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All Journal Jurnal Sain Veteriner Jurnal Ilmu Ternak Veteriner Indonesian Journal of Biotechnology BERITA BIOLOGI Jurnal Kedokteran Hewan
Didik Tulus Subekti
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Agriculture, Biological Sciences & Forestry Biochemistry, Genetics & Molecular Biology Immunology & microbiology Materials Science & Nanotechnology Medicine & Pharmacology Veterinary

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Detection of Brugia malayi microfilaria/Larvae in mosquito using Polimerase Chain Reaction. Haryuningtyas, Dyah; Subekti, Didik Tulus
Indonesian Journal of Animal and Veterinary Sciences Vol 13, No 3 (2008)
Publisher : Indonesian Animal Sciences Society

Show Abstract | Download Original | Original Source | Check in Google Scholar | Full PDF (203.014 KB) | DOI: 10.14334/jitv.v13i3.587

Abstract

Lymphathic filariasis that is also known as elepanthiasis is caused by infestation of 3 species nematode Wuchereria bancrofti, Brugia malayi and Brugia timori. In Indonesia 70% filariasis case caused by Brugia malayi. Mosquito species from genus Anopheles, Aedes, Culex, Mansonia and Armigeres are known as vector of this disease. Microfilaria detection on mosquito is one methode to know infection rate in vector population in endemic area.The objectives of the research were to study the ability of Hha1 repeat applicable to detect microfilaria/larvae in a pool of mosquitoes and to get description of adult mosquito night biting population lived in endemic area of filariasis brugian. Mosquito as positive control used in this research come from laboratory of parasitology of FKUI. Mosquito sample from the field was from Binawara and Kolam Kiri villages, South Kalimantan province. Mosquito were trapped then identified by its species. DNA of mosquitoes was extracted and then run by the PCR using Hha 1 repeat primer. Result of the research indicated that adult mosquitoes night biting from Binawara village consist of Culex, Mansonia, Anopheles genus and from Kolam Kiri village only from Mansonia genus. Hha 1 repeat primer is applicable to detect 1 mosquito infected with microfilaria/larvae in a pool of negative mosquitoes. Mosquito samplesfrom the two villages showing negative PCR.   Key Words: Filariasis, Brugia Malayi, Vector, Microfilaria, Filaria Larve, PCR
Cloning Gene Encoding Micronema 3 (Mic3) Protein of Tachyzoite Toxoplasma Gondii Local Isolate Artama1, Wayan T.; Dewi, Ni Nyoman Ayu; Subekti, Didik Tulus
Indonesian Journal of Biotechnology Vol 10, No 1 (2005)
Publisher : Universitas Gadjah Mada

Show Abstract | Download Original | Original Source | Check in Google Scholar | Full PDF (308.784 KB)

Abstract

Microneme 3 (MIC3) protein tachyzoites Toxoplasma gondii is one of protein which plays an important roleduring cell host invasion. Gene encoding MIC3 protein has been studied and it was suggested a potent vaccinecandidate against Toxoplasma gondii infection. The aim of this research is to clone and sequence the gene encodingMIC3 protein of tachyzoites Toxoplasma gondii local isolate by amplification using polymerase chain reaction withspecific primers. The amplified DNA fragment was cloned into pGEM-T and transformed into E. coli XL-1 Blue byheat shock method. Recombinant plasmids were isolated using alkali lysis method and analyzed by digestionusing restriction endonuclease enzymes PstI, HindIII, NcoI and EcoRV. The recombinant plasmids then sequencedto find out the nucleotide sequence of insert gene by ABIPRISM 377 DNA Sequencer. The DNA sequence thenwere analyzed by computer software for alignment. The result showed that transformation in E. coli XL-1 Blue bypGEM-T produced one clone that was encoding MIC3 protein. Analysis of 489 bp from 5’ and 447 from 3’ of genesequence showed 97-98% homology with gene encoding for MIC3 protein of RH isolate.Keywords: MIC3 protein, Toxoplasma gondii, tachyzoite, recombinant DNA
Cloning and Sequencing cDNA Encoding for Rhoptry-2 Toxoplasma Gondii Tachyzoite Local Isolate Artama, Wayan T.; Sari, Yulia; Subekti, Didik Tulus; Poerwanto, Soenarwan Hery; Subandono, Jarot
Indonesian Journal of Biotechnology Vol 10, No 2 (2005)
Publisher : Universitas Gadjah Mada

Show Abstract | Download Original | Original Source | Check in Google Scholar | Full PDF (338.765 KB)

Abstract

Rhoptry protein belongs to an excretory and secretory antigens (ESAs) that play an important role during activepenetration of parasite into the cell target. This protein an able Toxoplasma gondii to actively penetrate targetedcell, meanwhile ESAs protein stimulates intracellular vacuole modification. It is, therefore, after the parasitesuccessfully enter the cell target then Granule (GRA) proteins are responsible for the formation of parasitophorusvacuole, which is protect the fusion with other intracellular compartments such as lysosomal vacuole. Consequently,this parasite is being able to survive and multiply at the cell target. The current study was aimed to clone andsequens cDNA encoding for ROP-2 of local isolated T. gondii tachizoite through DNA recombinant technique.Total ribonucleic acid (RNA) was isolated from tachyzoites of local isolated T. gondii that were grown up in Balb/c mice. Messenger RNA was isolated from total RNA using PolyAtract mRNA Isolation System. Messenger RNA wasused as a template for synthesis cDNA using Riboclone cDNA Synthesis System AMV-RT. EcoRI adaptor fromRiboclone EcoRI Adaptor Ligation System was added to Complementary DNA and than ligated to pUC19. Recombinantplasmid was transformed into E. coli (XL1-Blue). The transformed E. coli XL-1 Blue were plated on LB agarcontaining X-Gal, IPTG and ampicillin. Recombinant clones (white colony) were picked up and grown up in theLB medium at 37oC overnight. Expression of recombinant protein was analysed by immunoblotting in order toidentify cDNA recombinant wich is express ESA of T. gondii local isolate. Recombinant plasmid were isolatedusing alkalilysis method and were elektroforated in 1% agarose gel. The isolated DNA recombinant plasmid wascut using Eco RI and then sequenced through Big Dye Terminator Mix AB1 377A Sequencer using M13 Forward andM13 Reverse primers. The conclusion of this results showed that the recombinant clone was coding for excretoryand secretory protein which has molecular weight of 54 kDa. The DNA alignments of sequence from the clonedgene showed 97% homology with gene encoding for ROP-2 of T. gondii RH isolate.Keywords: Toxoplasma gondii, tachizoite, ESA, complementary DNA, ROP2
PERBANDINGAN ANTARA ALANTOIN (5 UREIDOHYDANTOIN) DENGAN BETADINE® (POVIDONE IODINE) UNTUK PENGOBATAN LUKA BVSISI Subekti, Didik Tulus
BERITA BIOLOGI Vol 4, No 4 (1998)
Publisher : Research Center for Biology-Indonesian Institute of Sciences

Show Abstract | Download Original | Original Source | Check in Google Scholar | DOI: 10.14203/beritabiologi.v4i4.1265

Abstract

Study on the comparison between allantoin (5 ¢ ureidohydantoin) and Betadine ® (povidone iodine) was conducted to compare and evaluate their efficacy, especially to accelerate wound (incision) healing. Treatment divided into three groups, first group is Control (without therapy), second group is allantoin treatment and the last one is Betadine ® treatment. Allantoin obtained from cattle's urine by Meissner method. The solution made of 2,4 grams of allantoin in 600 milliliters aqueous solution. Treatments (therapies) were given three times a day topically. Results showed no significant difference between allantoin and Betadine ® treatments (p > 0,05), control and the other treatments i.e allantoin and Betadine ® therapies have significantly difference (p < 0,01).
EVALUATION OF B1 GENE TO DETECT Toxoplasma gondii: COMPARISON OF THREE SETS NESTED PCR PRIMER Ekawasti, Fitrine; Azmi, Zul; Subekti, Didik Tulus; desem, muhammad ibrahim; Nugraha, Arifin Budiman; Sadiah, Siti; Cahyaningsih, Umi
Jurnal Kedokteran Hewan Vol 17, No 2 (2023): June
Publisher : Universitas Syiah Kuala

Show Abstract | Download Original | Original Source | Check in Google Scholar | DOI: 10.21157/j.ked.hewan.v17i2.22251

Abstract

This study aimed to evaluate three sets of B1 gene DNA primer for the diagnosis of Toxoplasma gondii. The DNA of Toxoplasma gondii that stored on liquid nitrogen was isolated using DNAzol reagent. The first step of Polymerase Chain Reaction (PCRs) was performed using external and internal primer sets, respectively, and then nPCR. PCR products sequencing was performed by Apical Science. All sequences were analysed using CLC Sequence Viewer Version 8.0 software and compared to sequence database that deposited in ToxoDB (Toxoplasma gondii genome database) using BLAST (https://toxodb.org/toxo/app). Each B1 gene primer was evaluated by performing single PCR (forward and reverse) and nested PCR reactions. Three sets of B1 gene primer have different amplification precision. According to the results of amplicon sequencing, the primer set #2 has the best amplification precision of B1 gene.
POLYCLONAL ANTIBODY UTILIZATION FOR DETECTION OF JEMBRANA ANTIGEN AT BALI CATTLE IN WEST SUMATRA PROVINCE Helmi, Helmi; Purwati, Endang; Rahmadani, Ibnu; Subekti, Didik Tulus
Jurnal Kedokteran Hewan Vol 14, No 1 (2020): March
Publisher : Universitas Syiah Kuala

Show Abstract | Download Original | Original Source | Check in Google Scholar | DOI: 10.21157/j.ked.hewan.v14i1.15014

Abstract

The aims of this study were to detect Jembrana antigen with polyclonal antibody and to describe antigen distribution in the Bali cattle organ that positively infected with Jembrana disease. Spleens, lungs, and livers were harvested from 10 naturally infected Bali cattle whose infection was confirmed through positive Polymerase Chain Reaction (PCR) result for Jembrana virus. Immunohistochemistry test was performed using polyclonal antibodies produced in rabbits. The results showed that cells infected by Jembrana viruses displayed positive reaction with a reddish brown color. Immunohistochemistry methods using polyclonal antibodies can detect Jembrana antigens in the spleen, liver, and lung with the highest average detection score (P0.05) was found in spleen, followed by liver and lungs. There was significant difference in the distribution of Jembrana antigens between the spleen, liver, and lungs with spleen having the highest antigen density.
Optimization of Sybr Green Quantitative Real Time Polymerase Chain Reaction (qPCR) using Excreted-Secreted Antigens (ESAs) Genetik Marker for Detection Toxoplasma gondii Ekawasti, Fitrine; Winarsongko, Agus; Nepho, Farlin; Purwanto, Eko Setyo; Subekti, Didik Tulus; nuradji, harimurti; Dharmayanti, NLP Indi; Ahmad, Riza Zainuddin; Sa’diah, Siti; Cahyaningsih, Umi; Nurcahyo, Raden Wisnu
Jurnal Sain Veteriner Vol 42, No 1 (2024): April
Publisher : Faculty of Veterinary Medicine, Universitas Gadjah Mada bekerjasama dengan PB PDHI

Show Abstract | Download Original | Original Source | Check in Google Scholar | DOI: 10.22146/jsv.90867

Abstract

AbstractToxoplasma gondii is an obligate intracellular parasite, causing toxoplasmosis in almost all warm-blooded animals and humans worldwide. Toxoplasmosis is a zoonotic disease of serious public health concern. Host cell invasion by T. gondii tachyzoites has process involving the sequential secretion of Excreted-Secreted Antigens (ESAs). T. gondi ESAs could be a valuable candidate for the diagnosis of toxoplasmosis. Techniques to more accurately detection of T. gondii recently developed biotechnological methods that are currently being used, conventional and real time Polymerase Chain Reaction (RT-PCR). RT-PCR is more widely used because it is more sensitive and specific. The aims of this study were to optimize the Sybr Green RT-PCR in different region gene based on Excreted-Secreted Antigens (ESAs), tachyzoite surface antigen and bradhyzoite antige, then adapt the conventional PCR program to real-time PCR for detection Toxoplasma gondii. Optimization is necessary to get optimal condition of PCR to get the best results. T. gondii RH strains derived from liquid nitrogen and DNA extracted by DNAzol. The genetic marker used GRA1#1, GRA1#2, GRA7#1, GRA7#2, ROP1, MIC3, SAG1 and BAG1. The results of the optimization of multiple primer genes can adapt and be used optimal in RT-PCR by using the same cycle program simultaneously in one run. Overall, RT-PCR for the detection of T. gondii DNA demonstrated excellent agreement with conventional PCR. RT-PCR with melting curve analysis is rapid and simple that facilitates high throughput analysis to detect T. gondii. The optimal conditions obtained from the optimization results can facilitate further research to detect T. gondii.Keywords: Biotechnology molecular, Detection, excretory-secretory antigen, toxoplasmosis
Co-Authors Azmi, Zul Desem, Muhammad Ibrahim Dyah Haryuningtyas Endang Purwati RN Fitrine Ekawasti, Fitrine Helmi Helmi Jarot Subandono Nepho, Farlin Ni Nyoman Ayu Dewi NLP Indi Dharmayanti Nugraha, Arifin Budiman Nuradji, Harimurti Nurcahyo, Raden Wisnu Purwanto, Eko Setyo Rahmadani, Ibnu Riza Zainuddin Ahmad Sadiah, Siti Sa’diah, Siti Soenarwan Hery Poerwanto, Soenarwan Hery Tamaulina Br Sembiring Umi Cahyaningsih Wayan T. Artama Wayan T. Artama1, Wayan T. Winarsongko, Agus