inlJ Gene as a Potential Target Region for Detection of Listeria monocytogenes using real-time Polymerase Chain Reaction Jefferson Lynford Declan1,2, Muktiningsih Nurjayadi1,2, Gladys Indira Putri1,2, Dandy Akbar Juliansyah1,2, Ismaya Kridawati1,2, Irvan Maulana1,2, Maharanianska Azzahra1,2, Siti Fatimah2, Irma Ratna Kartika1,2, Fera Kurniadewi1,2, Vira Saamia3, I Made Wiranatha3, 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 andTechnology, 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
\(Listeria\) \(monocytogenes\) is a gram positive, rod-shape, and facultative anaerobic bacteria that cause food poisoning. These bacteria can cause listeriosis, with mild symptoms and even death with a mortality rate of up to 30%. This study aims to determine the potential of the \(inlJ\) gene fragment as a rapid detection with real-time Polymerase Chain Reaction for \(L.\) \(monocytogenes\). \(inlJ\) is a virulence factor of \(Listeria\) \(monocytogenes\) that contribute to attachment these bacteria to the host cells. This study used the DNA of \(L.\) \(monocytogenes\) with purity around 1.802 for A260/280 ratio and for amplification using concentration at 50ng/\(\mu\)L. The annealing temperature obtained from this research is 60 degrees Celsius based on the band in electrophoresis. The primer successfully amplifies the fragment of \(inlJ\) gene both PCR and rt-PCR with the amplicon size 200 bp and Ct at 15.95\(\pm\)0.3. This primer also can differentiate target and non-target bacteria based on the Ct and Tm values on rt-PCR. In addition, this primer success to detect DNA of \(L.\) \(monocytogenes\) as low as 71,3pg/\(\mu\)L. Based on this study, this primer has the potential to detect \(Listeria\) \(monocytogenes\). In the next step, it is necessary to examine the detection ability on artificial samples.
