The Potential of htrA Gene to Identify Enterococcus faecalis using real-time Polymerase Chain Reaction Irma Ratna Kartika1,2, Grace1,2, Ananda Indah Putri Sihombing1,2, Agus Setiawan1,2, Anisa Fitriyanti1,2, Helzi Angelina1,2, Gusti Angieta1,2, Puan Aqila Azizah1,2, Royna Rahma Musie1,2, Dandy Akbar Juliansyah1,2, Jefferson Lynford Declan1,2, Gladys Indira Putri1,2, Siti Fatimah2, Ayu Berkahingrum1,2, Fera Kurniadewi1,2, Bassam Abomoelak3, Hesham A. El Enshasy4,5,6, Muktiningsih Nurjayadi1,2
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.
3Arnold Palmer Hospital Pediatric Specialty Diagnostic Laboratory, Orlando, FL 32806, USA.
4Innovation Center in Agritechnology for Advanced Bioprocessing (ICA), Universiti Teknologi Malaysia (UTM), Pagoh, Johor, Malaysia.
5School of Chemical and Energy Engineering, Faculty of Engineering, Universiti Teknologi Malaysia (UTM), Skudai, Johor Bahru, Malaysia.
6City of Scientific Research and Technology Applications, New Burg Al Arab, Alexandria, Egypt.
Abstract
\(Enterococcus\) \(faecalis\) is known as the opportunistic pathogens that can cause a variety of diseases. These bacteria are capable of producing enterotoxins that lead to gastrointestinal symptoms. This study aims to develop a real-time Polymerase Chain Reaction (PCR) method that is more efficient for identifying \(Enterococcus\) \(faecalis\) by assessing the results of the confirmation and specificity test of the htrA primer. The concentration of isolated DNA is 107 ng/\(\mu\)L, and the purities of A260/280 are 2.02. \(htrA\) primer successfully amplified \(Enterococcus\) \(faecalis\) DNA fragments at an annealing temperature of 60-62 degrees Celsius with an amplicon length of 162 base pairs. These pairs of primers amplified target sequences at a Ct value of 17.62\(\pm\)0.11 and a Tm value of 81.21 degrees Celsius \(\pm\)0.06. Specificity tests showed that the \(htrA\) primer could distinguish between target and several non-target bacteria. Based on these results, it can be concluded that the \(Enterococcus\) \(faecalis\) can be efficiently identified by these primers utilizing the real-time PCR. In order to create sensitive and specific detection methods, the next procedure will involve generating \(htrA\) primers to identify \(Enterococcus\) \(faecalis\) in sensitivity testing and artificially contaminated samples.
