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

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

Detection of Salmonella typhimurium on artificially contaminated milk by real time PCR using STM4497 and fljB primers

Detection of food-borne bacterial pathogens was developed to overcome the limitations. The aim of this research was to develop Salmonella typhimurium detection by Real Time Polymerase Chain Reaction (RT-PCR) using two pairs of primers. The ability of primer pairs to detect S. typhimurium is seen from cycle threshold or Ct. Artificially contaminated milk sample with 24 ng each microliter can be detected with fljB (flagellin gene) primers on Ct 12,933 and with STM4497 (hypothetical protein code) primers on Ct 13,665. The specificity test of both primers showed that melting temperature (Tm) of fljB was 80,5 degree Celsius, and STM449 was 81,6 degree Celsius. FljB and STM4497 primers gave an average detection limit respectively of 11,75 Colony Forming Unit (CFU) each milliliter and 6,8 CFU each milliliter. The time needed throughout the detection process of S. typhimurium with fljB and STM4497 primers is faster than conventional methods. Based on the results it can be concluded that primers fljB and STM449 S. typhimurium can be applied to detection and quantification of S. typhimurium in milk samples

Primer concentration and Pre-denaturation Time Effect on

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