Development of Shigella flexneri Detection Method by Real-Time Polymerase Chain Reaction Targeting the sfmD Gene

Authors

• Muktiningsih Nurjayadi, Department of Chemistry, Faculty of Mathematics and Natural Science, Universitas Negeri Jakarta, Jakarta 13220, IndonesiaFollow
• Puan Aqila Azizah, Department of Chemistry, Faculty of Mathematics and Natural Science, Universitas Negeri Jakarta, Jakarta 13220, Indonesia
• Agus Setiawan, Department of Chemistry, Faculty of Mathematics and Natural Science, Universitas Negeri Jakarta, Jakarta 13220, Indonesia
• Ananda Indah Putri Sihombing, Department of Chemistry, Faculty of Mathematics and Natural Science, Universitas Negeri Jakarta, Jakarta 13220, Indonesia
• Anisa Fitriyanti, Research Center for Detection of Pathogenic Bacteria, Lembaga Penelitian dan Pengabdian Masyakarat, Universitas Negeri Jakarta, Jakarta 13220, Indonesia
• Grace Grace, Department of Chemistry, Faculty of Mathematics and Natural Science, Universitas Negeri Jakarta, Jakarta 13220, Indonesia
• Gusti Angieta Putri, Department of Chemistry, Faculty of Mathematics and Natural Science, Universitas Negeri Jakarta, Jakarta 13220, Indonesia
• Helzi Angelina, Department of Chemistry, Faculty of Mathematics and Natural Science, Universitas Negeri Jakarta, Jakarta 13220, Indonesia
• Royna Rahma Musie, Department of Chemistry, Faculty of Mathematics and Natural Science, Universitas Negeri Jakarta, Jakarta 13220, Indonesia
• Jefferson Lynford Declan, Department of Chemistry, Faculty of Mathematics and Natural Science, Universitas Negeri Jakarta, Jakarta 13220, Indonesia
• Gladys Indira Putri, Department of Chemistry, Faculty of Mathematics and Natural Science, Universitas Negeri Jakarta, Jakarta 13220, Indonesia
• Dandy Akbar Juliansyah, Department of Chemistry, Faculty of Mathematics and Natural Science, Universitas Negeri Jakarta, Jakarta 13220, Indonesia
• Siti Fatimah, Research Center for Detection of Pathogenic Bacteria, Lembaga Penelitian dan Pengabdian Masyakarat, Universitas Negeri Jakarta, Jakarta 13220, Indonesia
• Tiara Fahriza, Department of Chemistry, Faculty of Mathematics and Natural Science, Universitas Negeri Jakarta, Jakarta 13220, Indonesia
• Irma Ratna Kartika, Department of Chemistry, Faculty of Mathematics and Natural Science, Universitas Negeri Jakarta, Jakarta 13220, Indonesia
• Fera Kurniadewi, Department of Chemistry, Faculty of Mathematics and Natural Science, Universitas Negeri Jakarta, Jakarta 13220, Indonesia
• Novitasari Novitasari, Research Center for Testing Technology and Standard, National Research and Innovation Agency, Tangerang 15314, Indonesia
• Bassam Abomoelak, Arnold Palmer Hospital Pediatric Specialty Diagnostic Laboratory, FL 32806, USA
• Hesham Ali El Enshasy, Innovation Center in Agritechnology for Andvanced Bioprocessing, Universiti Teknologi Malaysia, Johor 84600, Malaysia
• Shyi-Tien Chen, Department of Safety, Health and Environmental Engineering, National Kaohsiung University of Science and Technology, Kaohsiung City 82445, Taiwan

Abstract

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.

Recommended Citation

Nurjayadi, Muktiningsih; Azizah, Puan Aqila; Setiawan, Agus; Sihombing, Ananda Indah Putri; Fitriyanti, Anisa; Grace, Grace; Putri, Gusti Angieta; Angelina, Helzi; Musie, Royna Rahma; Declan, Jefferson Lynford; Putri, Gladys Indira; Juliansyah, Dandy Akbar; Fatimah, Siti; Fahriza, Tiara; Kartika, Irma Ratna; Kurniadewi, Fera; Novitasari, Novitasari; Abomoelak, Bassam; El Enshasy, Hesham Ali; and Chen, Shyi-Tien (2025) "Development of Shigella flexneri Detection Method by Real-Time Polymerase Chain Reaction Targeting the sfmD Gene," Makara Journal of Science: Vol. 29: Iss. 4, Article 14.
DOI: 10.7454/mss.v29i4.2722
Available at: https://scholarhub.ui.ac.id/science/vol29/iss4/14