PENGEMBANGAN METODE DETEKSI KONTAMINASI BAKTERI PATOGEN Escherichia coli DALAM SUSU DENGAN REAL-TIME POLYMERASE CHAIN REACTION (RTi-PCR)

AMALIA MASTUROTUL MUWAFFAQOH, Dr. Tri Joko Raharjo, M. Si.

2014 | Skripsi | KIMIA

Penelitian deteksi kontaminasi bakteri patogen Escherichia coli dalam susu dengan Real-Time Polymerase Chain Reaction (RTi-PCR) dengan gen target tuf telah dilakukan. Penelitian ini bertujuan untuk mengembangkan metode deteksi kontaminasi bakteri E. coli dalam susu secara cepat. Tahapan penelitian dimulai dengan pengembangan metode yang meliputi peremajaan kultur E. coli, isolasi DNA, analisis konsentrasi DNA, serta amplifikasi dengan RTi-PCR yang dikombinasikan dengan Melting Curve Analysis. Kemudian penelitian dilanjutkan dengan validasi metode yang meliputi uji spesifisitas untuk membandingkan antara kontrol positif dan kontrol negatif, uji presisi yaitu perulangan metode pada 10 sampel kontrol positif, serta penentuan cut off deteksi. Tahap terakhir penelitian yaitu aplikasi metode pada sampel susu yang dijual di pasaran. Hasil penelitian menunjukkan bahwa metode RTi-PCR dengan primer TEco1553 dan primer TecoI754 spesifik untuk mendeteksi kontaminasi E. coli dalam susu dengan titik leleh puncak (melting peak point) yang khas pada 84,50 °C. Uji spesifisitas menunjukkan metode ini spesifik untuk mendeteksi kontaminasi E. coli dalam susu. Sedangkan pada uji presisi menunjukkan hasil kesepuluh kontrol positif dapat diamplifikasi dengan metode RTi-PCR. Metode RTi-PCR ini mampu mendeteksi kontamninasi E. coli dalam susu segar dengan cut off deteksi hingga 1,6 CFU/mL. Hasil analisis dari lima sampel susu di pasaran menunjukkan sampel susu yang dijual di pasar tradisional dan kedai susu positif terkontaminasi E. coli. Kata kunci : Escherichia coli, kontaminasi susu, Real-Time PCR.

Detection contaminant bacterial pathogen Escherichia coli in milk with Real-Time Polymerase Chain Reaction (RTi-PCR) have been done. The research aimed to develop detection method of E. coli contamination in milk rapidly. The prosedure of research was begun with development method which was consisted of E. coli culture enrichment, DNA isolation, analysis of DNA concentration, and RTi-PCR analysis that was combined with Melting Curve Analysis (MCA). Then, the research was continued by validation method which was consisted of specificity test to compare between positive control and negative control, precision test or repeatability method in 10 positive control, cut off detection determining, and the last was method application in commercial milk sample. The Results from this research showed that RTi-PCR method used TEco1553 and TecoI754 primer was right method to detect E. coli contamination in milk with typical melting peak point at 84,50 °C. Specificity test showed that this method was specific in detecting E. coli in milk. In precision test, all of positive control resulted melting peak with melting peak point between 84,00 °C – 84,50 °C. This RTi-PCR method could detected contamination of E. coli in milk with cut off detection until 1,6 CFU/mL. Five commercial milk samples that were analysed in application method showed a result that commercial milk samples which sold in traditional market and stall dairy was positive contaminated E. coli. Key word: Escherichia coli, milk contamination, Real-Time PCR.

Kata Kunci : Escherichia coli, kontaminasi susu, Real-Time PCR.