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Genotyping of Rotavirus by Using RT-PCR Methods Nirwati, Hera; Wibawa, Tri; Aman, Abu Tholib; Soenarto, Yati
Indonesian Journal of Biotechnology Vol 18, No 1 (2013)
Publisher : Universitas Gadjah Mada

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There is a great diversity of rotavirus genotypes circulating worldwide, with dominant genotypes changing from year to year. Rotavirus genotyping was performed by using reverse transcription PCR with type-specifi c-primers. Since rotavirus is a RNA virus that has high mutation rate, there was a possibility of technical diffi culty in genotyping due to mutation in the primer binding sites. During Indonesian rotavirus surveillance study 2006-2009, it was reported that 17% of samples subjected for G type and 21% of samplessubjected for P type were untypeable. The objective of this study was to identify genotypes of the samples that were untypeable previously using RT-PCR based on the method described by Das et al. (1994) and Gentsch et al. (1992). There were 30 samples subjected to G type and 61 samples subjected to P type to be re-typed using method described by Gouvea et al. (1990) and Simmond et al. (2008) for G and P typing, respectively. By using another set of primer, the genotype of all samples was identifi ed. This study highlights the importance of a constant reconsideration of primer sequences employed for the molecular typing of rotaviruses.Key words: rotavirus, G typing, P typing

Apoptosis and Phagocytosis Activity of Macrophages Infected by Mycobacterium tuberculosis Resistant and Sensitive Isoniazid Clinical Isolates Rachmawaty, Farida J.; Wibawa, Tri; Soesatyo, Marsetyawan H.N.E.
Indonesian Journal of Biotechnology Vol 11, No 2 (2006)
Publisher : Universitas Gadjah Mada

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Mycobacterium tuberculosis (M.tb) is the main causative pathogen that cause the pulmonary tuberculosis.Intracellular M.tb was reported able to induce macrophages apoptosis, which may have crucial role in the regulationof immun response against M.tb infection. As an intracellular bacteria, M.tb able to live and replicate withinmacrophages. Phagocytosis is the first step to achieved this condition. The induction of macrophages apoptosis byINH resistant and sensitive M.tb clinical isolates, and H37Rv was studied. The macrophages apoptosis level weremeasured using an Ag-capture ELISA for histone and fragmented DNA (Cell Death Detection ELISAplus, RocheDiagnostic GmBH). Phagocytosis activity also analyzed, after staining using fluorescence dye (AcriFluorTM, ScientificDevice Lab.). The results showed that there was no significantly different between INH resistant and sensitive M.tbclinical isolates in respect their ability to induce apoptosis. The phagocytosis activity among the clinical isolates wasshown to be strain dependent, and undistinguishable between the Mtb clinical isolates. There was no associationbetween macrophages apoptosis level and the phagocytosis activity. These data suggested that among the virulentMtb clinical isolates, the ability to induce macrophages apoptosis and phagocytosis were consistently in comparablelevelKeywords: Mycobacterium tuberculosis, apoptosis, phagocytosis, macrophages, isoniazid

Early Detection and Serotyping of Dengue Viruses Clinical Isolates Using Reverse Transcription Polymerase Chain Reaction (RT-PCR) 2 Primers Siregar, Abdul Rahman; Wibawa, Tri; Wijayanti, Nastiti
Indonesian Journal of Biotechnology Vol 16, No 2 (2011)
Publisher : Universitas Gadjah Mada

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Recently several methods for confirming Dengue Virus have been developed involve virus isolation, detection of virus antigen, and nucleic acid using PCR. It has been reported that rapid detection method for confirming DHF by Multiplex RT-PCR had been successfully developed. It was more effective than the other methods with a high sensitivity and specivicity were 100% at the early phase (day 1-3). This study was designed to develop rapid detection and serotyping methods for Dengue Virus using RT-PCR 2 primers (Dcon and preM) with annealing temperature was 57oC. The whole blood samples were collected from suspected dengue fever patients that had been confirmed with NS1 kit from R.S. Persahabatan DKI Jakarta and R.S. Prof. Dr. Sardjito DI Yogyakarta during Februari-August 2009. The PCR products showed that in 12 samples, 100 % were postitive with different pattern among the serotypes especially for DEN1 and DEN2, but not for DEN3 and Den4.  This method was also able to confirm the double infection DEN2-DEN3, but not for the other ones because of the unspecific pattern. From the results, it indicated that the 2 primers can be a promising early detection and serotyping method of Dengue Virus which infected the DHF patients. Key words: Dengue Virus, DHF, early detection, serotyping, RT-PCR 2 primers.

Effect of Oxidative Stress on AhpC Activity and Virulence in katG Ser315 Thr Mycobacterium tuberculosis Mutant Rintiswati, Ning; Wibawa, Tri; Asmara, Widya; Soebono, Hardyanto
Indonesian Journal of Biotechnology Vol 16, No 2 (2011)
Publisher : Universitas Gadjah Mada

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AbstractMycobacterium tuberculosis strains resistance to INH is mainly caused by the alteration in several genesencoding the molecular targets. Mutation of katG at codon 315 especially Ser315Thr are responsible forINH resistance in a large proportion of TB cases. The aim of this study is to evaluate the influence of stressoxidative on AhpC activity of katG Ser315Thr of M.tuberculosis, and to find out the relation of AhpC and thevirulence of this mutant. The study design was laboratoric experimental, subjects of study were M.tuberculosisINH resistance strains, and the treatment were serial dose of H2O2. Eighty five M.tuberculosis INH resistantclinical strain were screened for mutation of katGSer315Thr by PCR/RFLP and characterized on the basis ofphenotypic properties (catalase activity and AhpC activity). AhpC activity of katG Ser315Thr M.tuberculosisstrains in response to oxidative stress condition was evaluated by culturing the strains on liquid culturemedium containing 1mM H2O2. To ascertain role of AhpC in the virulence of katGSer315Thr mutant strains, themutants were infected into human macrophages culture, and several indicator of virulence were observed (i.e:replication competence, and apoptosis induction on human macrophages). The results showed that katG Ser315Thr were identified in 23 (27,05%) of 85 INH resistance strains, all mutant strains had decrease of catalaseactivity. AhpC activity of katG Ser315Thr of M.tuberculosis increased significantly with increase of hydrogenperoxide dose. In addition , it has been shown that increased AhpC activity related to replication ability ofmutant, and reduction of apoptosis macrophages induction significantly. We conclude that the productionof AhpC of katG Ser315Thr M.tuberculosis induced by oxidative stress. There was a role of AhpC in virulenceof the M.tuberculosis katG Ser315Thr strains by replication capability and macrophages apoptosis.Keywords : katG Ser315Thr Mycobacterium tuberculosis- oxidative stress - AhpC - virulence

Apoptosis and Phagocytosis Activity of Macrophages Infected by Mycobacterium tuberculosis Resistant and Sensitive Isoniazid Clinical Isolates Rachmawaty, Farida J.; Wibawa, Tri; Soesatyo, Marsetyawan H.N.E.
Indonesian Journal of Biotechnology Vol 11, No 1 (2006)
Publisher : Universitas Gadjah Mada

Show Abstract | Download Original | Original Source | Check in Google Scholar | Full PDF (43.035 KB)

Mycobacterium tuberculosis (M.tb) is the main causative pathogen that cause the pulmonary tuberculosis.Intracellular M.tb was reported able to induce macrophages apoptosis, which may have crucial role in the regulationof immun response against M.tb infection. As an intracellular bacteria, M.tb able to live and replicate withinmacrophages. Phagocytosis is the first step to achieved this condition. The induction of macrophages apoptosis byINH resistant and sensitive M.tb clinical isolates, and H37Rv was studied. The macrophages apoptosis level weremeasured using an Ag-capture ELISA for histone and fragmented DNA (Cell Death Detection ELISAplus, RocheDiagnostic GmBH). Phagocytosis activity also analyzed, after staining using fluorescence dye (AcriFluorTM, ScientificDevice Lab.). The results showed that there was no significantly different between INH resistant and sensitive M.tbclinical isolates in respect their ability to induce apoptosis. The phagocytosis activity among the clinical isolates wasshown to be strain dependent, and undistinguishable between the Mtb clinical isolates. There was no associationbetween macrophages apoptosis level and the phagocytosis activity. These data suggested that among the virulentMtb clinical isolates, the ability to induce macrophages apoptosis and phagocytosis were consistently in comparablelevelKeywords: Mycobacterium tuberculosis, apoptosis, phagocytosis, macrophages, isoniazid

Rapid Detection and Molecular Typing of Dengue Virus by Using Multiplex-Nested-RT-PCR Wijayanti, Nastiti; Wibawa, Tri; Nirwati, Hera; Haryanto, Aris; S, Sutaryo1
Indonesian Journal of Biotechnology Vol 11, No 2 (2006)
Publisher : Universitas Gadjah Mada

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world. We have evaluated the combination of one-step RT-PCR and multiplex nested PCR assays for detectingdengue viruses from clinical samples. Twelve patients were screened for the dengue virus, using a pair of primersthat conserve for several Flavivirus. The results showed that in 12 suspect patients, 100% were positive for Flavivirusand there are some genotypic variation among them, that indicated by several RT-PCR products higher than 511 bp,the expected product for RT-PCR. Further assay was performed to clarify the presence and serotypes of dengue virususing multiplex nested PCR. Serotyping results indicated that 83,3% of samples can be confirmed for dengue virus.Among the dengue virus positive 16,7 % are dengue-2, 16.7 % are dengue-3, and the most common 50% are dengue-4,whereas dengue-1 were not found among the patients. The combination of RT-PCR and multiplex nested PCR assaycan be used for rapid analysis dengue samples in early phase which is potentially useful for clinical, epidemiologyand also evolutionary studies.Key words: Flavivirus, dengue virus, serotype, RT-PCR, multiplex nested PCR

Candida albicans biofilm: formation and antifungal agents resistance Wibawa, Tri
Journal of the Medical Sciences (Berkala Ilmu Kedokteran) Vol 44, No 02 (2012)
Publisher : Universitas Gadjah Mada

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Candida sp are the most common fungal pathogens causing fatal health care associated infections.Among the genus of Candida, Candida albicans is the most frequent species isolated frompatients. The notorious C. albicans infection is the ability of this dimorphic fungus to formbiofilm. Biofilm has been pointed as a dynamic phenotypic switching in bacteria and fungi,which may result in higher morbidity and mortality in human beings. This review addresses thebasic explanation of biofilm formation which is characterized by the antifungal agents resistance.The factors that influence C. albicans biofim formation and antifungal agents resistance arediscussed.Key words: Candida sp â antifungal â resistance â biofilm - pathogenecity

Apoptosis and Phagocytosis Activity of Macrophages Infected by Mycobacterium tuberculosis Resistant and Sensitive Isoniazid Clinical Isolates Rachmawaty, Farida J.; Wibawa, Tri; Soesatyo, Marsetyawan H.N.E.
Indonesian Journal of Biotechnology Vol 11, No 1 (2006)
Publisher : Universitas Gadjah Mada

Show Abstract | Download Original | Original Source | Check in Google Scholar | Full PDF (43.035 KB)

Mycobacterium tuberculosis (M.tb) is the main causative pathogen that cause the pulmonary tuberculosis.Intracellular M.tb was reported able to induce macrophages apoptosis, which may have crucial role in the regulationof immun response against M.tb infection. As an intracellular bacteria, M.tb able to live and replicate withinmacrophages. Phagocytosis is the first step to achieved this condition. The induction of macrophages apoptosis byINH resistant and sensitive M.tb clinical isolates, and H37Rv was studied. The macrophages apoptosis level weremeasured using an Ag-capture ELISA for histone and fragmented DNA (Cell Death Detection ELISAplus, RocheDiagnostic GmBH). Phagocytosis activity also analyzed, after staining using fluorescence dye (AcriFluorTM, ScientificDevice Lab.). The results showed that there was no significantly different between INH resistant and sensitive M.tbclinical isolates in respect their ability to induce apoptosis. The phagocytosis activity among the clinical isolates wasshown to be strain dependent, and undistinguishable between the Mtb clinical isolates. There was no associationbetween macrophages apoptosis level and the phagocytosis activity. These data suggested that among the virulentMtb clinical isolates, the ability to induce macrophages apoptosis and phagocytosis were consistently in comparablelevelKeywords: Mycobacterium tuberculosis, apoptosis, phagocytosis, macrophages, isoniazid

Pengaruh Perbedaan Metode Ekstraksi Metabolit Sekunder Streptomyces sp. GMR22 terhadap Toksisitas pada Sel BHK-21 Mentari, Diani; Naima, Mirtani; Wulansari, Riska; Widada, Jaka; Nuringtyas, Tri Rini; Wibawa, Tri; Wijayanti, Nastiti
Pharmacon: Jurnal Farmasi Indonesia Vol 16, No 1 (2019)
Publisher : Universitas Muhammadiyah Surakarta

Show Abstract | Download Original | Original Source | Check in Google Scholar | DOI: 10.23917/pharmacon.v16i1.8032

Streptomyces sp. GMR22 is local isolate from Wanagama 1 Forest in Yogyakarta. They have the potential to be developed to produce active compounds because have PKS and NRPS genes.The active compounds from isolation are strongly influenced by various factors, one of them is extraction techniques. Effect difference of extraction technique will be affected by the quality of secondary metabolites produced.The purpose of this study was to compare the cytotoxicity effects of secondary metabolites of Streptomyces sp. GMR22 which have extracted with different stages from previous studies. The extraction technique was carried out by multilevel separatory funnel extraction methods, which was first extracted using non-polar solvent (n-hexane) and then extracted using semi-polar solvent (ethyl acetate). This research is important because in previous studies (separatory funnel only extracted using ethyl acetate) with the use of the lowest concentration in the dengue virus antiviral test (further test) caused 100% of deaths in BHK-21 cells.This study indicate that multilevel extraction result in lower CC50 value than previous studies. There are 49.160 µl/ml (n-hexane extract) and 284.56 µl/ml (ethyl acetate extract) while water extract is 464,38 µl/ml. FTIR compound analysis show that the three extracts produced have different spectrum patterns, especially in the n-hexane and ethyl acetate extract. Value of CC50 is not too high, it is expected that the secondary metabolites contained in the extracts can be used for further analysis such as antiviral testing because it is safe for normal host cells such as BHK-21 cells

Phenotype and Genotype of Enterococcus faecalis Isolated from Root Canal and Saliva of Primary Endodontic Patients Mubarak, Zaki; Asmara, Widya; Wibawa, Tri; Bachtiar, Boy
Journal of Dentistry Indonesia
Publisher : UI Scholars Hub

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Objective: To investigate the phenotype and genotype of E. faecalis isolated from the root canal and saliva of primary endodontic patients with periapical lesions. Methods: Eighteen adult male and female individuals suffering from primary endodontic infection, either with or without periapical lesions, were involved in this study. Root canal scraping and saliva were collected from each subject and used for bacterial quantitation using a real-time polymerase chain reaction (RT-PCR). Enterococci were isolated using ChromAgar medium and then identified using both biochemical (Gram staining and catalase tests) and molecular biology (conventional PCR) methods. Gelatinase activity, polysaccharide capsul profile and mRNA ace expression level were determined using microbiological, biochemical and molecular biology approach, respectively. Genotype of E. faecalis was determined based on nucleotide sequence of ace and gelE genes analyzed using web-based 3730xl DNA Analyze software. Results: The results showed that except for its proportion, no significant difference was found in phenotypes (gelatinase activity and mRNA ace expression levels) and genotypes (polymorphism of Cps operon and variation of ace and gelE nucleotide sequences) of E. faecalis isolated from the root canal and saliva of primary endodontic patients had or had no periapical lesions. Conclusion: It can be concluded that E. faecalis proportion had a role in the occurrence of periapical lesions in the primary endodontic patients, but not gelatinase activity, mRNA ace expression level, Cps operon polymorphism or ace and gelE nucleotide sequence variations.

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