Development of Detection Method of Escherichia coli Targeted yhaV Gene using real-time Polymerase Chain Reaction
Muktiningsih Nurjayadi1,2, Anisa Fitriyanti1,2, Royna Rahma Musie1,2, Gusti Angieta Putri1,2, Puan Aqila Azizah1,2, Helzi Angelina 1,2, Grace1,2, Ananda Indah Putri Sihombing1,2, Agus Setiawan1,2, Jefferson Lynford Declan1,2, Gladys Indira Putri1,2, Dandy Akbar Juliansyah1,2, Siti Fatimah2, Ayu Berkahingrum1,2, Irma Ratna Kartika1,2, Fera Kurniadewi1,2, Vira Saamia3, Shyi-Tien Chen4, Bassam Abomoelak5, Hesham A. El Enshasy6,7,8

1Department of Chemistry, Faculty of Mathematics and Natural Science, Universitas Negeri Jakarta, Gedung KH. Hasjim Asj^ari, 6th Floor, Jl. Rawamangun Muka, Jakarta Timur, 13220, Indonesia.
2Research Center for Detection of Pathogenic Bacteria, Lembaga Penelitian dan Pengabdian Kepada Masyarakat, Universitas Negeri Jakarta, Jl. Rawamangun Muka, Jakarta Timur, 13220, Indonesia.
3Center Forensic Laboratory of the Criminal Investigation, Police of the Republic of Indonesia, Cipambuan Babakan Madang, Bogor, 1681, Indonesia.
4Department of Safety, Health and Environmental Engineering, National Kaohsiung University of Science and Technology, No. 1 University Road, Yanchao District, Kaohsiung City 82445, Taiwan
5Arnold Palmer Hospital Pediatric Specialty Diagnostic Laboratory, Orlando, FL 32806, USA.
6Innovation Center in Agritechnology for Advanced Bioprocessing (ICA), Universiti Teknologi Malaysia (UTM), Pagoh, Johor, Malaysia.
7School of Chemical and Energy Engineering, Faculty of Engineering, Universiti Teknologi Malaysia (UTM), Skudai, Johor Bahru, Malaysia.
8City of Scientific Research and Technology Applications, New Burg Al Arab, Alexandria, Egypt.

Abstract

\(Escherichia\) \(coli\) is foodborne pathogenic bacteria that can cause diarrhea. The aims of this study are to determine the confirmation, specificity, and sensitivity test of the \(yhaV\) gene primer using real-time Polymerase Chain Reaction. The \(yhaV\) is a virulence gene that associated with the toxin-antitoxin system in \(E.\) \(coli\). This primer successfully to amplify DNA fragment with an amplicon length of 207 bp at an annealing temperature of 60 degrees Celsius by Gradient PCR. In addition, the primer pair produced Ct at 14.14\(\pm\)0.05 and showed one peak in melting curve with Tm value of 83.67 degrees Celsius \(\pm\)0.02. The \(yhaV\) primer also succeeded to distinguish target and non-target bacteria based on the differences in Ct and Tm values produced on the specificity test. The primer pair successfully detected \(Escherichia\) \(coli\) at the smallest concentration until 2.24 pg/\(\mu\)L with a Ct at 29.93, which has a detection limit of 31.5\(\times\)\(10^2\) CFU. Based on these results, it can be concluded that the \(yhaV\) primer can detect \(Escherichia\) \(coli\) using the real-time PCR method quick and accurate. In the next steps, these primers can be used to detect \(Escherichia\) \(coli\) bacteria in artificially contaminated and real food samples.

Keywords: Detection Method, Foodborne Pathogen, Escherichia coli, yhaV gene, real-time PCR

Topic: Chemistry