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Optimasi Duplex PCR untuk Deteksi Simultan Gen Penyandi Faktor Virulensi ompW dan ctxA Vibrio cholerae Praja, Rian Ka; Sukrama, I Dewa Made; Fatmawati, Ni Nengah Dwi; Hidayati, Wahyu
Indonesia Medicus Veterinus Vol 5 (5) 2016
Publisher : Faculty of Veterinary Medicine, Udayana University

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Abstract

Vibrio cholerae merupakan salah satu agen foodborne disease yang dapat ditularkan melalui seafood. Penelitian ini bertujuan untuk optimasi gen penyandi faktor virulensi outer membrane protein W (ompW) dan cholerae toxin subunit A (ctxA) menggunakan teknik Duplex Polymerase Chain Reaction (dPCR). Dua bakteri V. cholerae O1 serotipe Ogawa dan Inaba digunakan pada penelitian ini. Proses isolasi DNA dilakukan menggunakan metode Boil Cell Extraction (BCE). dPCR dilakukan menggunakan dua pasang primer (forward dan reverse) ompW-F, ompW-R dan ctxA-F, ctxA-R dengan panjang produk masing-masing 588 bp dan 302 bp. Tahap optimasi yang dilakukan dalam proses dPCR ini meliputi variasi suhu annealing, variasi konsentrasi primer serta variasi volume DNA template kemudian deteksi produk dPCR dilakukan dengan elektroforesis pada gel agarosa 1,5% dan divisualisasi menggunakan alat Gel DocTM XR (Bio-Rad). Hasil penelitian menunjukkan komposisi reaksi dPCR yang terbaik untuk mendeteksi gen ompW dan ctxA secara simultan terdiri dari PCR mix (Promega) 12,5 ?L, primer ompW-F, ompW-R 0,5 ?M, primer ctxA-F, ctxA-R 0,3 ?M, nuclease free water 6,5 ?L dan DNA template 2 ?L sehingga volume total menjadi 25 ?L. Kondisi mesin PCR terdiri dari pre-denaturasi 95oC selama 2 menit (1 siklus) diikuti oleh denaturasi 95oC selama 1 menit, annealing 53oC selama 1 menit, extension 72oC selama 1 menit (35 siklus), dan post-extension 72oC selama 5 menit (1 siklus). Dapat disimpulkan bahwa dPCR dapat digunakan untuk deteksi simultan gen penyandi faktor virulensi ompW dan ctxA V. cholerae.
Prevalensi Infeksi Escherichia coli O157:H7 pada Sapi Bali di Kecamatan Mengwi dan Kuta Selatan, Badung, Bali Praja, Rian Ka; Pinatih, Komang Januartha Putra; Suardana, I Wayan
Indonesia Medicus Veterinus Vol 4 (1) 2015
Publisher : Faculty of Veterinary Medicine, Udayana University

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Abstract

Escherichia coli O157:H7 merupakan agen zoonosis yang dapat menyebabkan hemorrhagic colitis dan hemolytic uremic syndrome (HUS) pada manusia. Sapi diketahui sebagai reservoir utama dari bakteri E. coli O157:H7. Penelitian ini bertujuan untuk mengetahui prevalensi infeksi E. coli O157:H7 pada sapi bali di Kecamatan Mengwi dan Kuta Selatan, Badung, Bali. Isolasi bakteri E. coli dilakukan pada media eosin methylene blue agar (EMBA) kemudian dilakukan pewarnaan Gram dan uji indol, methyl red, voges proskauer dan citrate (IMVIC). Identifikasi E. coli O157 dilakukan pada media selektif sorbitol Mac Conkey agar (SMAC) dan uji konfirmasi dengan uji aglutinasi lateks O157 serta uji antiserum H7 sebagai konfirmasi akhir dari E. coli O157:H7. Hasil penelitian ini menunjukkan bahwa dari 60 sampel feses sapi bali yang diambil di Kecamatan Mengwi, dua sampel (3,3%) positif E. coli O157:H7 dan lima sampel (8,3%) positif E. coli O157:H7 dari 60 sampel yang berasal dari Kuta Selatan. Hasil uji Chi Square menunjukkan nilai ?2 sebesar 0,243 yang berarti prevalensi infeksi E. coli O157:H7 pada sapi bali antara Kecamatan Mengwi dan Kecamatan Kuta Selatan tidak berbeda nyata (P > 0,05). Berdasarkan hasil penelitian ini dapat disimpulkan bahwa sapi bali di Kecamatan Mengwi dan Kuta Selatan terinfeksi E. coli O157:H7.
Detection of genes encoding ompW and ctxA of Vibrio cholerae isolated from shrimp and shellfish at Kedonganan fish market, Bali-Indonesia Kusuma Praja, Rian Arinta; Sukrama, I Dewa Made; Fatmawati, Ni Nengah Dwi
Oceana Biomedicina Journal Vol 2, No 1 (2019): Oceana Biomedicina Journal
Publisher : Universitas Hang Tuah

Show Abstract | Download Original | Original Source | Check in Google Scholar | Full PDF (251.1 KB) | DOI: 10.30649/obj.v2i1.23

Abstract

Contamination of pathogenic bacteria in food can lead to the emergence of foodborne disease. One of foodborne disease which often occurs in some developing countries such as Africa, Southeast Asia, and Latin America is cholera which is caused by Vibrio cholerae. The disease is transmitted through beverages and food, especially contaminated seafood. V. cholerae has several virulence factors including the outer membrane protein W (ompW) and cholerae toxin (ctx).The ompW acts as a protective barrier and can also be used as a marker specific species of V. cholerae and cholerae toxin is an enterotoxin responsible for the incidence of diarrhea in a cholera outbreak produced by pathogenic V. cholerae. This study was an observational study to determine the level of contamination of V. cholerae by detecting the outer membrane protein W (ompW) and cholerae toxin subunit A (ctxA) gene of V. cholerae in shrimp and shellfish sold at Kedonganan fish market. Samples were taken using total sampling technique and obtained 24 samples consisting of 14 shrimp samples and 10 shellfish samples. Samples were examined using culture methods and biochemical tests, and then further tested using Duplex Polymerase Chain Reaction (dPCR) to detect ompW and ctxA gene. The dPCR assay results showed 8 out of 14 (57.1%) samples from shrimp and 1 out of 10 (10%) samples from the shellfish positive carried ompW gene, and found no positive samples carrying the ctxA gene in samples derived from shrimp and shellfish. Chi square test analysis results indicated contamination of V. cholerae in shrimp was higher than shellfish based on ompW gene (p<0.05). It can be concluded that the shrimp and shellfish at Kedonganan fish market are contaminated by V. cholerae. Further research is needed to detect the virulence factors besides ompW and ctxA of V. cholerae in seafood. Keywords: Foodborne disease, Vibrio cholerae, ompW gene, ctxA gene, and Duplex Polymerase Chain Reaction (dPCR).
Molecular Mechanism of Cholerae Toxin (ctx) in Causing Diarrhea Praja, Rian Kusuma Arinta; Rosalina, Reny
Oceana Biomedicina Journal Vol 1, No 2 (2018): Oceana Biomedicina Journal
Publisher : Universitas Hang Tuah

Show Abstract | Download Original | Original Source | Check in Google Scholar | Full PDF (761.654 KB) | DOI: 10.30649/obj.v1i2.16

Abstract

Vibrio cholerae is one of the pathogenic bacteria transmitted through contaminated food, especially seafood and contaminated beverages. V. cholerae produces cholerae toxin (ctx) which is encoded by the ctx gene located within its chromosome. This toxin has been recognized as one of the toxins responsible for cholera outbreaks. The mechanism of ctx gene expression is induced by environmental signals such as pH, osmolarity, temperature, bile, amino acids, and CO2. These signals will be a positive transcriptional factor to the ToxR gene that regulates the biogenesis of cholerae toxin. After cholerae toxin has been successfully expressed, V. cholerae uses a type II secretion (T2S) pathway to deliver cholerae toxin to the extracellular environment. Cholerae toxin consists of A and B subunits. The B subunit plays a role in attaching to the receptor Manosialosyl Ganglioside (GM1 ganglioside) and the A subunit plays a role in catalyzing ADP-ribosylation of Gs (stimulatory) proteins and turning them into active condition. The Gs proteins will convert the inactive adenilate cyclase (AC) into active AC. The increase of AC activity will increase the cyclic adenosine 3'5'-monophosphate (cAMP) concentration along the cell membrane. The cAMP then causes the active secretion of sodium (Na+), chloride (Cl-), potassium (K+), bicarbonate (HCO3-), and water (H2O) out of the cell into the intestinal lumen, resulting in large fluid losses and electrolyte imbalances. Keywords: Vibrio cholerae, cholerae toxin (ctx), ToxR gene, type II secretion (T2S), GM1 ganglioside, adenilate cyclase.
The infection of Vibrio parahaemolyticus in shrimp and human Praja, Rian Ka
Oceana Biomedicina Journal Vol 1, No 1 (2018): Oceana Biomedicina Journal
Publisher : Universitas Hang Tuah

Show Abstract | Download Original | Original Source | Check in Google Scholar | Full PDF (258.508 KB) | DOI: 10.30649/obj.v1i1.6

Abstract

Vibrio parahaemolyticus is an aquatic zoonotic agent that can threaten human and aquaculture animal health. Humans can be infected by consuming contaminated raw seafood or wound-related infections. Generally infection of V. parahemolyticus is orally transmitted and causes gastroenteritis in humans while in aquaculture animals especially shrimp can cause Acute Hepatopancreatic Necrosis Disease (AHPND) or Early Mortality Syndrome (EMS) with a very high mortality rate and cause economic losses. Shrimp species susceptible to infection are Litopenaeus vannamei, Penaeus monodon, and P. chinensis. V. parahaemolyticus produces several toxins in human disease such as thermostable direct hemolysin (TDH), TDH-related haemolysin (TRH), and thermolabile hemolysin (TLH). Meanwhile, Photorabdus insect-related (Pir) toxins consisting of PirAvp and PirBvp are the toxins associated with AHPND in shrimp. The genes that encode the toxin are used as targets to diagnose V. parahaemolyticus pathogens molecularly. Until now the treatment of V. parahaemolyticus infection is using antibiotics and fluid therapy, but there were V. parahaemolyticus isolates from aquaculture that have been resistant to antibiotics so that the use of antibiotics in aquaculture must be controlled and the use of alternative therapy are very important to be developed to control V. parahaemolyticus infection. Keywords: V. parahaemolyticus, zoonotic, gastroenteritis, Acute Hepatopancreatic Necrosis Disease (AHPND), Early Mortality Syndrome (EMS).
THE EXISTENCE OF VIBRIO CHOLERAE IN INDONESIA: FROM ENVIRONMENTAL TO CLINICAL ASPECTS (A CONCISE REVIEW) Ka Praja, Rian; Pusporini, Anggita Ratri; Rosalina, Reny; Arta, I Wayan Muda Suta; Sukrama, I Dewa Made; Fatmawati, Ni Nengah Dwi
Jurnal PPI Dunia Vol 4 No 1 (2021)
Publisher : OISAA

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Abstract

Vibrio cholerae is an infectious agent causing cholera disease with a high prevalence in various developing countries. V. cholerae is a pathogen with broad spectrum host that can infect humans and animals, especially aquaculture. The existence of this disease in Indonesia has long been identified in several outbreaks. Various reports in Indonesia have succeeded in finding the existence of V. cholerae in the environment, aquaculture, food and beverage, as well as in clinical cases of V. cholerae infection. The presence of V. cholerae in environment such as water source is commonly related with contamination. However, V. cholerae can be found in aquatic environment as this environment is natural habitat for V. cholerae. Thus, aquaculture is prone to be infected with V. cholerae because the presence of this pathogen is abundant in aquatic environment. Contaminated food and beverage are associated with hygiene and sanitation and human is commonly infected after consuming contaminated food or beverage. This brief review has the main focus to discuss the existence of V. cholerae from environmental to clinical aspects found in Indonesia.
Perancangan primer gen lktB pada Fusobacterium necrophorum untuk analisis PCR Praja, Rian Ka; Rosalina, Reny
Jurnal Sains dan Teknologi Peternakan Vol 2 No 2 (2021): Vol 2 No 2 (2021): Jurnal Sains dan Teknologi Peternakan
Publisher : Universitas Sulawesi Barat

Show Abstract | Download Original | Original Source | Check in Google Scholar | DOI: 10.31605/jstp.v2i2.960

Abstract

Fusobacterium necrophorum is a pathogen causing disease in animals, especially cattle, goats, and sheep. F. necrophorum infection can result in a variety of necrotic conditions (necrobacillosis). This study aimed to design a pair of primers for detecting the leukotoxin B (lktB) gene expressed by F. necrophorum as diagnostic support. The lktB gene sequence was obtained from GenBank NCBI with accession number AF312861.3:685-2337. Furthermore, the sequence was used as a template for in silico primer design using Primer-BLAST. Primer candidates successfully designed were then analyzed for their secondary structure using NetPrimer. The results showed that forward primer set 6 (5'-TCGGATGCTGGAATGCTACTT-3') and reverse primer set 6 (5'-GGGCTCCCAAATCCTTACGA-3') were a favorable primer set with a product size of 228 bp. However, laboratory experiments need to be carried out to determine the optimal conditions for this primer set.