Hotma Hutapea, Hotma
Kementerian Kesehatan Republik Indonesia

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Diterima: 8 April 2013 Direvisi: 6 Mei 2013 Disetujui: 20 Agustus 2013 59 Kloning Fragmen DNA Pengkode Integrase (int) HIV (Human Immunodeficiency Virus) 1 Pada Escherichia Coli JM109 Hutapea, Hotma; Oktavian, Antonius
Jurnal Biotek Medisiana Indonesia Vol 2, No 2 (2013)
Publisher : Central Basic Biomedical and Health Technology

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Abstract

Human Immunodeficiency Virus (HIV) is an RNA virus. It is a lentivirus, a retrovirus member family which causes Acquired Immunodeficiency Syndrome (AIDS). An early event in every retroviral multiplication is the integration of the viral double-stranded DNA genome into the host chromosome. The integration is facilitated by the activation of integrase.The goal of this research is to obtain the HIV integrase encoding gene. The obtained integrase ORF was intended to be cloned into cloning vector pJETclone and to transform Escherichia coli JM109. The cDNA synthesis was conducted by reverse transcription by converting the RNA genome to DNA. The obtained cDNA was amplified by Polymerase Chain Reaction (PCR) technique to obtain integrase encoding gene. The PCR product was inserted into the plasmid using blunt ended cloning system and was characterized using DNA gel electrophoresis and nucleotide analysis. The DNA gel electrophoresis of PCR product showed the expected band. Further characterization was conducted using nucleotide sequencing showed that the PCR product was homologue to HIV-1 integrase from Indonesia. The PCR was performed on the cloned showed DNA insert on expected size. The nucleotide analysis was conducted on the pure recombinant plasmid, the DNA was read properly.Key words: HIV-1,Iintegrase, E.coli JM109 AbstrakHIV (Human Immunodeficiency Virus) adalah virus RNA, dan termasuk ke dalam lentivirus, anggota kelompok retrovirus yang menyebabkan penyakit AIDS Acquired Immunodeficiency Syndrome. Tahap awal multiplikasi setiap retrovirus adalah integrasi genom DNA untai ganda virus ke dalam kromosom inang. Tahap integrasi ini difasilitasi oleh aktivasi enzim integrase. Tujuan penelitian ini adalah untuk memperoleh DNA pengkode integrase HIV. Kerangka baca terbuka atau Open Reading Frame (ORF) pengkode integrase yang diperoleh diklon ke vektor kloning pJETclone dan digunakan untuk mentransformasi Escherichia coli JM109. Sintesis cDNA dilakukan dengan mentranskripsi balik genom RNA menjadi DNA yang selanjutnya digunakan sebagai templat untuk amplifikasi ORF pengkode integrase dengan teknik reaksi polimerisasi berantai atau Polymerase Chain Reaction (PCR). Produk PCR selanjutnya disisipkan kedalam plasmid menggunakan sistem kloning blunt-ended dan dikarakterisasi dengan elektroforesis DNA dan analisis nukleotida. Analisis dengan elektroforesis DNA menunjukkan bahwa produk PCR berukuran sesuai dengan ukuran teoritis. Karakterisasi lebih lanjut dengan teknik analisis nukleotida menunjukkan produk PCR tersebut homolog dengan integrase HIV-1 dari Indonesia. Teknik PCR juga dilakukan terhadap klon, dan adanya produk PCR berukuran sesuai dengan ukuran teoritis menunjukkan adanya DNA sisipan. Analisis nukleotida dilakukan pada plasmid rekombinan murni dan DNA terbaca dengan baik.Keywords: HIV-1,Integrase, E.coli JM109
Teknik Long Polymerase Chain Reaction (LPCR) Untuk Perbanyakan Kerangka Baca Terbuka Gen Pengkode Polimerase Virus Hepatitis B Hutapea, Hotma; Retnoningrum, Debbie; Rahman, Ernawati Giri; Rostinawati, Tina
JURNAL PLASMA Vol 1, No 2 Jun (2015)
Publisher : Balai Penelitian dan Pengembangan Biomedis Papua

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Abstract

Teknik PCR dapat dikembangkan untuk mengamplifikasi potongan DNA dengan ukuran panjang. Salah satu teknik yang digunakan adalah Long PCR (LPCR). Teknik LPCR sering digunakan untuk mengamplifikasi gen atau potongan DNA yang berukuran lebih panjang ketika PCR standar tidak dapat diaplikasikan untuk amplifikasi tersebut. Tujuan dari penelitian ini adalah untuk memperoleh kerangka baca terbuka gen pengkode polimerase virus hepatitis B (PolHBV) menggunakan metode LPCR, dan menentukan urutan nukleotida potongan DNA tersebut. Amplifikasi dilakukan menggunakan Taq polimerase. Kerangka baca terbuka gen pengkode PolHBV telah berhasil diamplifikasi dengan LPCR yang telah dimodifikasi. Hal tersebut diindikasikan dengan keberadaan pita DNA berukuran  pasangan basa (bp) pada elektroforesis gel agarosa yang mendekati ukuran teoritisnya yaitu 2532 bp. Hasil penentuan urutan nukleotida parsial menunjukkan bahwa potongan DNA yang diperoleh memiliki homologi 98% dengan gen PolHBV yang dideposit di GenBank.Technique of PCR can be improved to permit the amplification of longer DNA fragment. One of this technique is Long PCR (LPCR). LPCR is often used to amplify larger genes or large segment of DNA which standard PCR is not applicable. The objective of this study is to obtain the open reading frame of gene encoding polymerase of HBV (polHBV) using LPCR method, and to determine the nucleotide sequence of DNA fragment encoding polHBV. The amplification was conducted using Taq polymerase. The open reading frame of gene encoding polHBV was successfully amplified using modified LPCR method. It  was  identified by  a  band  between  2000 dan  3000  base  pairs  (bp) DNA marker.  The determination of nucleotide sequence informed that DNA fragment obtained was 98% homologue to the gene encoding PolHBV deposited on GenBank. .
Hubungan Jumlah Cluster of Differentiation 4 (CD4) dengan Infeksi Oportunistik Pada Pasien HIV/AIDS di Rumah Sakit Umum Daerah (RSUD) DOK II Jayapura Widiyanti, Mirna; Hutapea, Hotma
JURNAL BIOLOGI PAPUA Vol 7, No 1 (2015)
Publisher : JURNAL BIOLOGI PAPUA

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Abstract

Human Immunodeficiency Virus (HIV) is an infection that attacks and weakens the immune system. HIV infection causes a decrease in the number of Cluster Differentiation 4 (CD4) thereby increasing the progression of the disease and lead to high risk of opportunistic infections (OI). The purpose of this study was to examine the relationship between CD4 cell count with opportunistic infections in patients infected with HIV/AIDS. Analytical research methods using cross-sectional design, by taking medical records. The population in this study were 67 patients with HIV/AIDS in the VCT Clinic Dok II Hospital Jayapura 2014. Data were processed with the Chi Square test hypotheses. Based on the results of hypothesis testing of 67 patients, there were 21 people have opportunistic infections. Tuberculosis is an opportunistic infection that is most common (17.9%). Significance of the relationship seen in the low CD4 counts (< 350 cells/mm3) and found value of 0.02 (CI 95%) which indicates that there is a relationship if p
GAMBARAN KASUS MUTASI TERKAIT RESISTENSI ANTIRETROVIRAL PADA ORANG DENGAN HIV-AIDS (ODHA) DI TIGA KABUPATEN/KOTA DI PROVINSI PAPUA Hutapea, Hotma
Buletin Penelitian Kesehatan Vol 46 No 3 (2018)
Publisher : Sekretariat Badan Penelitian dan Pengembangan Kesehatan

Show Abstract | Download Original | Original Source | Check in Google Scholar | Full PDF (324.692 KB) | DOI: 10.22435/bpk.v46i3.902

Abstract

Antiretroviral (ARV) therapy has been found effective to decrease HIV-1 infected cases, however increase the resistances due to nucleotide mutation. The mutation causes virus to be resistant to ARV, making the therapy is no longer effective. The ARV therapy District Nabire, Jayapura, and Jayawijaya was Reverse Transcriptase Inhibitor (RTI). The goal of this study was to obtain the data of mutation associated to viral resistance to ARV RTI and protease inhibitor group. Plasma samples were obtained from 252 subjects purposively in HIV/AIDS population from corresponding Care, Support, and Therapy clinic. The genotyping, measurement of CD4 and viral load were performed to all samples. DNA analysis was performed by genotyping method. Among 252 samples, 89 samples (35.32%) had CD4 count &lt;350 sel/ul. Twenty three samples (8.73%) had viral load &gt;10.000 copies/mL. There were 15 (5,95%) samples were identified as mutant related to RTI resistance, and none for protease. The most frequent mutation motive was M184V/I, 12 samples (80,00%). This data provided important information about the continuous need of ARV therapy monitoring to suppress transmission drug resistance. &nbsp; &nbsp; Abstrak &nbsp; Penemuan antiretroviral (ARV) secara signifikan telah menjadi bagian penting dalam penanggulangan HIV/AIDS. Terapi ARV dilakukan untuk menurunkan kasus HIV-1, namun dapat menyebabkan resistensi virus terhadap ARV tersebut. Data prevalensi HIV-1 resisten ARV pada Orang Dengan HIV/AIDS (ODHA) di Papua, khususnya Kabupaten Nabire, Kab./Kota Jayapura, dan Kab. Jayawijaya belum tersedia. Tujuan penelitian ini adalah untuk mendapatkan data prevalensi mutasi terkait resistensi virus terhadap ARV golongan penghambat rtase dan protease. Sebanyak 84 responden yang sudah diterapi minimal 6 bulan diambil secara purposive dari setiap lokasi penelitian. Genotyping, pemeriksaan nilai CD4 dan viral load dilakukan terhadap semua sampel. Analisis DNA dilakukan dengan metode genotyping. Dari 252 sampel, sebanyak 89 responden (35,32%) memiliki nilai CD4 &lt;350 sel/ul darah. Sebanyak 23 responden (8,73%) memiliki nilai viral load &gt;10.000 salinan/mL. Sebanyak 15 sampel (5,95%) teridentifikasi mengalami mutasi pada DNA target ARV golongan penghambat rtase, dan tidak ditemukan mutasi terkait resistensi pada gen protease. Motif mutasi yang paling banyak adalah M184V/I, yaitu sebanyak 12 sampel (80,00%). Penelitian ini mengindikasikan bahwa pemantauan terapi ARV secara berkesinambungan tetap diperlukan untuk menekan penulan HIV resisten ARV. &nbsp;