Potensi DNA Aptamer Poliklonal untuk Deteksi Secara Bersamaan Bakteri Patogen Staphylococcus aureus, Streptococcus agalactiae, dan Escherichia coli

Abstract

Mastitis adalah peradangan pada ambing yang umumnya disebabkan oleh infeksi mikroba. Mastitis subklinis sulit dideteksi karena tidak ada gejala klinis pada ambing dan fisik susu, kecuali adanya penurunan produksi susu yang menyebabkan kerugian ekonomi pada peternak. Berbagai patogen penyebab mastitis diantaranya bakteri, mikoplasma, virus dan cendawan. Bakteri penyebab mastitis pada sapi perah diantaranya yaitu Staphylococcus aureus, Streptococcus agalactiae, dan Escherichia coli. Deteksi dan identifikasi patogen penyebab mastitis perlu dilakukan untuk diagnosis mastitis. Deteksi mastitis subklinis secara langsung melalui penghitungan jumlah sel somatik dan secara tidak langsung dengan uji CMT, IPB-1 mastitis test, konduktivitas elektrik, dan deteksi enzim (LDH, NAGase). Identifikasi agen patogen penyebab mastitis dapat dilakukan dengan pemeriksaan mikrobiologi dan metode molekuler berbasis PCR. Patogen penyebab mastitis semakin cepat terdeteksi maka semakin baik untuk pengobatan mastitis. Aptamer telah banyak dikembangkan untuk diagnostik pada berbagai penyakit hewan. Aptamer digunakan untuk mendeteksi virus, bakteri, dan parasit patogen penyebab penyakit. Berbagai penelitian telah berhasil memanfaatkan aptamer untuk deteksi masing-masing bakteri S. aureus, Streptococcus sp., dan E. coli. Penggunaan aptamer untuk deteksi secara bersamaan bakteri S. aureus, S. agalactiae, dan E. coli belum pernah dilaporkan dan belum tersedia komersial. Penelitian ini bertujuan untuk isolasi dan seleksi DNA aptamer poliklonal yang memiliki reaktivitas pada ketiga bakteri patogen mastitis S. aureus, S. agalactiae, dan E. coli dengan menggunakan metode sequential toggle cell - systematic evolution of ligands by exponential enrichment (STC-SELEX). Metode STC-SELEX dipilih karena pada proses seleksi in vitro dapat digunakan lebih dari satu target sehingga dihasilkan aptamer yang mampu mengikat ke beberapa target. DNA aptamer dari hasil SELEX 9, SELEX 12, dan SELEX 15 dilakukan kloning ke plasmid pGEM-T easy. Hasil transformasi SELEX 9, SELEX 12, dan SELEX 15 dengan metode heat shock diperoleh banyak klon yang tumbuh pada media seleksi. Sebanyak 40 klon transforman berwarna putih dipilih secara acak untuk diseleksi dengan metode PCR koloni yang dilanjutkan isolasi plasmid (29 klon terpilih), dan sekuensing (22 klon terpilih). Hasil analisis sekuensing diperoleh 14 DNA aptamer poliklonal (S9K12, S9K13, S9K19, S9K22, S12K2, S12K6, S12K7, S12K10, S15K3, S15K4, S15K6, S15K13, S15K15, S15K20) yang memiliki constant region benar dan berukuran 81 pb. Sekuen 14 DNA aptamer poliklonal dikarakterisasi secara in silico untuk mengetahui hubungan filogenetik dan memprediksi struktur sekunder. Program MEGA11 dengan metode statistik maximum parsimony dan bootstrap 1000 ulangan digunakan dalam pembuatan pohon filogenetik. Hasil analisis menunjukkan terdapat pembagian pohon filogenetik dalam 2 klad besar. Program Mfold digunakan untuk prediksi struktur sekunder menunjukkan bahwa 14 DNA aptamer poliklonal memiliki struktur sekunder dengan struktur stem dan loop yang bervariasi. Program QGRS Mapper digunakan untuk menentukan urutan Gquadruplex menunjukkan terdapat 9 DNA aptamer poliklonal (S9K12, S12K2, S12K6, S12K10, S15K3, S15K4, S15K6, S15K13, dan S15K20) mempunyai sekuen G-quadruplex dan 5 DNA aptamer poliklonal tidak terdapat sekuen Gquadruplex (S9K13, S9K19, S9K22, S12K7, S15K15). Analisis kapasitas pengikatan, afinitas pengikatan, dan spesifisitas pengikatan DNA aptamer poliklonal dilakukan dengan metode qPCR. Hasil analisis menunjukkan kapasitas pengikatan tertinggi aptamer S12K6 terhadap S. agalactiae (nilai Cq 8,40), aptamer S12K2 terhadap S. aureus BPA-12 (nilai Cq 7,94), dan aptamer S9K22 terhadap E. coli EPEC 4 (nilai Cq 8,38). Aptamer S15K3 menunjukkan kapasitas pengikatan yang tinggi pada ketiga bakteri target (S. agalactiae, S. aureus BPA-12, E. coli EPEC 4) dengan nilai Cq (10,01; 9,86; 10,18). Uji afinitas pengikatan pada 6 DNA aptamer poliklonal (S15K3, S15K6, S15K13, S15K15, S12K10, dan S9K19) menunjukkan hasil aptamer S15K3 (Kd 6,84 nM), S15K13 (Kd 6,53 nM), dan S12K10 (Kd 6,70 nM) memiliki afinitas pengikatan yang tinggi pada S. agalactiae. Aptamer S15K6 (Kd 17,01 nM) memiliki afinitas pengikatan tertinggi pada S. aureus BPA-12. Aptamer S15K13 (Kd 5,21 nM) dan S15K15 (Kd 8,89 nM) memiliki afinitas pengikatan tinggi pada E. coli EPEC 4. Uji spesifisitas pengikatan DNA aptamer poliklonal (S15K3, S15K13, dan S9K19) menunjukkan hasil ketiganya memiliki kemampuan mengikat yang tinggi pada ketiga bakteri target (S. aureus BPA-12, S. agalactiae, E. coli EPEC 4) dengan nilai Cq kecil sekitar 10. Ketiga DNA aptamer poliklonal masih mampu mengikat S. aureus BPA-6 meskipun dengan nilai Cq yang besar (15,32; 16,32; 16,06) dan tidak dapat mengikat bakteri E coli MHA 6 dan L. monocytogenes yang ditunjukkan dengan tidak munculnya nilai Cq.

Mastitis is an inflammation of the mammary glands caused by a microbial infection. Clinical mastitis can be detected easily due to the clinical symptoms. On the contrary, sub-clinical mastitis is difficult to identify since the udder and milk do not exhibit any clinical signs. However, the milk production will decrease which causes economic loss for the farmers. Various pathogens such as bacteria, mycoplasma, viruses and fungi can cause mastitis. The common bacteria causing this infection in dairy farms are Staphylococcus aureus, Streptococcus agalactiae, and Escherichia coli. Detection and identification of pathogens causing mastitis are carried out to establish the diagnosis of this infection. Detection of sub-clinical mastitis was carried out directly by counting the somatic cells and indirectly by CMT test, IPB-1 mastitis test, electrical conductivity, and detection of enzymes (LDH, NAGase). Identification of pathogenic agents causing mastitis can be done by microbiological examination and PCR-based molecular method. Early detection of pathogen which cause mastitis will benefit the treatment. Aptamers in animals have been developed for diagnostic purposes in various diseases. Aptamers are used to detect various pathogenic agents that cause disease, including viruses, bacteria, and parasites. Various studies using aptamers for the detection of S. aureus, Streptococcus sp., and E. coli have been successfully conducted. However, the use of aptamer for simultaneous detection of S. aureus, S. agalactiae, and E. coli bacteria has not been reported. This study aimed to isolate and characterize polyclonal DNA aptamer with broad reactivity to S. aureus, S. agalactiae, and E. coli bacteria using a sequential toggle cell-SELEX (STC SELEX) method. The STC-SELEX method was chosen because in the in vitro selection process more than one target can be used to produce an aptamer that capable of binding to several targets. The DNA aptamer pool from SELEX 9, 12, and 15 was cloned into the pGEM-T easy plasmid. The transformation results of SELEX 9, SELEX 12, and SELEX 15 using heat shock method obtained many clones that grow on the selection media. A total of 40 white transformant clones were randomly selected for selection by colony PCR method followed by plasmid isolation (29 selected clones), and sequencing (22 selected clones). The results of the sequencing analysis were obtained 14 polyclonal DNA aptamers (S9K12, S9K13, S9K19, S9K22, S12K2, S12K6, S12K7, S12K10, S15K3, S15K4, S15K6, S15K13, S15K15, S15K20) with a constant region and the expected size of 81 bp. The sequence of 14 polyclonal DNA aptamers from in vitro selection of STC SELEX were characterized by in silico to determine phylogenetic relationships and predict secondary structure. MEGA11 software with maximum parsimony statistical method and bootstrap 1000 replicates were used to construct the phylogenetic tree. Phylogenetic analysis showed that the phylogenetic tree was devided into 2 major clades. The Mfold software used for secondary structure prediction showed 14 polyclonal aptamer DNAs had secondary structures with varying stem and loop structures. QGRS Mapper software was used to determine the G-quadruplex sequence showing 9 polyclonal DNA aptamers (S9K12, S12K2, S12K6, S12K10, S15K3, S15K4, S15K6, S15K13, and S15K20) had G-quadruplex sequences and 5 polyclonal DNA aptamers (S9K13, S9K19, S9K22, S12K7, S15K15) without G-quadruplex sequences. Analysis of binding capacity, binding affinity, and binding specificity of polyclonal aptamer DNA was carried out using qPCR method. The results indicated aptamer S12K6 has the highest binding ability into S. agalactiae (Cq value 8.40), aptamer S12K2 performed binding capacity most to S. aureus BPA-12 (Cq value 7.94), and aptamer S9K22 has the highest capacity of binding to E. coli EPEC 4 (Cq value 8.38). Aptamer S15K3 showed high binding capacity against the three target bacteria (S. agalactiae, S. aureus BPA-12, E. coli EPEC 4) with Cq values (10.01; 9.86; 10.18). The binding affinity assay for 6 polyclonal DNA aptamers (S15K3, S15K6, S15K13, S15K15, S12K10, and S9K19) showed that the aptamers S15K3 (Kd 6,84 nM), S15K13 (Kd 6,53 nM), and S12K10 (Kd 6,70 nM) had high binding affinity against S. agalactiae. Aptamer S15K6 (Kd 17.01 nM) had the highest binding affinity against S. aureus BPA-12. Aptamer S15K13 (Kd 5.21 nM) and S15K15 (Kd 8.89 nM) had high binding affinity against E. coli EPEC 4. The binding specificity assay of polyclonal DNA aptamers (S15K3, S15K13, and S9K19) showed that all three had high binding ability on the three target bacteria (S. aureus BPA-12, S. agalactiae, E. coli EPEC 4) with a small Cq value of about 10. The three polyclonal DNA aptamers were still able to bind S. aureus BPA-6 even though with large Cq values (15.32; 16.32; 16.06) and could not bind E. coli MHA 6 and Listeria monocytogenes bacteria.