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Journal : Makara Journal of Science

Detection Method for Escherichia coli Using Real-Time Polymerase Chain Reaction Targeting the yhaV Gene Nurjayadi, Muktiningsih; Fitriyanti, Anisa; Musie, Royna Rahma; Putri, Gusti Angieta; Azizah, Puan Aqila; Angelina, Helzi; Grace, Grace; Sihombing, Ananda Indah Putri; Setiawan, Agus; Declan, Jefferson Lynford; Putri, Gladys Indira; Juliansyah, Dandy Akbar; Fatimah, Siti; Berkahingrum, Ayu; Kartika, Irma Ratna; Kurniadewi, Fera; Saamia, Vira; Chen, Shyi-Tien; Aboemolak, Bassam; Enshasy, Hesham Ali El
Makara Journal of Science Vol. 29, No. 3
Publisher : UI Scholars Hub

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Abstract

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.
Development of Shigella flexneri Detection Method by Real-Time Polymerase Chain Reaction Targeting the sfmD Gene 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; Chen, Shyi-Tien
Makara Journal of Science Vol. 29, No. 4
Publisher : UI Scholars Hub

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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.
Co-Authors Abdul Hakam Barok Aboemolak, Bassam Abomoelak, Bassam Abumoelak, Bassam Achmad Ridwan Achmad Ridwan, Achmad Ade Danova Adinda Myra Amalia Putri Adinda Myra Amalia Putri Agung Purwanto AGUS SETIAWAN Ahmad Yudianto Aina Q Marof Aisyafitri, Afifah Akbar, Dandy Albana, Fadhila Shofi Alin Mardiah Aliyah, Elvira Zulfah Amalia, Ira Nur Ananda Indah Putri Sihombing Andika, Andika Prasetya Angelina, Helzi Anggraeni, Rosita Gio Anisa Fitriyanti Annisa Auliya Annisa Maharani Annisa, Cindy Ermintia Aqilah, Amadita Shafa Arief Rahmad Maulana Akbar Arieztania Rahmadhani Arif Rahman Asri Sulfianti Athiyah Layla Atikah Nur Rahmawati Atin Supiyani Aulia Siti Pathoni Aulia Widowati Aurellia, Dema Griseldis Axel Mareta Ayu Berkahingrum Azahra, Nur Fadillah Adelia Azidi Irwan Azizah, Puan Aqila Azzahra, Maharanianska Bassam Abomoelak Bassam Abomoelak Berkahingrum, Ayu Cahyani, Septia Dwi Chen, Shyi-Tien Dahlia Dahlia Dalia Sukmawati Dandy Akbar Juliansyah Dandy Akbar Juliansyah Dandy Akbar Juliansyah Daniel Joe Dailin Danova, Ade Darwis, Darsef Desi Lestari Desi, Desi Handayani Dessy Natalia Devi Aliefiyardi Dian Larasati Diana Vivanti Diat Nurhidayat Dudi Hardianto Dwi Ana Oktaviani El Enshasy, Hesham El Enshasy, Hesham Ali El-Enshasy, Hesham Ali Elenshasy, Hesham Ali Elsa Septiani Enshasy, Hesham Ali El Erdawati Erliasna, Emia Ezzaty NM Razif Fahriza, Tiara Farid Agouillal Fathya Putri Fajriani Fatmawati, Tri Kurnia Fera Kurnia Dewi Fera Kurnia Dewi Fera Kurnia Dewi Fera Kurniadewi Fera Kurniadewi Fera Kurniadewi Fernita Puspasari Fitri W Fitri, Ika Keumala Fitriyanti, Anisa Furqoni, Abdul Hadi Geta Putri Mentari Giri Alviyanto Gladys Indira Putri Gladys Indira Putri Gladys Indira Putri Grace Grace, Grace Gusti Angieta Putri Hairishah, Nazrisya Hati, Futi Kusuma Helzi Angelina Hermawati, Elvira Hesham A. El Enshasy Hesham Ali Al-Enshashy Hesham Ali El Enshasy Hesham Ali El-Enshasy I Made Wiranatha I Made Wiranatha I Nyoman Ruja IMAM SANTOSO Imanuelle Orchidea R P Irma Ratna Kartika Irvan Maulana Irvan Maulana Irwan Saputra Isfasona Bunga Falana Ismaya Krisdawati Ismaya Krisdawati Ismaya Krisdawati Izzatul Ilma Chairinnisa Jefferson Lynford Declan Johanes Erwin Tantaruna Juliansyah, Dandy Akbar Kartika, Irma Ratna Kinanti Istantia Chantika Krisdawati, Ismaya Kurnia Agustini Kurnia Agustini Kurniadewi, Fera Lanasari, Rossy Lilis Septyarini Lim J. R Listya Ayu Saraswati Maharani Azka Azzahra Maharanianska Azzahra Maria Paristiowati Masturdin, Masturdin Maulana, Irvan Mellyna Fitriani Meyliana Wulandari Musie, Royna Rahma Nabilla Anisa Putrie Nasywa Fhelia Salta Nida Nur Afifah Nita Aresanti Novi Permanasari Novi Wulandari Novianti Mantasha NOVITASARI Novitasari Novitasari Nurhayati Muslimah Nurullaili Malanul Hikmah Nur’aini, Ruhul Aflah Oni Soesanto Soesanto Pardiana Permatasari, Nur Azmi Priyani, Valda Tsabitha Puan Aqila Azizah Purnama, Yaktiva Dwi Putri Novitasari Putri, Adinda Myra Amalia Putri, Gladys Indira Putri, Gusti Angieta Putri, Nabilah Mustika Raden Haryo Bimo Setiarto Rahma, Wanda Amelia Rahmat Yunus Yunus Rahmawati, Atikah Nur Raihanah, Dinnah Ramadhan, Fitri Nur Rara Seruni Rara Seruni Retno Dwi Cahayningrum Rheva Amadea Rina Agustina Ririn Gustini Rizkahana Syehfia Rizki Fitria Roshanida A. Rahman Rosi Nurhujaimah Rosita Gio Anggraeni Royna Rahma Musie S, Restu.N S Saamia, Vira Saamia4, Vira Salsabila, Nadia Saputro, Dwi Anna Oktaviani Sara E Gomaa Sarah Adilisa Kartini Sari, Ely Puspita Setia Budi Shangkara, Muhammad Arkent Sheila Nurul Fadillah Octaviany Shyi-Tien Chen Sihombing, Ananda Indah Putri Sintia Mardita Siti Azizah Siti Fatimah SITI FATIMAH Siti Munawaoh Siti Munawaroh Siti Namiran Hadis Siti Zahroni Sondang N. Sihombing SRI RAHAYU Sri Rahayu Sulastri, Sindy Kania Sulistiyani Susilo, Ahmad Dwi Susilo, Tanto Budi Syamsul Bachri Tiara Fahriza Toton Sutono Tri Hastuti Budi Utami Tri Susanti Ucu Cahyana Ulfah Choiriyah Uswatul Nisa Vira Saamia Vira Saamia Vira Saamia Wahyuningtiyas, Kartika Putri Wella, Wella Aulia Putri Wiranatha, I Made Yohana Caroline Sihombing Yohannes Eka Cordias Buulolo Yuli Rahmawati Zarani M Taher Zeral, Lauzer