Primer concentration and Pre-denaturation Time Effect on cyt-K Bacillus cereus Detection using Real-Time PCR Method

¹ Department 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
² Research Center for Detection of Pathogenic Bacteria, Lembaga Penelitian dan Pengabdian Kepada Masyarakat, Universitas Negeri Jakarta, Jl. Rawamangun Muka, Jakarta Timur 13220, Indonesia
³ Department of Biology, Faculty of Mathematics and Natural Science, Universitas Negeri Jakarta, Gedung KH. Hasjim Asj’ari, 6th Floor, Jl. Rawamangun Muka Jakarta Timur, 13220, Indonesia.
⁴ Center for Agricultural Quarantine Standards Test, Jl. Pemuda Rawamangun, Jakarta Timur, 13220, Indonesia
⁵ Center Forensic Laboratory of the Criminal Investigation, Police of the Republic of Indonesia, Cipambuan, Babakan Madang, Bogor, West Java 16810
⁶ Institute of Bioproduct Development (IBD), Universiti Teknologi Malaysia (UTM), Skudai, Johor Bahru, Johor, Malaysia.
⁷ School of Chemical and Energy Engineering, Universiti Teknologi Malaysia (UTM), Skudai, Johor Bahru, Johor, Malaysia.
⁸ City of Scientific Research and Technology Applications, New Burg Al Arab, Alexandria, Egypt.
⁹ Arnold Palmer Hospital Paediatric Speciality Diagnostic Laboratory, Orlando, FL 32806, USA

^* Corresponding author: muktiningsih@unj.ac.id

Abstract

Foodborne disease is a global threat that can affect all sections of society, both in developed or developing countries. Bacillus cereus is a Gram-positive bacteria that can cause food poisoning disease in humans. [2] Real-Time PCR detection method is one of the molecular marker methods that has been widely recognized as a fast, reliable, sensitive and specific detection tool for detecting pathogenic bacteria. In previous studies, the optimum condition and formulas applied for cyt-K 2 primer pairs have been obtained using Real-Time PCR. The purpose of this study is to find out the best conditions work of the primer pair cyt-K Bacillus cereus on detecting bacteria target using variations of pre-denaturation time and primer concentration with Real-Time PCR method. The annealing temperature used for PCR is at 60°C with sample concentration 50 ng/µL of B. cereus. Real-time PCR detection of variations in pre-denaturation time and primer concentration obtained the best conditions for primer pair cyt-K work at minute 4 with a primer concentration of 10 pmol and successfully amplifying the target by producing a Ct value of B. cereus at 13.04. Based on the results of the study, the primer pair cyt-K were reproducible in detecting the target gene and in the further step, this research can be continued to developed a prototype detection kit for foodborne pathogen bacteria using Real-Time PCR method.