Assessing the ecpA Gene as a Target for Detecting Klebsiella pneumoniae Using real-time Polymerase Chain Reaction Method
Muktiningsih Nurjayadi1,2, Agus Setiawan1,2, Ananda Indah Putri Sihombing1,2, Anisa Fitriyanti1,2, Grace1,2, Gusti Angieta Putri1,2, Helzi Angelina1,2, Puan Aqila Azizah1,2 , Royna Rahma Musie1,2, Jefferson Lynford Declan1,2, Gladys Indira Putri1,2, Dandy Akbar Juliansyah1,2, Siti Fatimah2, Atikah Nur Rahmawati 1,2, Irma Ratna Kartika1,2, Fera Kurniadewi1,2, Irwan Saputra1,2, Bassam Abomoelak3, Hesham A. El Enshasy4,5,6

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

\(Klebsiella\) \(pneumoniae\) is the most nosocomial pathogenic bacteria found in the hospital that causes infections such as pneumonia, liver abscesses, and urinary tract infections. The \(ecpA\) gene is one of the target genes of \(Klebsiella\) \(pneumoniae\) that functions in biofilm development to increase the ability of bacteria to colonize and as the main subunit of fimbriae. This study aims to determine the confirmation, sensitivity, and specificity test of pathogenic bacteria by real-time Polymerase Chain Reaction method. The\(ecpA\) primer has an amplicons length of 170 bp and has an annealing temperature of 60 degrees Celsius. These primer pair produced Ct test at 16.78\(\pm\)0.04 and Tm value of 86.33 degrees Celsius\(\pm\)0.12. The specificity test of the \(ecpA\) gene successfully differentiated between target and non-target bacteria based on Ct and Tm value. Therefore, these primers can detect \(Klebsiella\) \(pneumoniae\) bacteria at the smallest concentration until 1.06 pg/\(\mu\)L equivalent to 1.47\(\times\)\(10^4\) CFU. Based on these results, the \(ecpA\) primer successfully detected \(Klebsiella\) \(pneumoniae\) bacteria quickly, specifically and sensitively using the rt-PCR method. In the next step, these primer pairs can detect the artificial contaminated samples using rt-PCR method.

Keywords: Klebsiella pneumoniae, Pathogenic Bacteria, ecpA Gene, real-time Polymerase Chain Reaction.

Topic: Chemistry