Muktiningsih Nurjayadi, Anisa Fitriyanti, Royna Rahma Musie, Gusti Angieta Putri, Puan Aqila Azizah, Helzi Angelina, Grace, Ananda Indah Putri Sihombing, Agus Setiawan, Jefferson Lynford Declan, Gladys Indira Putri, Dandy Akbar Juliansyah, Siti Fatimah, Ayu Berkahingrum, Irma Ratna Kartika, Fera Kurniadewi, Vira Saamia, Shyi-Tien Chen, Bassam Aboemolak, Hesham A. El Enshasy
Escherichia coli is a foodborne pathogenic bacterium that can cause diarrhea, while yhaV is a virulence-associated gene linked to the toxin–antitoxin system in E. coli. This study was aimed at evaluating the confirmation, specificity, and sensitivity of a yhaV gene primer using real-time polymerase chain reaction. The yhaV-targeting PCR successfully amplified a DNA fragment with an amplicon length of 207 bp (base pairs) under an annealing temperature optimized to a range of 54 °C to 62 °C via gradient PCR. The PCR using the primer pair produced a consistent Ct (cycle threshold) of 14.14 ± 0.05 and showed a single peak in the melting curve at a Tm (melting temperature) of 83.67 °C ± 0.02. The specificity test indicated that the yhaV primer effectively distinguished E. coli from nontarget bacteria on the basis of differences in Ct and Tm values. The sensitivity analysis showed that the PCR directed toward the primer pair successfully detected E. coli at a minimum concentration of 2.24 pg/µL, with a Ct value of 29.93 and a detection limit of 31.5 × 102 CFU. These results suggest that yhaV-based real-time PCR quickly and accurately identifies E. coli. Primer designs that target yhaV have the potential to be developed as components of a rapid, specific, and sensitive kit for detecting E. coli in food samples. © 2025, Universitas Indonesia. All rights reserved.
Department of Chemistry, Faculty of Mathematics and Natural Science, Universitas Negeri Jakarta, Jakarta Timur, 13220, Indonesia; Research Center for Detection of Pathogenic Bacteria, Lembaga Penelitian dan Pengabdian Kepada Masyarakat, Universitas Negeri Jakarta, Jakarta Timur, 13220, Indonesia; Center Forensic Laboratory of the Criminal Investigation, Police of the Republic of Indonesia, Bogor, 16810, Indonesia; Department of Safety, Health and Environmental Engineering, National Kaohsiung University of Science and Technology, Kaohsiung City, 82445, Taiwan; Arnold Palmer Hospital Pediatric Specialty Diagnostic Laboratory, 32806, FL, United States; Innovation Center in Agritechnology for Advanced Bioprocessing, Universiti Teknologi Malaysia, Johor, 81310, Malaysia; School of Chemical and Energy Engineering, Faculty of Engineering, Universiti Teknologi Malaysia, Johor, 81310, Malaysia; City of Scientific Research and Technology Applications, Alexandria, 21934, Egypt
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