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Journal : METAMORFOSA Journal of Biological Sciences

OPTIMASI DIGESTI ENZIM RESTRIKSI SacII PADA ISOLAT Mycobacterium tuberculosis H37Rv UNTUK DETEKSI MUTASI PROMOTER inhA PADA KASUS MDR-TB DENGAN METODE PCR-RFLP Ida Ayu Ratih Dwi Nugraha Putri; Sagung Chandra Yowani; I Nengah Wirajana
Metamorfosa: Journal of Biological Sciences Vol 4 No 1 (2017)
Publisher : Prodi Magister Ilmu Biologi, Fakultas MIPA, Universitas Udayana

Show Abstract | Download Original | Original Source | Check in Google Scholar | DOI: 10.24843/metamorfosa.2017.v04.i01.p14

Abstract

The aim of this study was to optimize the digestion process of SacII restriction enzyme in Mycobacterium tuberculosis H37Rv isolate. SacII enzyme that have CCGC^GG restriction site could be used to detect the mutation at -24 promoter region of inhA M. tuberculosis. The mutation in this position is one of the causes of isoniazid resistance. The methods of this study were conducted in several step, respectively: DNA isolation of M. tuberculosis H37Rv, amplification of inhA promoter region of M. tuberculosis H37Rv using PCR method, and digest optimization using SacII. In this study, optimization of SacII digestion was conducted for formulaion and incubation time. The formulation was optimized by GM-ONE Buffer and GM-Buffer IV, meanwhile the incubation time was optimized in 1 hour, 2 hour, and 3 hour. The result of this study showed that SacII work optimally in GM-Buffer IV compared to GM-ONE Buffer and incubate for 3 hours. Under the optimal digest condition, SacII enzyme could be used to detect the mutation at -24 promoter region of inhA in the case of MDR-TB using PCR-RFLP method.
DESAIN TAQMAN PROBE SECARA IN SILICO SEBAGAI PENDETEKSI MUTASI PADA KODON 516 GEN rpoB Mycobacterium tuberculosis UNTUK METODE REAL-TIME PCR Dek Pueteri Dewi Suryani; Putu Sanna Yustiantara; Sagung Chandra Yowani
Metamorfosa: Journal of Biological Sciences Vol 4 No 1 (2017)
Publisher : Prodi Magister Ilmu Biologi, Fakultas MIPA, Universitas Udayana

Show Abstract | Download Original | Original Source | Check in Google Scholar | DOI: 10.24843/metamorfosa.2017.v04.i01.p04

Abstract

The aim of this research was to in silico design of TaqMan probe. Design of TaqMan probe were conducted using software Clone Manager Suite 6. As a template, the rpoB gene of M. tuberculosis H37Rv (accession number U12205.1) was used. The results of this research were 8 sequences such as, R516MV-2, R516MV-3, R516MV-4, R516MV-5, R516MV-7, R516MV-8, R516MV-11, R516MV-13. These sequences were met the criteria of TaqMan probe, such as length of nucleotide (23-28 nucleotide), Tm value (72ÂșC), %GC (50-58%), runs and repeats (?4 bases), dimer structure in accordance to the requirements and does not form hairpin structures. In addition, these sequences were met labeling criteria of TaqMan probe which are including the location of G bases and the number of G-C bases in sequences. Therefore, these sequences could be labeled by FAM (reporter) at 5' end and TAMRA (quencher) at 3' end. The conclusion of this research, the sequences were met the criteria of TaqMan probe. Therefore, it could be targeted to detect mutations at codon 516 with a change of aspartic acid into valine (GAC ? GTC) by using real-time PCR method.
Co-Authors A. W. Dwiputri Ade Ari Sundari Ade Ari Sundari Amelia Putri, Ni Luh Dian Saptari Arifani Siswidiasari Asmara A. A. R. Astuti, M.A.P. Ayu Nyoman Chandra Yustiana Ayu Putu Chika Iswari Anjani, Sang Cista Dewi, Ni Putu Fiona Dek Pueteri Dewi Suryani Deniariasih, N.W. Devi, Ni Wayan Antika Sri Devita Kusdianingrum Dewa Ayu Swastini Dewi Andayani Farmawati Dwicandra, N.M.O. Dyah Subadrika Warma Dewi Endang Kumolosasi Gede Bayu Surya Ekayana, I Heni Pujiastuti I Gede Ketut Sajinadiyasa I Gusti A. A. Santhi Rahmaryani I Gusti Ayu Agung Septiari I Ketut Juniarta I Ketut Tunas I Made Dira Swantara I Nengah Wirajana I Nyoman Gede Budiana I Wayan Kasa Ida Ayu Gendari Ida Ayu Ratih Dwi Nugraha Putri Ida Bagus Nyoman Putra Dwija Indra Juana Adikara Jennifer Tamara Jennifer Tamara Kadek Ratna Sari Dewi Kadek Widya Yuli Hartati Ketut Ratnayani Ketut Widyani Astuti Liangky Syane S Luh Vida Sasmitha Luk Ketut Budi Maitriani M. A. Pratiwi Made Dharmesti Wijaya Made Rai Dwitya Wiradiputra Marfu'ah, Nurul Marlia Singgih Wibowo Mirawati N.K.W. Ni Kadek Ari Cipta Pratiwi Ni Kadek Ariani Ni Ketut Sri Anggreni Ni Komang Sasi Ani Ni Made Febrianti Ni Made Yustikarini Ni Nyoman Pradnya Utari Ni Putu Ariantari Ni Putu Intan Satya Dewi Ni Putu Monica Rosdiana Dewi Paramitha Ni Wayan Sukma Pramitha Sari1 Osamu Iitsuka Pradnyaniti, D.G Putri, Ni Kadek Sri Adnyani Alit Putu Ayu Indrayathi Putu Lita Astriani Rasmaya Niruri Ratna Sari Dewi, Kadek Rini Noviyani Sang Ayu Putu Chika Iswari Anjani Saputra, Gusti Nyoman Oka Sari Dewi, Kadek Ratna Sudarma, I Wayan Ari Syamsul Arifin Tasya Pramiswari Tirtawati, Ni Putu Ayu Wijayakusuma, I Gusti Ngurah Lanang Yayanasri Yustiantara, Putu Sanna