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Konsentrasi dan kemurnian ekstraksi DNA metode sonikasi dan spin column dari sampel dahak penderita tuberkulosis Saputra, Fitrianingsih; Indra, Asep Iin Nur; Djuminar, Ai; Merdekawati, Fusvita; Nurhayati, Betty
Current Biomedicine Vol. 2 No. 2 (2024): July
Publisher : School of Veterinary Medicine and Biomedical Sciences, IPB University, Bogor, Indonesia

Show Abstract | Download Original | Original Source | Check in Google Scholar | DOI: 10.29244/currbiomed.2.2.84-92

Abstract

Background: The Polymerase Chain Reaction (PCR) method can identify Mycobacterium tuberculosis in a sputum sample of a patient with TB (TB). One crucial step to ensure accurate PCR results is the DNA extraction process. Objective: The research aims to compare the concentration and purity of DNA from the sputum of TB patients using ultrasound and spin column extraction techniques. Methods: The research uses descriptive study designs with post-only design strategies. The primary data was derived from 18 sputum specimens from TB patients. Concentration measurement and DNA purity testing using a nanodrop spectroscopic photometer. Results: DNA extraction by ultrasound method has an average concentration of 18.9 ± 8.5 ng/L, with a peak of 37.6 ng/ L. The spin column method produces an average of 55.5 ± 27.9 ng/μL; the peak is 105.0 ng/ μL. The purity value of the DNA extract is in the range of 1.8 ± 2.0 with the ultrasound method of 61% and the spin column of 78%. Conclusion: The sonication method has a lower average concentration and a higher percentage of purity than the spin column method, and there are differences in concentrations and purity values between the two methods.
OPTIMASI VARIASI KONSENTRASI AGAROSE DAN WAKTU ELEKTROFORESIS TERHADAP KUALITAS PITA DNA Mycobacterium tuberculosis RESISTAN ISONIAZID Halimah, Ina; Firman Solihat , Mohamad; Djuminar, Ai; Rismiati, Zuri
Jurnal Kesehatan Siliwangi Vol. 4 No. 3 (2024): JURNAL KESEHATAN SILIWANGI
Publisher : Politeknik Kesehatan Kemenkes Bandung

Show Abstract | Download Original | Original Source | Check in Google Scholar | DOI: 10.34011/jks.v4i3.1854

Abstract

Mycobacterium tuberculosis resistan isoniazid (INH) terjadi akibat mutasi gen dimana sebagian besar terjadi pada gen katG. Pada penelitian ini digunakan sampel dengan primer untuk mendeteksi mutasi gen katG untuk MRD TB pada kodon 315 dan 463 dengan panjang produk primer yang bervariasi diantaranya 101 bp (untuk mutasi S315T), 141 bp (mutasi S315N), 200 bp (mutasi S315I), 247 bp (mutasi S315R), 290 bp (mutasi S315G), dan 400 bp (mutasi R463L). Penelitian ini bertujuan untuk mengetahui konsentrasi dan lama waktu elektroforesis Mycobacterium tuberculosis resistan isoniazid (INH) pada gel agarose gel agarose yang optimum. Jenis penelitian yang digunakan adalah eksperimen mengenai perbandingan variasi konsentrasi agarose 1%, 1,5% dan 2% terhadap variasi lama waktu elektroforesis pada 30 menit, 45 menit dan 60 menit. Analisa pita DNA hasil elektroforesis menggunakan aplikasi imageJ. Pada penelitian ini sampel yang digunakan adalah pull DNA Mycobacterium tuberculosis resistan isoniazid hasil produk real time PCR. Hasil penelitian menunjukkan bahwa pita DNA Mycobacterium tuberculosis resistan isoniazid (INH) gen katG untuk MRD TB pada kodon 315 dengan ukuran produk 101 bp S315T, 141 bp S315N, 200 bp S315I, dan 290 bp S315G diperoleh optimal pada konsentrasi agarose 2% dan waktu elektroforesis selama 45 menit. Pita DNA Mycobacterium tuberculosis resistan isoniazid (INH) gen katG untuk MRD TB pada kodon 315 dengan ukuran produk 247 bp S315R dan gen katG untuk MRD TB pada kodon 463, ukuran 400 bp R463L optimal pada konsentrasi agarose 2% dengan waktu elektroforsis selama 60 menit.
Comparison of Purity and Concentration Values of Mycobacterium tuberculosis DNA Extraction Result from the Boiling and Spin Column Method Febriyanti, Intan; Djuminar, Ai; Merdekawati, Fusvita; Indra , Asep Iin Nur
JURNAL INDONESIA DARI ILMU LABORATORIUM MEDIS DAN TEKNOLOGI Vol 5 No 2 (2023): Combatting Bacterial and Fungal Infections: The Critical Role of Advanced Researc
Publisher : Universitas Nahdlatul Ulama Surabaya

Show Abstract | Download Original | Original Source | Check in Google Scholar | DOI: 10.33086/ijmlst.v5i2.4771

Abstract

The Polymerase Chain Reaction (PCR) technique is extensively employed in molecular biology to precisely detect Mycobacterium tuberculosis. Prior to conducting PCR, extracting of high-quality genomic Deoxyribonucleic Acid (DNA) is crucial to ensure accurate and reliable results. The primary objective of this study is to conduct a comparative analysis of the purity and concentration of M. tuberculosis DNA acquired through the utilization of the boiling method and the spin column extraction methods. A descriptive comparative research design was employed, utilizing a sample of 16 sputum specimens that had previously been confirmed as positive for M. tuberculosis through Acid-Fast Bacteria (AFB) examination and Molecular Rapid Test (MRT). The extraction of DNA was carried out using the boiling method and the spin column method. Subsequently, the concentration and purity of the extracted DNA were assessed using the NanoDrop Spectrophotometer, and the results were compared. The obtained yield of M. tuberculosis DNA isolates through the boiling method ranged from 9.6 ng/µL to 1258.7 ng/µL, with an average purity value of 1.23. Conversely, for the spin column method, the concentration of M. tuberculosis DNA isolates ranged from 8.7 ng/µL to 207.8 ng/µL, with an average purity value of 1.83. In conclusion, there is a significant difference between the purity and concentration of M. tuberculosis DNA extraction results using the boiling method and spin column methods.
Analysis of Purity and Concentration Escherichia coli DNA by Boiling Method Isolation with Addition of Proteinase-K and RNase Lesiani, Bunga Rossa; Abror, Yogi Khoirul; Merdekawati, Fusvita; Djuminar, Ai
JURNAL INDONESIA DARI ILMU LABORATORIUM MEDIS DAN TEKNOLOGI Vol 5 No 2 (2023): Combatting Bacterial and Fungal Infections: The Critical Role of Advanced Researc
Publisher : Universitas Nahdlatul Ulama Surabaya

Show Abstract | Download Original | Original Source | Check in Google Scholar | DOI: 10.33086/ijmlst.v5i2.4773

Abstract

Escherichia coli is a leading cause of Urinary Tract Infections (UTIs) in Indonesia, with approximately 180,000 cases reported annually. The more cases of UTIs, the more PCR diagnosis is needed with an accurate, fast, simple, and economical DNA isolation method. However, currently, there is no DNA purification stage from protein and RNA contaminants in the boiling DNA isolation method. This study aimed to investigate the impact of incorporating Proteinase-K and RNase into the boiling DNA isolation method on the purity and concentration of E. coli’s DNA during isolation. The boiling method involved heating to 95°C – 100°C bring to cell lysis and release of cellular components, including DNA. Urine samples were artificially contaminated with E. coli at different McFarland standards (0.25, 0.5, and 1). The boiling DNA isolation method was then performed and then analyzed for purity and concentration using a NanoDrop spectrophotometer. This study demonstrated a positive correlation between Proteinase-K and RNase concentrations used in the boiling DNA isolation method and the subsequent increase in DNA purity and concentration. An increase in DNA purity and concentration was obtained even though it was not statistically significant compared to that without Proteinase-K and RNase addition, with p-values of 0.245 for DNA purity and 0.353 for DNA concentration. Further research is recommended with higher Proteinase-K and RNase concentrations in the boiling DNA isolation method to achieve improved purity and concentration of E. coli DNA. Such enhancements could improve PCR amplification and help diagnose E. coli-related UTIs.
Optimization of Concentration and Staining Duration of Methyl Green in The Examination of Escherichia coli DNA Bands Using Agarose Gel Electrophoresis Adrian Prasetya, Rifky; Merdekawati, Fusvita; Iin Nur Indra, Asep; Djuminar, Ai
Mukhtabar Journal of Medical Laboratory Technology Vol 2 No 2 (2024): Mukhtabar: Journal of Medical Laboratory Technology (October 2024)
Publisher : LPPM STIKes Muhammadiyah Ciamis

Show Abstract | Download Original | Original Source | Check in Google Scholar | DOI: 10.52221/mjmlt.v2i2.696

Abstract

Background & Objective: Nowadays, many alternative dyes are used for staining DNA during electrophoresis, one of which is Methyl Green. Methyl Green has several advantages, including being cheaper than EtBr, having low toxicity, being non-carcinogenic, and as a cationic dye, Methyl Green is resistant to photobleaching. This study aims to determine the optimal concentration and staining duration of Methyl Green as a DNA dye for examining Escherichia coli DNA bands using agarose gel electrophoresis. Method: The research method used is experimental. Amplified Escherichia coli 16S rRNA gene DNA, sized 584 bp, which has undergone electrophoresis, was stained with Methyl Green dye at concentrations of 0.10%, 0.15%, 0.20%, 0.25%, 0.00015%, 0.00020%, 0.00025%, and 0.00030% with varying immersion times of 10, 15, 20, and 25 minutes. Result: The resulting DNA bands were analyzed or measured for surface area using ImageJ software. The mean value for each experimental group was calculated. The highest mean value was used as the basis for determining the most optimal condition. Conclusion: This study concludes that the optimal concentration and staining duration of Methyl Green, based on the highest mean value of 19,844,845, is 0.00030% Methyl Green with a staining duration of 25 minutes.