MAHARANIANSKA AZZAHRA, . (2022) VALIDASI FORMULA KIT DETEKSI BAKTERI PATOGEN Bacillus cereus & Listeria monocytogenes PENYEBAB KERACUNAN PANGAN DENGAN REAL-TIME POLYMERASE CHAIN REACTION. Sarjana thesis, UNIVERSITAS NEGERI JAKARTA.
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Abstract
Penyakit akibat pangan (foodborne disease) merupakan salah satu ancaman global yang dapat mempengaruhi semua bagian masyarakat, baik di negara berkembang ataupun maju. Bakteri Bacillus cereus dan Listeria monocytogenes termasuk ke dalam bakteri Gram-positif penyebab keracunan pangan yang dapat mengakibatkan penyakit pada manusia. Metode deteksi Real-Time PCR merupakan salah satu metode penanda molekuler yang banyak dikembangkan sebagai metode deteksi cepat yang akurat, sensitif dan spesifik dalam mendeteksi bakteri patogen. Penelitian ini bertujuan untuk memvalidasi hasil penelitian sebelumnya sehingga dikembangkan formula prototype kit deteksi bakteri Bacillus cereus dan Listeria monocytogenes penyebab keracunan pangan yang bersifat reprodusibel dengan Real-time PCR. Validasi dilakukan dengan membandingkan data dan mengkonfirmasi kembali kondisi pada penelitian sebelumnya sebagai landasan pengembangan prototype kit deteksi. Hasil amplifikasi bakteri B. cereus dan L. monocytogenes menghasilkan amplikon sesuai target penelitian sebelumnya, secara berturut sebesar 114 bp dan 158 bp dengan konsentrasi isolat 50 ng/μL pada suhu annealing 60°C. Validasi Real-time PCR pasangan primer gen cyt-K 2 dan gen hly berhasil mengamplifikasi target dengan menghasilkan nilai Ct 17,62 pada bakteri B. cereus dan 19, 11 pada L. monocytogenes. Optimasi Real-time PCR dengan variasi waktu pra-denaturasi dan konsentrasi primer didapatkan kondisi optimum kedua pasangan primer cyt-K 2 dan hly berturut-turut pada waktu pra-denaturasi 4 menit dengan konsentrasi primer 10 pmol dan 5 menit dengan konsentrasi primer 15 pmol. Hasil kurva melt curve menunjukkan hasil produk spesifik dengan munculnya satu puncak pada nilai Tm 78,42°C gen cyt-K 2 dan 79,51°C gen hly. Uji konfirmasi sampel pangan basi dan fresh pada ketiga sampel susu, kaldu jamur dan kaldu daging, B. cereus dan L. monocytogenes terdeteksi positif terkandung dalam semua sampel pangan dengan perhitungan CFU (Colony Forming Unit) diatas nilai LOD (Limit of Detection) dan data nilai BSN sebagai standar. Berdasarkan hasil penelitian, pasangan primer cyt-K 2 dan hly bersifat reprodusibel dalam mendeteksi gen target menggunakan metode Real-time PCR. ******************************************************* Foodborne disease is a global threat that can affect all sections of society, both in developing and developed countries. Bacillus cereus and Listeria monocytogenes are Gram-positive food poisoning bacteria that can cause disease in humans. The Real-Time PCR detection method is one of the molecular marker methods that has been widely developed as a fast, accurate, sensitive and specific detection method for detecting pathogenic bacteria. This study aims to validate the results of previous studies that a prototype kit formula for the detection of Bacillus cereus and Listeria monocytogenes bacteria is developed are reproducible with Real-time PCR. Validation is done by comparing the data and reconfirming the conditions in previous studies as the basis for developing a detection kit prototype. The results of the amplification of B. cereus and L. monocytogenes bacteria produced amplicons according to the target of the previous study, 114 bp and 158 bp, respectively, with an isolate concentration of 50 ng/μL at an annealing temperature of 60°C. Real-time PCR validation of the cyt-K 2 gene and hly gene primer pair successfully amplified the target by producing a Ct value of 17.62 in B. cereus and 19.11 in L. monocytogenes. Optimization of Real-time PCR with variations of pre-denaturation time and primer concentration obtained optimum conditions for both pairs of primers cyt-K 2 and hly respectively at pre-denaturation time of 4 minutes with a primer concentration of 10 pmol and 5 minutes with a primer concentration of 15 pmol. The results of the melt curve show specific product yields with the appearance of a peak at the Tm value of 78.42°C for the cyt-K 2 gene and 79.51°C for the hly gene. Confirmation test of stale and fresh food samples on the three samples of milk, mushroom broth and meat broth, B. cereus and L. monocytogenes was detected positively contained in all food samples by calculating CFU (Colony Forming Unit) above the LOD (Limit of Detection) value and with BSN data value as standard. Based on the results of the study, the primer pair cyt-K 2 and hly were reproducible in detecting the target gene using the Real-time PCR method.
| Item Type: | Thesis (Sarjana) |
|---|---|
| Additional Information: | 1). Prof. Dr. Muktiningsih Nurjayadi M.Si.; 2). Dwi Ana Oktaviani Saputro S.Si. |
| Subjects: | Sains > Kimia |
| Divisions: | FMIPA > S1 Kimia |
| Depositing User: | sawung yudo |
| Date Deposited: | 08 Nov 2024 07:55 |
| Last Modified: | 08 Nov 2024 07:55 |
| URI: | http://repository.unj.ac.id/id/eprint/52031 |
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