Potensi DNA Aptamer Poliklonal untuk Deteksi Secara Bersamaan Bakteri Patogen Staphylococcus aureus, Streptococcus agalactiae, dan Escherichia coli
Date
2022Author
Kusumawati, Arizah
Wibawan, I Wayan
Setiyono, Agus
Sudarwanto, Mirnawati
Mustopa, Apon
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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.
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