Validation of real-time PCR for the detection of sfmD and gspA Shigella flexneri gene fragments and sifA Salmonella typhi gene
Muktiningsih Nurjayadi1,2, Ananda Indah Putri Sihombing1,2, Gusti Angieta Putri1,2, Puan Aqila Azizah1,2, Anisa Fitriyanti1,2, Royna Rahma Musie1,2, Helzi Angelina1,2, Grace1,2, Agus Setiawan1,2, Jefferson Lynford Declan1,2,Gladys Indira Putri1,2, Dandy Akbar Juliansyah1,2, Siti Fatimah1,2, Rosita Gio Anggraeni1,2, , Irwan Saputra1,2, Irma Ratna Kartika1,2, Fera Kurniadewi1,2, 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

\(Shigella\) \(flexneri\) and \(Salmonella\) \(typhi\) are foodborne bacteria that can cause to severe illness even death. This research aimed to validate the confirmation test for the primer pair of the \(sfmD\) and \(gspA\) genes of \(S.\) \(flexneri\), and the \(sifA\) gene of \(S.\) \(typhi\) using rt-PCR. Previous research using the mic rt-PCR Cycler instrument showed that the \(sfmD\) primer amplified the target gene with a Ct of 14.72 and a Tm of 82.39 degrees Celsius, the \(gspA\) primer with a Ct of 14.80 and a Tm of 79.34 degrees Celsius, and the \(sifA\) primer with a Ct of 17.08 and a Tm of 82.25 degrees Celsius. In this study, a validation test was conducted using the ViiA-7 rt-PCR System. It shows that the \(sfmD\) primer pair amplified the target gene with a Ct of 16.535 and a Tm of 81.7 degrees Celsius, the \(gspA\) primer with a Ct of 17.097 and a Tm of 78.231 degrees Celsius, and the \(sifA\) primer with a Ct of 17.002 and a Tm of 82.111 degrees Celsius. These primer pairs successfully amplified the target bacteria using the rt-PCR method with the ViiA-7 rt-PCR System. The next steps could involve specificity and sensitivity testing validation.

Keywords: real-time PCR, Shigella flexneri, Salmonella typhi, sfmD gene, gspA gene, sifA gene

Topic: Chemistry