence information from the National Center for Biotechnology Information (NCBI) database. The selection of primers and probes is based on good primer and probe criteria, where the criteria are determined in-silico using the Beacon Designer and Primer3Plus websites. The performance of the primer and TaqMan probe was measured based on three parameters, namely the specificity test for six different types of animal meat, the limit of detection test with serial dilutions of dog DNA from 10-10-5 ng/μL and the levels of dog meat mixed with beef, and the test towards commercial meatball products. The test results of the TaqMan Probe�based qPCR method are known to be specific for the DNA of raw dog species and dog meat in a mixture of beef. The designed primers and probes can detect dog DNA up to a concentration of 10-5 ng/μL and the detection limit for dog meat in beef mixtures is up to 0.01%. This method produces linearity of 0.99 and PCR efficiency of 106%. Application of the method to 5 branded meatballs and 5 bulk meatballs, two samples produced Ct values detected above the 35th cycle. Based on these results, the dog DNA amplification method with primers and TaqMan Probe that has been designed needs to be validated again, such as testing commercial meatball products
| Item Type: | Thesis (Sarjana) |
|---|---|
| Identification Number: | 052410 |
| Uncontrolled Keywords: | Anjing, Primer Cytochrome b, Real-Time PCR, TaqMan Probe - Dog, Cytochrome b Primer, Real-Time PCR, TaqMan Probe |
| Divisions: | Fakultas Teknologi Pertanian > Teknologi Hasil Pertanian |
| Depositing User: | Sugeng Moelyono |
| Date Deposited: | 11 Nov 2024 02:51 |
| Last Modified: | 11 Nov 2024 02:51 |
| URI: | http://repository.ub.ac.id/id/eprint/228509 |
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