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All Journal HAYATI Journal of Biosciences VALENSI JSMARTech : Journal of Smart Bioprospecting and Technology
Anggraeni, Rosita Gio
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Agriculture, Biological Sciences & Forestry Biochemistry, Genetics & Molecular Biology Chemistry Earth & Planetary Sciences Materials Science & Nanotechnology Medicine & Pharmacology Neuroscience

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Development of Bacillus subtilis Detection Method Targeting Genes codY, narH, and ureC Using Polymerase Chain Reaction Nurjayadi, Muktiningsih; Berkahingrum, Ayu; Putri, Adinda Myra Amalia; Rahmawati, Atikah Nur; Anggraeni, Rosita Gio; Fahriza, Tiara; Declan, Jefferson Lynford; Putri, Gladys Indira; Krisdawati, Ismaya; Juliansyah, Dandy Akbar; Azzahra, Maharanianska; Maulana, Irvan; Kartika, Irma Ratna; Kurniadewi, Fera; Sukmawati, Dalia; Rahayu, Sri; Saamia, Vira; Wiranatha, I Made; Abomoelak, Bassam; El Enshasy, Hesham
JSMARTech: Journal of Smart Bioprospecting and Technology Vol. 5 No. 1 (2024): JSMARTech Volume 5, No. 1, 2024
Publisher : JSMARTech

Show Abstract | Download Original | Original Source | Check in Google Scholar | DOI: 10.21776/ub.jsmartech.2024.005.01.26

Abstract

Bacillus subtilis, a causative agent of foodborne illness in bread, cakes, cereals, and cheese leading to symptoms like diarrhea and nausea, was investigated for its role in nosocomial infections, including endocarditis, bacteremia, septicemia, and meningitis. The research focused on three genes: codY, narH, and ureC, which respectively contribute to bacterial survival through biofilm and spore formation, acid resistance, and adaptation to anaerobic conditions. The objective of this study was to evaluate the codY, narH, and ureC genes using the Gradient PCR method for Bacillus subtilis detection in bread and cheese through Real-Time PCR. During the research, bacterial growth was observed with an OD600 of 1.438 in Tryptic Soy Broth (TSB), and colonies of medium size, smooth, cream-coloured, and round shape were successfully isolated on Tryptic Soy Agar (TSA). The DNA template used had a 71.5 ng/µL concentration with an A260/280 ratio of approximately 1.830. The annealing temperature for Gradient PCR used was 53-62°C. The primers successfully amplified codY (175 bp), narH (222 bp), and ureC (153 bp) gene amplicons. The optimal annealing temperature for the primers used was 60°C, as indicated by the presence of a single bright band in electrophoresis. Using these three different genes, testing can also be conducted with the Multiplex PCR method. The next step involves developing a detection kit using optimized primers and annealing temperatures for the identification of Bacillus subtilis in bread and cheese samples using Real-Time PCR.
Detection of the Yersinia enterocolitica Bacteria Targeting the myfA and ystA Genes in Contaminated Vegetable Samples using Real-Time PCR to Develop Rapid Detection of Food Poisoning Bacteria Nurjayadi, Muktiningsih; Anggraeni, Rosita GIo; Putri, Gladys Indira; Declan, Jefferson Lynford; Juliansyah, Dandy Akbar; Fahriza, Tiara; Putri, Adinda Myra Amalia; Berkahingrum, Ayu; Rahmawati, Atikah Nur; Kartika, Irma Ratna; Kurniadewi, Fera; Sukmawati, Dalia; Rahayu, Sri; Saamia, Vira; Wiranatha, I Made; Abomoelak, Bassam; El-Enshasy, Hesham Ali
HAYATI Journal of Biosciences Vol. 32 No. 4 (2025): July 2025
Publisher : Bogor Agricultural University, Indonesia

Show Abstract | Download Original | Original Source | Check in Google Scholar | DOI: 10.4308/hjb.32.4.989-1002

Abstract

Yersinia enterocolitica is a pathogenic bacterium with the ability to survive and multiply in food in a low-temperature environment that can cause death in humans. In previous studies, the optimum annealing temperature of ymoA, ystA, and ail gene primers with amplicons of 185 bp, 123 bp, and 192 bp, respectively, was successfully found. This study aims to develop a pathogenic bacteria detection kit with confirmation, sensitivity, and specificity of myfA and ystA primers in detecting Yersinia enterocolitica bacteria quickly and accurately using the real-time Polymerase Chain Reaction method. The results showed that myfA and ystA primers have optimum annealing temperatures at 60°C with amplicon lengths of 181 bp and 123 bp, respectively. Primer myfA was able to amplify the target with real-time PCR at Ct 12.07±1 and Tm 81±1°C, while the ystA primer at Ct 12.38±1 and Tm 83±1°C. myfA and ystA primers were also able to distinguish target and non-target bacteria based on Ct or Tm. The designed primers successfully detected Yersinia enterocolitica bacteria with the smallest concentration of 0.000439 ng/µL equivalent to 7.024 × 102 CFU. The detection limit obtained is smaller than the contamination threshold set by the Food and Drug Administration (BPOM). Primer myfA and ystA Yersinia enterocolitica also successfully detected the target bacteria in cabbage and lettuce samples artificially. Based on these results, myfA and ystA primers successfully detected Yersinia enterocolitica in vegetable samples using real-time PCR quickly, sensitively, specifically, and accurately.
Efficacy Test of Prototype Kit for Detection Bacillus cereus and Listeria monocytogenes in Processed Meat using Real-time PCR Method Nurjayadi, Muktiningsih; Fahriza, Tiara; Putri, Adinda Myra Amalia; Rahmawati, Atikah Nur; Berkahingrum, Ayu; Anggraeni, Rosita Gio; Putri, Gladys Indira; Declan, Jefferson Lynford; Akbar, Dandy; Maulana, Irvan; Azzahra, Maharanianska; Shangkara, Muhammad Arkent; Kartika, Irma Ratna; Kurniadewi, Fera; Sukmawati, Dalia; Rahayu, Sri; Saamia, Vira; Wiranatha, I Made; Abumoelak, Bassam; El Enshasy, Hesham
Jurnal Kimia Valensi Vol. 11 No. 1 (2025): Jurnal Kimia VALENSI
Publisher : Department of Chemistry, Faculty of Science and Technology Syarif Hidayatullah Jakarta State Islamic University

Show Abstract | Download Original | Original Source | Check in Google Scholar | DOI: 10.15408/jkv.v11i1.44280

Abstract

According to the World Health Organization (WHO), harmful agents such as Bacillus cereus and Listeria monocytogenes are responsible for 600 million cases of disease and 420,000 deaths annually. This research aims to test the effectiveness of the real-time PCR method for developing a prototype kit to detect pathogenic bacteria in processed meat. As a comparison, the and conventional PCR methods were used to obtain the accuracy, specificity, sensitivity, and effectiveness of the real-time PCR method. All the samples were cultured in solid media agar, performed amplification using specific primers cyt-K 2 and hly using PCR and real-time PCR. Meatballs, nuggets, and sausages, five samples each, were found to be positive positively contaminated with all targeted bacteria. However, they did not provide specific results using solid media culture and the PCR method. In addition, the real-time PCR method using prototype kit formulas accomplished that all contaminated samples had a Ct value smaller than the negative control, NTC (No Template Control), and had a similar melting curve to the positive control. This establishes that the real-time PCR method clarifies that all samples were contaminated with target bacteria. A formula was developed in the prototype kit with real-time PCR methods that can be used and applied on a commercial scale efficiently and precisely.
Co-Authors Abomoelak, Bassam Abumoelak, Bassam Akbar, Dandy Azzahra, Maharanianska Berkahingrum, Ayu Dalia Sukmawati El Enshasy, Hesham El-Enshasy, Hesham Ali Fahriza, Tiara Jefferson Lynford Declan Juliansyah, Dandy Akbar Kartika, Irma Ratna Krisdawati, Ismaya Kurniadewi, Fera Maulana, Irvan Muktiningsih Nurjayadi Putri, Adinda Myra Amalia Putri, Gladys Indira Rahmawati, Atikah Nur Saamia, Vira Shangkara, Muhammad Arkent SRI RAHAYU Wiranatha, I Made