Development of Shigella flexneri Detection Method by real-time Polymerase Chain Reaction targeting the sfmD gene
Muktiningsih Nurjayadi1,2, Puan Aqila Azizah1,2, Agus Setiawan1,2, Ananda Indah Putri Sihombing1,2, Anisa Fitriyanti1,2, Grace1,2, Gusti Angieta Putri1,2, Helzi Angelina1,2, Royna Rahma Musie1,2, Jefferson Lynford Declan1,2, Gladys Indira Putri1,2, Dandy Akbar Juliansyah1,2, Siti Fatimah2, Tiara Fahriza 1,2, Irma Ratna Kartika1,2, Fera Kurniadewi1,2, Novitasari3, Bassam Abomoelak5, Hesham A. El Enshasy5,6,7

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.
3Research Center for Testing Technology and Standard, National Research and Innovation Agency (BRIN), Jl. Raya Puspitek Serpong, Tangerang Selatan 15314, Indonesia.
4Arnold Palmer Hospital Pediatric Specialty Diagnostic Laboratory, Orlando, FL 32806, USA.
5Innovation Center in Agritechnology for Advanced Bioprocessing (ICA), Universiti Teknologi Malaysia (UTM), Pagoh, Johor, Malaysia.
6School of Chemical and Energy Engineering, Faculty of Engineering, Universiti Teknologi Malaysia (UTM), Skudai, Johor Bahru, Malaysia.
7City of Scientific Research and Technology Applications, New Burg Al Arab, Alexandria, Egypt.

Abstract

\(Shigella\) \(flexneri\) can cause shigellosis, which can cause high fever, vomiting, diarrhea, and death. This study aims to develop a detection method using real-time PCR by targeting the \(sfmD\) gene of \(Shigella\) \(flexneri\). The virulence factor of the gene enables adhesion to the host surface. This method consists of primer design, DNA isolation, optimization of primer annealing temperature, confirmation, specificity, and sensitivity tests. The isolated DNA concentration and purity values were in the concentration of 142 ng/\(\mu\)L and purity of 1.92. Based on the optimization of primer in the range of 54-62 degrees Celsius, this study used \(sfmD\) primer with amplicons length of 155 bp and annealing temperature of 60 degrees Celsius. These pairs of primers amplified target sequences at Ct 15.11\(\pm\)0.38 with Tm 82.41 degrees Celsius\(\pm\)0.01. Primer specificity test obtained the results that primer can distinguish \(Shigella\) \(flexneri\) from non-target bacteria. The findings reveal that primer can identify \(Shigella\) \(flexneri\) up to a detection limit of 16 pg/\(\mu\)L at Ct 26.68 and equivalent to 2.79\(\times\)\(10^2\) CFU. This study concludes that \(sfmD\) primer amplified target DNA \(Shigella\) \(flexneri\). Further study can be conducted by detecting \(Shigella\) \(flexneri\) in artificially contaminated food samples.

Keywords: Shigella flexneri, sfmD gene, real-time PCR, Detection Method

Topic: Chemistry