Budiman Bela
Institute of Human Virology and Cancer Biology, Faculty of Medicine, Universitas Indonesia, Jakarta

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In vitro transcription of HIV-1 RNA for standard RNA Yasmon, Andi; Bela, Budiman; Ibrahim, Fera; Syahruddin, Elisna
Medical Journal of Indonesia Vol 20, No 3 (2011): August
Publisher : Faculty of Medicine Universitas Indonesia

Show Abstract | Download Original | Original Source | Check in Google Scholar | Full PDF (119.131 KB) | DOI: 10.13181/mji.v20i3.447

Abstract

Background: The quantitative assays are important tests in the management of patients with HIV-1/AIDS. The important step in developing the assay is the availability of the standard HIV-1 RNA. For this purpose, we optimized in vitro HIV-1 RNA transcription to produce the standard HIV-1 RNA.Methods: The HIV-1 DNA was amplified from pNL43 by PCR using a primer pair that was specific for conserved region of HIV-1 Gag gene. The PCR product was further cloned into pBluescript II KS. The recombinant plasmid was restricted with EcoRI enzyme. Then, the linearized plasmid was used as template for RNA transcription. RT-PCR and PCR were performed simultaneously for confirmation of synthesized RNA fragment.Results: A 115 bp DNA of HIV-1 Gag gene has been cloned into pBluescript II SK with the exact true orientation. The reaction of the RNA transcription was also successfully performed. The RNA transcripts have been confirmed and showed the accuracy of the transcripts.Conclusion: We successfuly constructed the recombinant plasmid containing a conserved region of HIV-1 Gag gene, and the HIV-1 RNA has been transcribed in vitro as well. (Med J Indones. 2011; 20:185-9)Keywords: HIV-1/AIDS, Quantitative assay, RNA transcription
A second generation of RT-PCR assay for detection of human immunodeficiency virus type 1 (HIV-1) infection Yasmon, Andi; Fatmawati, Ni N.D.; Ibrahim, Fera; Bela, Budiman
Medical Journal of Indonesia Vol 19, No 3 (2010): August
Publisher : Faculty of Medicine Universitas Indonesia

Show Abstract | Download Original | Original Source | Check in Google Scholar | Full PDF (120.308 KB) | DOI: 10.13181/mji.v19i3.397

Abstract

Aim A spesific and rapid diagnosis such as RT-PCR assay is the most needed to minimize transmission of HIV-1 infection. Therefore, in this study we developed the RT-PCR assay that was spesific against the gag gene of HIV-1.Methods The developed RT-PCR assay was evaluated against 46 specimens that were obtained from voluntary counseling and testing for HIV (VCT) in Rumah Sakit Umum Pemerintah (RSUP) Sanglah, Bali. To get the sensitivity and specificity of RT-PCR assay, the results of assays were compared with the results of commercially serologic teststhat were commonly used in Indonesia.Results The RT-PCR assay could detect 21 of 26 serologic test-positive specimens and showed 19 negative results of 20 serologic test-negative specimens. There was one specimen that was positive in RT-PCR but negative in serologic assay, which might depict a true yield at particular condition when the serologic assay was unable to detect. Five serologic positive-test specimens were negative by RT-PCR that was possibly caused by low detection level of the assay.Conclusion The RT-PCR assay is potential to be used for the detection of HIV-1 infection with a sensitivity and specificity of 80.8% and 95.0% respectively. (Med J Indones 2010;19:154-7)Key words: AIDS, diagnosis, PoL, sensitivity, specificity
Optimizations of expression and purification of recombinant HIV-1 CRF01_AE p24 protein in Escherichia coli for development of immunodiagnostic assay Simaremare, Ade P.R.; Bela, Budiman; Yasmon, Andi; Ibrahim, Fera
Medical Journal of Indonesia Vol 24, No 1 (2015): March
Publisher : Faculty of Medicine Universitas Indonesia

Show Abstract | Download Original | Original Source | Check in Google Scholar | Full PDF (1028.215 KB) | DOI: 10.13181/mji.v24i1.1166

Abstract

Background: Conventional method for confirmation of HIV infection is Western blot. However, it has limitations because of contamination by human cellular antigen and genetic diversity among the HIV-1 subtypes that show indeterminate result and inaccuracy for the diagnosis of different strains. Most of Western blot developed are based on HIV-1 B subtype. In Indonesia HIV-1 CRF01_AE subtype is dominantly circulated. Therefore, we optimized the expression, purification of the recombinant HIV-1 CRF01_AE p24 protein for development of immunodiagnostic assay.Methods: Optimization of protein expression in Escherichia coli strain BL21CP was performed including induction time, isopropyl-1-thio-d-galactopyranoside (IPTG) and immidazole consentrations, and induction temperature. Purification of the recombinant p24 protein was used by using Ni-NTA (Qiagen) purification system in native condition. Results: Expression and purification of HIV-1 CRF01_AE p24 protein have been performed. Confirmation of the recombinant protein by Western blot showed the expression and purification of recombinant p24 protein has been optimized well and reactive with sera of patients with HIV-1 CRF01_AE subtype positive.Conclusion: The recombinant HIV-1 CRF01_AE p24 protein has been expressed and purified successfully, and it is potential to be used as antigen for immunodiagnostic assay.
Simultaneous detection of Legionella species and Legionella pneumophila by duplex PCR (dPCR) assay in cooling tower water samples from Jakarta, Indonesia Yasmon, Andi; Yusmaniar, Yusmaniar; Anis, Anis; Bela, Budiman
Medical Journal of Indonesia Vol 19, No 4 (2010): November
Publisher : Faculty of Medicine Universitas Indonesia

Show Abstract | Download Original | Original Source | Check in Google Scholar | Full PDF (158.177 KB) | DOI: 10.13181/mji.v19i4.408

Abstract

Aim: Since culture method is time-consuming and has low  sensitivity, we developed a duplex PCR (dPCR) assay for the detection of Legionella sp. and L. pneumophila in cooling tower samples. We used culture method as a gold standard.Methods: Optimization of dPCR method was performed to obtain an assay with high sensitivity and specifi city. The optimized method was used to detect Legionella sp. dan L. pneumophila in 9 samples obtained from 9 buildings in Jakarta. For culture method, the bacteria were grown or isolated on selective growth factor supplemented-buffered charcoal yeast extract (BCYE) media.Results: Of 9 samples tested by dPCR assay, 6 were positive for Legionella species,1 was positive for L. pneumophila, and 2 showed negative results. For the same samples, no Legionella sp. was detected by the culture method.Conclusion: dPCR assay was much more sensitive than the culture method and was potentially used as a rapid, specifi c and sensitive test for routine detection of Legionella sp. dan for L. pneumophila in water samples. (Med J Indones 2010; 19:223-7)Keywords: BCYE media, mip gene, 16S-rRNA gene
Teknik reverse transcription – polymerase chain reaction (RT-PCR) dan hibridisasi dot blot dengan pelacak DNA untuk deteksi human immunodeficiency virus (HIV) dalam serum darah Rosilawati, Maria Lina; Bela, Budiman
Universa Medicina Vol 26, No 3 (2007)
Publisher : Faculty of Medicine, Trisakti University

Show Abstract | Download Original | Original Source | Check in Google Scholar | DOI: 10.18051/UnivMed.2007.v26.111-119

Abstract

LATAR BELAKANGTeknik biologi molekuler seperti teknik reverse transcription – polymerase chain reaction (RT-PCR) dot blot hybridization dengan pelacak DNA berlabel biotin dapat mendeteksi human immunodeficiency virus (HIV) dalam serum darah. Teknik ini selanjutnya dapat diterapkan untuk skrining HIV donor jaringan biologi terutama dari Bank Jaringan Riset Badan Tenaga Nuklir Nasional (BATAN), seperti amnion, allograft steril radiasi, melalui darahnya. Penelitian ini bertujuan untuk membandingkan sensitivitas metode RT-PCR hibridisasi dot blot dengan pelacak DNA berlabel biotin dan RT-PCR elektroforesis gel agarosa untuk deteksi HIV.METODEPenelitian ini menggunakan serum darah dari Rumah Sakit Ketergantungan Obat (RSKO) Fatmawati. Jumlah serum yang dipakai sebanyak 55 sampel terdiri dari 5 sampel negatif HIV hasil uji serologi dengan rapid test dan 50 sampel dengan enzyme linked immunoassay (ELISA). Ekstraksi RNA HIV sampel darah dilaksanakan menggunakan kit RNA viral extraction sedangkan teknik one step RT-PCR digunakan untuk amplifikasi DNA. HASILHasil penelitian menunjukkan pada 55 sampel yang diuji baik dengan teknik RT-PCR elektroforesis gel agarosa maupun RT-PCR hibridisasi dot blot, 43 sampel positif mengandung HIV. Hasil RT-PCR hibridisasi dot blot jauh lebih jelas dibanding dengan RT-PCR-elektroforesis gel agarosa. Hal ini terlihat munculnya dot hitam tebal pada film sedangkan pada gel agarosa pita DNA tampak tipis untuk beberapa sampel positif HIV yang sama KESIMPULANTeknik RT-PCR hibridsasi dot blot dengan pelacak DNA berlabel biotin lebih sensitif dibanding dengan RT-PCR elektroforesis gel agarosa untuk mendeteksi HIV.
Antiproliferative activity and caspase enhancement properties of Annona muricata leaves extract against colorectal cancer cells Indrawati, Lili; Ascobat, Purwantyastuti; Bela, Budiman; Abdullah, Murdani; Surono, Ingrid S.; Pramono, Suwijiyo
Medical Journal of Indonesia Vol 25, No 3 (2016): September
Publisher : Faculty of Medicine Universitas Indonesia

Show Abstract | Download Original | Original Source | Check in Google Scholar | Full PDF (498.684 KB) | DOI: 10.13181/mji.v25i3.1449

Abstract

Background: The prevalence of colorectal cancer is rising in Asia including Indonesia. Annona muricata tea leaves, that is traditionally used for maintaining health, and lately being used by cancer patients. The objectives of this study is to investigate its effects in human colorectal cancer cell in vitro and ex vivo.Methods: Thirty patients with colorectal cancer (CRC) were enrolled in a randomized double-blind placebo-controlled trial. They were equally divided into two groups: those treated with 300 mg A. muricata leaf extract and placebo daily for 8 weeks. Serum from supplemented CRC patients of both groups was compared for caspase 9 and caspase 8 enhancement activity. Antiproliferative effect of water extract of A. muricata leaves and its fractions were evaluated against colorectal cancer cell line (DLD-1 and COLO 205) compared with 5-fluorouracil and placebo, the dose range was 62.5-2,000 µg/mL. Method used was 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Data were analyzed by Mann-Whitney U test. The p value was set at 0.05.Results: Ethanol-soluble fraction of A. muricata leaves extract water extract (ESFAM) leaves extract had cytotoxicity effects on DLD-1 as well as COLO 205 cell line, as shown by the lower IC50 compared to 5-fluorouracil and placebo, 20.59 μg/mL and 654.9μg/mL, respectively. Serum of subjects supplemented with extract significantly induced caspase 9 (p=0.001) activity of DLD-1 colorectal cancer cell line, but not for caspase 8 activity (p=0.372).Conclusion: The study's results suggest the cytotoxicity potential of  A. muricata  leaves extract  in in vitro and ex vivo studies.