NABILLA ALYA PRAMUDIYASIH, . (2022) VALIDASI FORMULA KIT DETEKSI BAKTERI PATOGEN ESCHERICHIA COLI DAN SHIGELLA FLEXNERI PENYEBAB KERACUNAN PANGAN MENGGUNAKAN REAL-TIME POLYMERASE CHAIN REACTION. Sarjana thesis, UNIVERSITAS NEGERI JAKARTA.
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Abstract
Bakteri Escherichia coli dan Shigella flexneri merupakan bakteri penyebab kasus keracunan pangan. Banyaknya jumlah kasus keracunan makanan yang terjadi di Indonesia menjadikan perlu dikembangkannya metode deteksi yang cepat, spesifik, dan akurat. Kondisi dan formula reaksi metode deteksi bakteri foodborne pathogen menggunakan metode Real-Time PCR telah ditemukan pada penelitian sebelumnya. Metode Real-Time PCR dapat melakukan proses amplifikasi sekaligus dengan kuantifikasi sampel DNA secara cepat, spesifik, dan akurat. Penelitian ini merupakan tahap validasi kondisi dan formula reaksi yang ditemukan pada penelitian sebelumnya, untuk memperoleh prototype kit deteksi yang bersifat konsisten dan reprodusibel. Validasi menggunakan suhu annealing 60oC menghasilkan gen fim-C Escherichia coli membentuk amplikon berukuran 121 bp (base pair) dan gen ipaH Shigella flexneri membentuk amplikon berukuran 188 bp. Hasil validasi dengan Real-Time PCR menunjukkan bahwa primer fim-C mampu mengamplifikasi DNA bakteri target E. coli pada Ct (cycle threshold) 15,18 dengan Tm (temperature melting) 80 . Primer ipaH mampu mengamplifikasi DNA bakteri target S. flexneri pada Ct 11,92 dengan Tm 80 sesuai dengan standar yang sudah ditemukan pada penelitian sebelumnya menggunakan konsentrasi template DNA 50 ng. Optimasi kondisi primer fim-C E. coli dan ipaH S. flexneri optimum pada waktu pra-denaturasi 5 menit dengan konsentrasi primer yang stabil 5 pmol untuk fim-C E. coli dan 10 pmol untuk ipaH S.flexneri. Uji coba skala laboratorium menggunakan sampel pangan susu sapi non-pasteurisasi, sampel kaldu jamur, dan sampel kaldu daging dalam dua kondisi yaitu fresh dan basi. Susu fresh, susu basi, kaldu daging fresh, kaldu daging basi, dan kaldu jamur basi terindikasi adanya cemaran bakteri Escherichia coli. Sampel kaldu jamur basi juga terindikasi adanya cemaran bakteri Shigella flexneri. Sampel pangan dianggap positif tercemar bakteri karena melebihi batas SNI. Berdasarkan data yang diperoleh dapat disimpulkan bahwa validasi kondisi dan formula reaksi prototype kit deteksi menghasilkan data yang konsisten dengan penelitian sebelumnya dan bersifat reprodusibel. **************************************** Escherichia coli and Shigella flexneri are bacteria that cause food poisoning cases. The large number of cases of food poisoning that occur in Indonesia makes it necessary to develop fast, specific, and accurate detection methods. The conditions and reaction formulas for detecting foodborne pathogen bacteria using the Real-Time PCR method have been found in previous studies. The Real-Time PCR method can simultaneously carry out the amplification process by quantifying DNA samples quickly, precisely, and accurately. This research is a stage of validating the conditions and reaction formulas found in previous studies to obtain a detection kit prototype that is consistent and reproducible. Validation using an annealing temperature of 60℃ resulted in the fim-C gene of Escherichia coli forming an amplicon measuring 121 bp (base pair) and the ipaH gene Shigella flexneri forming an amplicon measuring 188 bp. The validation results using Real-Time PCR showed that the fim-C primer could amplify the DNA of the target bacteria E. coli at Ct (cycle threshold) 15.18 with Tm (temperature melting) ±80℃. The ipaH primer was able to amplify the DNA of the target bacterium S. flexneri at Ct 11.92 with a Tm of ±80℃ according to the standard found in previous studies using a template DNA concentration of 50 ng. Optimization of primary conditions for fim-C E. coli and ipaH S. flexneri was optimum at a pre-denaturation time of 5 minutes with stable primer concentrations of 5 pmol for fim-C E. coli and 10 pmol for ipaH S.flexneri. Laboratory-scale trials used non-pasteurized cow's milk food samples, mushroom broth samples, and meat broth samples in two conditions: fresh and stale. Fresh milk, stale milk, fresh meat broth, thick meat broth, and thick mushroom broth indicated the presence of Escherichia coli bacteria contamination. Stale mushroom broth samples also indicated the presence of Shigella flexneri bacterial contamination. The food sample is considered positive for bacterial contamination because it exceeds the SNI limit. Based on the data obtained, it can be concluded that the condition validation and reaction formula of the detection kit prototype produced data consistent with previous research and reproducible.
| Item Type: | Thesis (Sarjana) |
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| Additional Information: | 1). Prof. Dr. Muktiningsih Nurjayadi, M. Si. ; 2). Vira Saamia, S.Si, M. |
| Subjects: | Sains > Kimia |
| Divisions: | FMIPA > S1 Kimia |
| Depositing User: | sawung yudo |
| Date Deposited: | 12 Nov 2024 01:39 |
| Last Modified: | 12 Nov 2024 01:39 |
| URI: | http://repository.unj.ac.id/id/eprint/52066 |
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