Andi Yasmon
Department of Microbiology, Faculty of Medicine, Universitas Indonesia, Jakarta

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Applicability of an oligonucleotide probe in radioisotope 32P-based dot blot hybridization for detection of hepatitis C virus in large sample numbers: a preliminary study Rosilawati, Maria L.; Yasmon, Andi
Medical Journal of Indonesia Vol 21, No 2 (2012): May
Publisher : Faculty of Medicine Universitas Indonesia

Show Abstract | Download Original | Original Source | Check in Google Scholar | Full PDF (597.065 KB) | DOI: 10.13181/mji.v21i2.480

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Background: This study aimed to design and analyze the applicability of an oligonucleotide probe in radioisotope 32P-based dot blot hybridization for detection of hepatitis C virus.Methods: Forty-six of plasma samples were used. The plasma was extracted to obtain viral RNA genome as template for RT-PCR and the amplicon was used for nested PCR. Twenty-four HCV genomes were retrieved from GeneBank DNA sequence and alignment was performed by Bio Edit Software version 7.0.9.0. An oligonucleotide probe was designed based on a highly conserved region that is located on internal sequence between two primers used for nested PCR. Blast analysis on GeneBank was performed to obtain homology of the oligonucleotide for HCV. The oligonucleotide was then labeled with 32P and dot blot hybridization was applied for nested PCR products. DNA Sequencing was performed to confirm the amplicon and dot blot hybridization results.Results: Blast analysis showed high homology (100%) for HCV. Nested PCR resulted in three patterns of DNA fragments representing HCV genotypes 1, 2, and 3, respectively. The primers used in nested PCR were not specific and resulted in DNA fragments difficult to be interpreted. Dot blot hybridization using the designed oligonucleotide showed high intensity dots. All nested PCR fragments showed the dot blot positive. The dot blot results were in accordance with DNA sequencing that confirmed three patterns of DNA fragments as different HCV genotypes.Conclusion: The oligonucleotide showed excellent bioinformatically criteria. 32P-based dot blot hybridization yielded high intensity dots and was easier to be interpreted than nested PCR assay. (Med J Indones. 2012;21:71-6)Keywords: Dot blot hybridization, hepatitis C virus, oligonucleotide, radioisotope
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

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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
Development of multiplex-PCR assay for rapid detection of Candida spp. Tarini, Ni Made A.; Wahid, Mardiastuti H.; Ibrahim, Fera; Yasmon, Andi; Djauzi, Samsuridjal
Medical Journal of Indonesia Vol 19, No 2 (2010): May
Publisher : Faculty of Medicine Universitas Indonesia

Show Abstract | Download Original | Original Source | Check in Google Scholar | Full PDF (108.411 KB) | DOI: 10.13181/mji.v19i2.387

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Aim Candida spp. infection commonly occur in immunocompromised patients. Biochemical assay for identification of Candida spp. is time-consuming and shows many undetermined results. Specific detection for antibody, antigen and metabolites of Candida spp. had low sensitivity and specificity. In this study, we developed a rapid diagnostic method, Multiplex-PCR, to identify Candida spp.Methods Five Candida spp. isolates were cultured, identifi ed with germ tube and API® 20 C AUX (BioMerieux® SA) kit. Furthermore, DNA was purified by QIAamp DNA mini (Qiagen®) kit for Multiplex-PCR assay.Results DNA detection limit by Multiplex-PCR assays for C. albicans, C. tropicalis, C. parapsilosis, C. krusei and C. glabrata were 4 pg, 0.98 pg, 0.98 pg, 0.5 pg and 16 pg respectively. This assay was also more sensitive than culture in that Multiplex-PCR could detect 2.6-2.9 x 100 CFU/ml, whereas culture 2.6-2.9 x 102 CFU/ml.Conclusion Multiplex-PCR is much more sensitive than culture and thus, can be recommended as a sensitive and specific assay for identification of Candida spp. (Med J Indones 2010; 19:83-7)Keywords: Candida spp., multiplex-PCR
Optimizing real-time PCR method to detect Leptospira spp. in human blood and urine specimens Karuniawati, Anis; Yasmon, Andi; Ningsih, Ika
Medical Journal of Indonesia Vol 21, No 1 (2012): February
Publisher : Faculty of Medicine Universitas Indonesia

Show Abstract | Download Original | Original Source | Check in Google Scholar | Full PDF (925.644 KB) | DOI: 10.13181/mji.v21i1.472

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Background: Leptospirosis is an acute infectious disease in humans caused by Leptospira spp. and classified as a zoonosis. Clinical symptoms of leptospirosis are nonspecific and the current available laboratory method for detecting Leptospira spp. is difficult, which resulted to the misdiagnosis of this disease. Therefore, the rapid and accurate method is needed to diagnose the disease. This study was aimed to optimize molecular diagnostic test using real-time PCR assay as a rapid, sensitive and specific method for the detection of pathogenic Leptospira spp. in humans.Methods: Bacterial DNA was extracted by DNA extraction kit according to the manufacturer’s instructions. Primers and probes used in this study was based on previous and published research. The assay is performed using PCR-IQTM5, iCycler Multicolor real-time PCR detection system. Specificity of the primer used was evaluated towards some bacterial pathogens.Results: Limit detection of the DNA was 0.375 fg/ml and the primers used does not cross-react with the genomes of the pathogens tested. Limit detection of DNA in blood is 150 fg/μl, and in urine is 1470 fg/μl.Conclusion: Real-time PCR test is a rapid and accurate method for detecting pathogenic Leptospira spp. in human specimens. Further research is needed to determine the sensitivity and specificity of real-time PCR tests compared with other diagnostic methods in clinical settings. (Med J Indones 2012;21:13-7)Keywords: Leptospirosis, Leptospira, optimization, real-time PCR
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

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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
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

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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
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

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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.
Multiplex nested polymerase chain reaction for Treponema pallidum using blood is more sensitive than using serum Effendi, Ida; Rosana, Yeva; Yasmon, Andi; Indriatmi, Wresti
Universa Medicina Vol 37, No 1 (2018)
Publisher : Faculty of Medicine, Trisakti University

Show Abstract | Download Original | Original Source | Check in Google Scholar | DOI: 10.18051/UnivMed.2018.v37.75-84

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BackgroundSyphilis is a multistage disease transmitted primarily through sexual intercourse. Nowadays, the polymerase chain reaction (PCR) test for Treponema pallidum has been widely used and is expected to overcome problems in diagnostic tests for syphilis. The Treponema pallidum PCR is influenced by type of specimens, PCR methods and target genes. This study aimed to assess the use of blood and serum in multiplex nested PCR for Treponema pallidum, targeting the 23S rRNA.MethodsA cross-sectional study was conducted from April 2015 - April 2016. Sampling was carried out consecutively among patients with clinical features of secondary syphilis who came to Sexually Transmitted Disease (STD) clinics in Jakarta. All sera were also tested with Rapid Plasma Reagin (RPR) and Treponema pallidum Hemagglutination Assay (TPHA) assay, which was considered as the gold standard for this study. We determined the sensitivity and specificity of the multiplex nested PCR for Treponema pallidum using blood and serum.ResultsPCR test was performed on 122 clinical specimens (61 blood and 61 serum). The positive results of PCR test on blood was 22.95% and serum was 6.56%, while the positive results of serology was 68.85%. The sensitivity of Treponema pallidum multiplex nested PCR on blood was 30.95% compared to serum 9.52% (p=0.006). PCR test on blood is able to detect 3.25 times higher than serum. ConclusionThe use of blood has a higher proportion of positives compared to serum in Treponema pallidum multiplex nested PCR using 23S rRNA target gene.
Detection and identification of azithromycin resistance mutations on Treponema pallidum 23S rRNA gene by nested multiplex polymerase chain reaction Gultom, Desy A.; Rosana, Yeva; Efendi, Ida; Indriatmi, Wresti; Yasmon, Andi
Medical Journal of Indonesia Vol 26, No 2 (2017): June
Publisher : Faculty of Medicine Universitas Indonesia

Show Abstract | Download Original | Original Source | Check in Google Scholar | Full PDF (551.425 KB) | DOI: 10.13181/mji.v26i2.1543

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Background: Azithromycin-resistant strains of Treponema pallidum is associated with the mutation of 23S rRNA gene of T. pallidum. Although these strains are now prevalent in many countries, there is no laboratory test kit to detect and identify these mutations. Thus, in this study we developed a nested multiplex polymerase chain reaction (PCR) to detect and identify A2058G and A2059G mutations in 23S rRNA gene.Methods: Three primer sets were designed for nested PCR reactions. To obtain maximum PCR reaction, all parameters were optimized. The specificity of the primer sets was evaluated towards some microorganisms. A sensitivity test was conducted to get detection limit of deoxyribonucleic acid (DNA). Forty-five whole blood specimens were tested by PCR, and positive results were confirmed by the DNA sequencing.Results: The assay could detect at least 4,400 DNA copy number and showed no cross reaction with other microorganisms used in the specificity test. A total 13 of 45 whole blood specimens were PCR positive for T. pallidum, and no single mutations (either A2058G or A2059G) were detected. Two positive specimens were confirmed by the DNA sequencing and showed no mutation.Conclusion: Nested multiplex PCR developed in this study showed a specific and sensitive test for the detection and identification of A2058G and/or A2059G mutations of 23S rRNA T. pallidum gene.
DETEKSI GEN MSG UNTUK PENINGKATKAN KETEPATAN DIAGNOSIS PNEUMOCYSTIS JIROVECII PADA PASIEN HIV DENGAN PNEUMONIA Tjampakasari, Conny Riana; Yasmon, Andi; Sudarmo, Fitrahwati
Indonesian Journal of Biotechnology and Biodiversity Vol 2, No 1 (2018): Indonesian Journal of Biotechnology and Biodiversity
Publisher : Universitas Esa Unggul

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Abstract

AbstractPneumocystis jirovecii is the cause of opportunistic infections in the lower respiratory tract of immunocompromised patients, especiallyin human immunodeficiencyvirus (HIV). Until now the case of P. jirovecii in Indonesia is not known with certainty because the data obtained only based on patient’s clinical condition. Culture is still on going research while microscopic as a gold standard has some limitation, among others, takes a long time in the process, requires special kills to work and interpret results. The diagnosis of P.jirovecii infection in Indonesia is still based on clinical and microscopic examination, which takes a long time, is less sensitive and specific. Based on this reason then developed rPCR that  more sensitive and specific. In addtionthe results obtained are less sensitive and spesific. Based on that, in this study developed real-time PCR (rPCR) molecular test that more sensitive and apesific using gene MajorSurface Glicoprotein (MSG). MSG is detection target of its presence in the fungus cell wall is very abundant and has a sustainable sequence. The rPCR was successfully optimized with a minimum DNA detection capability of 6.55 cop / μl and did not cross-react with other microorganisms tested in this study. Obtauned 50 clinical sampels consisting of sputum and sputum induction. The result of comparison between microscopic test and rPCR show that rPCR increase positive results up to 20%. The rPCR test can detect P.jirovecii on clinical samples of sputum and induced sputum from HIV patients with pneumonia with CD4 +> 200 or ≤ 200 cells.  Keywords : real time PCR, HIV, pneumocystis jirovecii. AbstrakPneumocystisjiroveciiadalah penyebab infeksi oportunistik di saluran pernapasan bawah pada individu dengan sistem kekebalan tubuh yang lemah, terutama pada pasien HIV. Pemeriksaan infeksi P. jirovecii di Indonesia masih berdasarkan pemeriksaan klinis dan mikroskopis, yang memerlukan waktu yang cukup lama, kurang sensitif dan spesifik. Berdasarkan hal tersebut maka dalam penelitian ini dikembangkan uji molekuler real time PCR (rPCR) yang lebih sensitif dan spesifik. Uji rPCR telah berhasil dioptimasi dengan kemampuan deteksi minimum DNA 6,55 copy/μl dan tidak bereaksi silang dengan mikroorganisme yang diuji pada penelitian ini. Dibandingkan dengan uji mikroskopis, uji rPCR memberikan hasil positif 20% lebih tinggi daripada uji mikroskopis. Uji rPCR dapat mendeteksi P.jiroveciipada sampel klinis sputum dan sputum induksi dari pasien HIV dengan pneumonia denganjumlah sel CD4+> 200 maupun ≤ 200. Oleh karena itu, uji rPCR yang telah dioptimasi dalam studi ini dapat mendeteksi P.jirovecii pada sampel klinis sputum dan sputum induksi dari pasien HIV dengan pneumonia dengan jumlah sel CD4+> 200 maupun ≤ 200. Kata Kunci : real time PCR, HIV, pneumocystis jirovecii.