Deteksi gen eae, bfpA, espA Escherichia coli isolat penderita diare

HARTI, Agnes Sri, Dr. Susi Iravati, Apt

2006 | Tesis | S2 Bioteknologi

Salah satu penyebab kasus diare pada anak-anak dan bayi terutama di negara berkembang, termasuk Indonesia adalah strains dari kelompok E. coli diaregenik yaitu Enteropathogenic Escherichia coli (EPEC). Faktor virulen yang berperan penting dalam patogenesis EPEC antara lain gen eae (effacing and attaching ), bfpA (bundle-forming pilus A) dan espA (encoding secreted protein A). Di Indonesia, penggunaan probe DNA dalam metode hibridisasi untuk deteksi terhadap gen target pada diaregenik Escherichia coli belum biasa dilakukan. Penggunaan probe DNA eae, bfpA dan espA dalam metode hibridisasi DNA dapat digunakan untuk deteksi EPEC pada isolate Escherichia coli penderita diare. Metode penelitian yang dilakukan yaitu isolasi DNA sampel dari isolat E. coli penderita diare anak-anak dari beberapa Puskesmas di Yogyakarta. Pembuatan probe DNA eae, bfpA dan espA dari hasil amplifikasi fragmen DNA secara PCR menggunakan primer spesifik terhadap isolat standart EPEC (Biofarma). Selanjutnya, probe DNA eae, bfpA dan espA tersebut digunakan dalam metode hibridisasi untuk deteksi EPEC dengan non EPEC. Hasil penelitian pada 49 sampel isolat Escherichia coli diperoleh 25 isolat (51,0 %) merupakan strain EPEC. Dari 25 isolat EPEC tersebut diantaranya 20 isolat (80 %) merupakan EPEC tipikal. Hasil analisis profil protein sekresi secara SDSPAGE menunjukkan adanya pita-pita protein sekresi dengan berat molekul 40 kDa, diduga sebagai protein sekresi (sekreton).

The Enteropathogenic Escherichia coli (EPEC) is one of pathogenic strain of diarrheagenic E. coli group cause in children and infant diarrhea that occurs in developing countries. The significant virulence factor in pathogenic EPEC among others are eae (E. coli attaching-effacing ), bfpA (bundle-forming pilus) and espA (encoding secreted protein A). The use of DNA probe in hybridization technique to detect of gene target diarrheagenic Escherichia coli in Indonesia is unusually. The DNA probes of eae, bfpA and espA on hybridization technique is used to detect EPEC and non EPEC of Escherichia coli in stool specimens. The method used in the experiment was by isolation the DNA samples of diarrheagenic E. coli specimen from Public Health Center in Yogyakarta. The DNA probes of eae, bfpA and espA from amplification DNA fragment of PCR product was as spesific primer on gold standard of EPEC (Biofarma). The DNA probes of eae, bfpA and espA in hybridization technique could be used to identify EPEC and non EPEC of E. coli in stool specimens. The result of the experiment was 49 isolates of E. coli, 25 isolates (51 %) were EPEC strain. Therefore among 25 isolates of EPEC, 20 isolates (80 %) were typical EPEC strain. The profile analysis of secretion protein by SDS-PAGE was detected as bands secretio n protein with 40 kDa molecular weight, and was predicted as secretion protein.

Kata Kunci : Probe DNA,eae,Deteksi EPEC,Diare, probe DNA, eae, bfpA, espA, Enteropathogenic Escherichia coli (EPEC)