Perbandingan Kuantitas dan Kualitas Isolat DNA Candida

albicans Antara Metode Filter Based Kit, Alkaline Lysis, dan

Heat Treatment

SKRIPSI
Untuk Memenuhi Persyaratan
Memperoleh Gelar Sarjana Kedokteran

Oleh :

AULIA MAHYA FARADISA

21701101065

PROGRAM STUDI PENDIDIKAN DOKTER

FAKULTAS KEDOKTERAN
UNIVERSITAS ISLAM MALANG
2021
 Perbandingan Kuantitas dan Kualitas Isolat DNA
Candida albicans Antara Metode Filter based kit, Alkaline
lysis, dan Heat treatment

TUGAS AKHIR
Untuk Memenuhi Persyaratan
Memperoleh Gelar Sarjana Kedokteran

Oleh
AULIA MAHYA FARADISA

21701101065

PROGRAM STUDI KEDOKTERAN

FAKULTAS KEDOKTERAN
UNIVERSITAS ISLAM MALANG
2021
 Perbandingan Kuantitas dan Kualitas Isolat DNA
Candida albicans Antara Metode Filter based kit, Alkaline
lysis, dan Heat treatment

TUGAS AKHIR
Untuk Memenuhi Persyaratan
Memperoleh Gelar Sarjana Kedokteran

Oleh

AULIA MAHYA FARADISA

21701101065

PROGRAM STUDI KEDOKTERAN

FAKULTAS KEDOKTERAN
UNIVERSITAS ISLAM MALANG
2021
 RINGKASAN

Aulia Mahya Faradisa. Fakultas Kedokteran, Universitas Islam Malang, 28

Oktober 2021. PERBANDINGAN KUANTITAS DAN KUALITAS ISOLAT
DNA Candida albicans ANTARA FILTER BASED KIT, ALKALINE LYSIS DAN
HEAT TREATMENT. Pembimbing 1: dr. Hj. Noer Aini, M.Kes, Pembimbing 2:
Rio Risandiansyah, S.Ked, MP., PhD

Pendahuluan: Prevalensi kasus kandidiasis invasif oleh Candida albicans) masih

menjadi permasalahan yang penting. Penegakan diagnosis kandidiasis
menggunakan preparat kultur membutuhkan waktu yang lama. Polymerase Chain
Reaction (PCR) merupakan metode diagnosis yang cepat berbasis
Deoxyribonucleic Acid (DNA). Metode isolasi DNA yang umum digunakan adalah
Filter Based Kit (FBK), namun, terdapat metode lebih sederhana yaitu Alkaline
Lysis (AL) dan Heat Treatment (HT). Penelitian ini bertujuan mengetahui
perbandingan kuantitas dan kualitas isolasi DNA C. albicans dengan menggunakan
metode AL dan HT dibandingkan dengan FBK.

Metode: Penelitian ini merupakan eksperimental laboratorium in vitro.

Kuantifikasi C. albicans dilakukan dengan haemocytometer dan didapatkan jumlah
stok sampel 109 sel/mL. Metode FBK, AL, dan HT dilakukan pada konsentrasi 108,
109 sel/mL dan normal saline (NS). Perhitungan kuantitas dan kualitas DNA
dilakukan menggunakan Spektrofotometer NanoDrop. Analisa statistik
menggunakan One Way Analysis of Variance (ANOVA) dan Least Significant
Difference (LSD) pada SPSS versi 22.0.

Hasil: Hasil kuantitas DNA C. albicans pada konsentrasi 109 sel/mL terhadap
kontrol pada metode FBK, AL, HT yaitu 61,60±3,78, 8,20±1,92, 165±1,58, dan
berbeda signifikan (p< 0.05). Sedangkan pada konsentrasi C. albicans 108 sel/mL
terhadap kontrol, kuantitas DNA metode FBK, AL, HT yaitu 2,60±0,54, 3,40±1,14,
dan 36,8±5 µg/mL, dan berbeda signifikan (p< 0.05). Kualitas DNA (A260/A280)
konsentrasi C. albicans 109 sel/mL terhadap kontrol pada metode FBK, AL, HT
1,87±0,05, 0±0, dan 1,35±0,05 dan berbeda signifikan (p< 0.05). Demikian pula
pada konsentrasi DNA C. albicans 108 sel/mL 2,10±0,54, 0±0, dan 1,80±0,54
µg/mL.

Kesimpulan: Isolasi DNA C. albicans metode HT menghasilkan kuantitas dan

kualitas DNA yang lebih baik bila dibandingkan metode FBK dan AL.

Kata Kunci: Isolasi DNA; Polymerase Chain Reaction; diagnosis.

 SUMMARY

Aulia Mahya Faradisa. Faculty of Medicine, Islamic University of Malang,

October 28, 2021. QUANTITY AND QUALITY COMPARISON OF DNA
ISOLAT Candida albicans BETWEEN FILTER BASED KIT, ALKALINE
LYSIS AND HEAT TREATMENT METHODS. Supervisor 1: dr. Hj. Noer Aini,
M.Kes, Supervisor 2: Rio Risandiansyah, S.Ked, M.P., Ph.D

Introduction: The prevalence of invasive candidiasis caused by Candida albicans

(C. albicans) is still a problem. Diagnosis of candidiasis using culture requires a
long time. Polymerase Chain Reaction (PCR) is a rapid diagnosis based on
Deoxyribonucleic Acid (DNA). Commonly, the DNA isolation method is Filter
Based Kit (FBK), but there are simpler methods including alkaline lysis (AL) and
heat treatment (HT). This study aims to compare the quantity and quality of C.
albicans DNA using AL and HT compared with the FBK method.

Methods: This research is an in vitro laboratory experiment. Quantification of C.

albicans was done using haemocytometer where a stock sample of 109 cells/mL was
obtained. The FBK, AL, and HT methods were carried out at the concentrations of
108, 109 cells/mL and each of method was compared with normal saline (NS).
Calculation of the quantity and quality of DNA were conducted using a NanoDrop
Spectrophotometer. Data were analysed using One Way Analysis of Variance
(ANOVA) and followed by least significant difference (LSD) on SPSS version
22.0. it considered significant at p < 0.05.

Results: The quantity of C. albicans DNA at 109 cells/mL compared to control on

FBK, AL, HT methods were 61.60±3.78, 8.20±1.92, and 165±1.58 µg/mL,
respectively, and significantly different (p<0.05). The quantity of C. albicans DNA
at stock sample of 108 cells/mL compared to control on FBK, AL, HT methods were
2.60±0.54, 3.40±1.14, and 36.8±5.26 µg/mL, respectively, and significantly
different (p<0.05). The quality of DNA (A260/A280) at 109 cells/mL were
101.87±0.05, 0±0, and 1.35±0.05, and at 108 cells/mL were 2, 10±0.54, 0±0, and
1.80±0.54. All groups were significant both at 109 cells/mL and 108 cells/mL
(p<0.05).

Conclusion: HT method provided better quantity and quality C. albicans DNA

isolation in comparison with FBK and AL methods.

Keywords: DNA Isolation; Polymerase Chain Reaction; diagnosis.

 BAB I

PENDAHULUAN

1.1 Latar Belakang

Prevalensi kasus infeksi jamur yang disebabkan oleh Candida albicans

masih menjadi penyebab mortalitas dan morbiditas terutama pada pasien invasive

candidiasis (Wahyuningsih et al., 2021; Puspitasari et al., 2019). Data perkiraan

global tercatat kurang lebih 700.000 kasus invasive candidiasis. Salah satu jenis

invasive candidiasis yakni candidemia penyebab utamanya adalah Candida

albicans yang merupakan spesies mewakili rata-rata global 66% dari Candida spp

(Puspitasari et al., 2019). Pada penelitian sebelumnya, prevalensi pasien invasive

candidiasis di RS Cipto Mangunkusumo tercatat 12.3% dan menjadi penyebab

mortalitas cukup besar yakni 64,8% yang disebabkan oleh Candida albicans.

Peningkatan jumlah kematian tiap tahun pada pasien kandidiasis invasif

membutuhkan diagnosis cepat untuk penegakan diagnosa (Arastehfar et al., 2020).

Gold standard untuk penegakan diagnosa candidiasis adalah dengan pemeriksaan

kultur jamur dan histopatologi (Pitarch et al., 2018) Metode kultur membutuhkan

waktu 2 - 5 hari kerja untuk mencapai hasil yang akurat dan kurang sensitif sehingga

dibutuhkan diagnosis cepat yang memiliki sensitifitas dan spesifisitas yang baik

(Sidharta et al., 2021).

Salah satu metode diagnosis cepat yang memiliki sensitivitas dan

spesifisitas yang baik adalah Polymerase Chain Reaction (PCR). Metode ini sudah

banyak digunakan untuk deteksi infeksi yang disebabkan oleh mikrooganisme

patogen, baik bakteri, virus maupun jamur. PCR dalam mendeteksi infeksi jamur
memiliki sensitifitas dan spesifisitas kurang lebih 85% (Sidharta et al., 2021).

Deteksi dengan PCR membutuhkan sampel DNA yang baik melalui proses isolasi

DNA (Sundari, 2018)

Isolat DNA didapatkan melalui metode isolasi DNA yang sudah banyak

dikembangkan (Barbosa et al., 2016). Salah satunya adalah metode filter based kit,

merupakan metode yang umum digunakan untuk mengisolasi DNA. Metode ini

sudah banyak digunakan karena cepat dan cukup mudah digunakan sebab dalam

bentuk kit komersial (Mutia et al., 2018). Kekurangan dari metode ini adalah alat

yang digunakan tidak bisa didapatkan dengan mudah dan harganya yang relatif

mahal (Yahya et al., 2017). Maka dari itu dibutuhkan metode yang lebih sederhana

yang menghasilkan kuantitas dan kualitas DNA yang baik yaitu alkaline lysis dan

heat treatment. Metode tersebut memiliki prinsip yang sederhana yaitu memiliki

tahapan yang lebih sedikit sehingga lebih efektif dan efisien baik dari segi biaya

maupun waktu. (Mülhardt dan Beese, 2007). Metode alkaline lysis dan heat

treatment memikiki kelebihan yaitu bahan yang digunakan tidak beracun,

prosedurnya sederhana, biayanya murah, membutuhkan persiapan yang mudah dan

cepat, serta menghasilkan kemurnian yang bagus (Mulhardt dan Beese, 2007;

Warsinah et al., 2011)

Uji isolasi DNA dengan parameter kuantitas dan kualitas DNA

menggunakan metode filter based kit, alkaline lysis dan heat treatment belum

pernah dilakukan. Penelitian ini akan melakukan uji perbandingan kuantitas dan

kualitas dari DNA Candida albicans dengan menggunakan metode isolasi DNA

alkaline lysis dan heat treatment dibandingkan dengan metode filter based kit.
1.2 Rumusan Masalah

1.2.1 Bagaimana perbandingan kuantitas DNA Candida albicans menggunakan

metode isolasi DNA alkaline lysis dan heat treatment dibandingkan dengan

metode filter based kit ?

1.2.2 Bagaimana perbandingan kualitas DNA Candida albicans menggunakan

metode isolasi DNA alkaline lysis dan heat treatment dibandingkan dengan

metode filter based kit ?

1.3 Tujuan Penelitian

1.3.1 Mengetahui perbandingan kadar kuantitas DNA Candida albicans antara

metode sederhana alkaline lysis dan heat treatment dibandingkan metode

standart filter based kit pada beberapa konsentrasi.

1.3.1 Mengetahui perbandingan kadar kuantitas DNA Candida albicans antara

metode sederhana alkaline lysis dan heat treatment dibandingkan metode

standart filter based kit beberapa konsentrasi.

1.4 Manfaat Penelitian

1.4.1 Manfaat Teoritis

Penelitian ini diharapkan dapat digunakan sebagai landasan ilmiah tentang

perbandingan kuantitas dan kualitas DNA Candida albicans antara metode

filter based kit dengan metode alkaline lysis, dan heat treatment.

1.4.2 Manfaat Praktis

Mengetahui efektifitas antara metode filter based kit, alkaline lysis dan heat

treatment dalam isolasi DNA jamur untuk diagnosis berbasis DNA.

 PENUTUP

7.1 Kesimpulan

1. Metode heat treatment menghasilkan kuantitas yang tinggi dan kualitas DNA

Candida albicans yang baik pada konsentrasi 108 sel/mL dibandingkan

dengan metode filter based kit dan alkaline lysis.

2. Metode filter based kit menghasilkan kuantitas DNA yang tinggi dan kualitas

DNA Candida albicans yang baik pada konsentrasi 109 sel/mL dibandingkan

metode heat treatment dan alkaline lysis.

7.2 Saran

1. Membandingkan metode isolasi DNA pada dosis infektif untuk beberapa

jenis penyakit yang disebabkan oleh Candida albicans, yaitu pada 103

hingga 106 CFU/ml.

2. Melakukan penelitian lanjutan seperti uji PCR pada hasil isolat DNA yang

didapatkan sehingga hasilnya dapat terkonfirmasi secara aplikatif.

3. Melakukan modifikasi metode alkaline lysis dengan meningkatkan

konsentrasi NaOH (1,5 - 2N) untuk mendapatkan kuantitas dan kualitas

DNA yang baik.

4. Melakukan modifikasi metode heat treatment dengan menambahkan

tahapan presipitasi dan purifikasi.

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