pipB Gene as Detection Target for Development of Detection Method of Pathogenic Bacteria Salmonella typhi Using Real-Time PCR
Muktiningsih Nurjayadi1,2, Gusti Angieta Putri1,2, Ananda Indah Putri Sihombing1,2, Puan Aqila Azizah1,2, Anisa Fitriyanti1,2, Royna Rahma Musie1,2, Helzi Angelina1,2, Grace1,2, Agus Setiawan1,2, Dandy Akbar Juliansyah1,2, Jefferson Lynford Declan1,2, Gladys Indira Putri1,2, Siti Fatimah2, Adinda Myra Amalia Putri1,2, Vira Saamia3, Irma Ratna Kartika1,2, Fera Kurniadewi1,2, 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

\(Salmonella\) \(typhi\) is bacteria that lead to typhoid fever and one of the causes of death due to bacterial infections. In Indonesia, typhoid fever occurs around 1.100 cases per 100.000 population per year with a mortality rate of 3.1-10.4 percent. It is necessary to develop a rapid and accurate detection of \(S.\) \(typhi\). This study aims to test primers targeting the \(pipB\) gene. The method used primer design, DNA isolation, annealing temperature optimization, confirmation, specificity, and sensitivity test. The \(pipB\) gene has a function as an autophagia inhibitor in human. The \(pipB\) primer can amplified a DNA fragment of 196 bp at the optimum annealing temperature between 55-61 degrees Celsius. Confirmation test with real-time PCR found that \(pipB\) primers amplified at cycle to 13.11\(\pm\)0.07 with a Tm value of 84.13 degrees Celsius\(\pm\)0.07. The specificity test of these primers could distinguish target bacteria from non-target bacteria based on their Ct and Tm values. The sensitivity test of the primer pair obtained a LoD value of 55.78\(\times\)\(10^2\) CFU equivalent to 3.2 pg/\(\mu\)L. In conclusions, \(S.\) \(typhi\) \(pipB\) primers successfully detected \(S.\) \(typhi\) bacterial DNA by real-time PCR method. The next study can involve detecting \(S.\) \(typhi\) in artificially contaminated foods.

Keywords: detection method, pipB gene, real-time PCR, Salmonella typhi, typhoid

Topic: Chemistry