Muktiningsih Nurjayadi, Puan Aqila Azizah, Agus Setiawan, Ananda Indah Putri Sihombing, Anisa Fitriyanti, Grace, Gusti Angieta Putri, Helzi Angelina, Royna Rahma Musie, Jefferson Lynford Declan, Gladys Indira Putri, Dandy Akbar Juliansyah, Siti Fatimah, Tiara Fahriza, Irma Ratna Kartika, Fera Kurniadewi, Novitasari, Bassam Abomoelak, Hesham A. El Enshasy, Shyi-Tien Chen
Shigella flexneri can cause shigellosis, which results in high fever, vomiting, diarrhea, or death. This study developed a detection method based on real-time polymerase chain reaction that targets the sfmD gene of S. flexneri given that the virulence factor encoded by this gene is thought to facilitate adhesion to a host surface. The proposed method involves primer design, DNA isolation, the optimization of primer annealing temperature, and confirmation, specificity, and sensitivity tests. A sfmD primer with an amplicon length of 155 bp was annealed to the target DNA at an optimized temperature of 60 ℃ (range 54 °C-62 °C). This primer pair amplified the target sequences at a cycle threshold (Ct) of 15.11 ± 0.38 and a melting temperature of 82.41 ± 0.01 ℃. The primer specificity test showed that the primer distinguished S. flexneri from nontarget bacteria. The findings also revealed that the primer detected S. flexneri down to a limit of 16 pg/μL at a Ct of 26.68, equivalent to 2.79×102 CFU. Overall, the sfmD primer can effectively amplify S. flexneri DNA. Future research can be directed toward the detection of S. flexneri in artificially contaminated food samples to validate the real-food applicability and strengthen its potential use in food safety monitoring and clinical diagnostics. © 2025, Universitas Indonesia. All rights reserved.
Department of Chemistry, Faculty of Mathematics and Natural Science, Universitas Negeri Jakarta, Jakarta, 13220, Indonesia; Research Center for Detection of Pathogenic Bacteria, Lembaga Penelitian dan Pengabdian Masyakarat, Universitas Negeri Jakarta, Jakarta, 13220, Indonesia; Department of Chemistry, Faculty of Mathematics and Natural Sciences, Universitas Indonesia, Depok, 16424, Indonesia; Research Center for Testing Technology and Standard, National Research and Innovation Agency, Tangerang, 15314, Indonesia; Arnold Palmer Hospital Pediatric Specialty Diagnostic Laboratory, 32806, FL, United States; Innovation Center in Agritechnology for Andvanced Bioprocessing, Universiti Teknologi Malaysia, Johor, 84600, Malaysia; School of Chemical and Energy Engineering, Faculty of Engineering, Universiti Teknologi Malaysia, Johor Bahru, 81310, Malaysia; City of Scientific Research and Technology Applications, Alexandria, 21935, Egypt; Department of Safety, Health and Environmental Engineering, National Kaohsiung University of Science and Technology, Kaohsiung City, 82445, Taiwan
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