The Potential of grxB Gene for Detection of C. sakazakii in Infant Formula Milk Using Real-Time Polymerase Chain Reaction

Cronobacter sakazakii is one of the bacteria that causes food poisoning that contaminates infant formula. This pathogen causes necrotizing enterocolitis, sepsis, and meningitis in infants or neonates with reported case fatality rates ranging from 40% to 80%. Therefore, it is necessary to develop fast and accurate detection of C. sakazakii in infant formula milk. This research aims to develop a method for detecting C. sakazakii bacteria using real-time PCR with high sensitivity, specificity, and accuracy. A rapid detection method using real-time PCR with the target gene grxB successfully detects the presence of C. sakazakii DNA in artificially contaminated formula milk. The results of the real-time PCR test showed that C. sakazakii DNA with a concentration of 53 ng/µL could be amplified by the grxB gene primer pair with a Ct value of 12 and a Tm value of 85.8ºC. The specificity test showed that the grxB primer could differentiate between target and some non-target bacteria. The sensitivity test showed the ability of the grxB primer to detect the smallest concentration of 3,392 pg/µL with a Ct of 24,06. Based on the results obtained, it can be concluded that the grxB primer has the potential to be used as rapid detection method for C. sakazakii bacteria in infant formula using real-time PCR

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

Validation of the Detection Kit for Pathogenic Bacteria Salmonella typhi Causes Food Poisoning with Real Time Polymerase Chain Reaction

Salmonella typhi is a gram-negative bacteria that causes food poisoning. Salmonella typhi bacteria can cause typhus, which can produce lipopolysaccharide complex endotoxin, which plays an important role in the pathogenesis oftyphoid fever. Cases of food poisoning are still common and are one of the causes of death and death in Indonesia. In 2019, there were 77 cases of food poisoning in Indonesia. A fast, accurate, and specific detection method is needed to detect poisoning. In previous studies, the optimum conditions and formulas for detecting these bacteria have been obtained using Real Time PCR. The results of previous studies are used as the basis for the development of a prototypedetection kit. In this study, validation was carried out, which aimed to confirm the results of previous studies so that a reproducible and accurate product for the detection of Salmonella typhi bacteria could be obtained. The results of this study showed that fim-C primers for Salmonella typhi were amplified at 95 base pairs (bp) with an annealing temperature of 60ºC and a standard DNA concentration of 50 ng/µL. The results of the Real Time PCR confirmation test of Salmonellatyphi bacteria at a pre-denaturation of 3 minutes with a concentration of 10 pmol obtained the Ct value according to thestandard with previous studies. The Ct value obtained was 13.96 for S. typhi bacteria. Based on the results obtained, it can be conclude that the condition validation stage for pure cultures was successfully carried out by producing consistentand reproducible data

Validation of the Detection Kit for Pathogenic Bacteria Salmonella typhi Causes Food Poisoning with Real Time Polymerase Chain Reaction

Salmonella typhi is a gram-negative bacteria that causes food poisoning. Salmonella typhi bacteria can cause typhus, which can produce lipopolysaccharide complex endotoxin, which plays an important role in the pathogenesis of typhoid fever. Cases of food poisoning are still common and are one of the causes of death and death in Indonesia. In 2019, there were 77 cases of food poisoning in Indonesia. A fast, accurate, and specific detection method is needed to detect poisoning. In previous studies, the optimum conditions and formulas for detecting these bacteria have been obtained using Real Time PCR. The results of previous studies are used as the basis for the development of a prototype detection kit. In this study, validation was carried out, which aimed to confirm the results of previous studies so that a reproducible and accurate product for the detection of Salmonella typhi bacteria could be obtained. The results of this study showed that fim-C primers for Salmonella typhi were amplified at 95 base pairs (bp) with an annealing temperature of 60ºC and a standard DNA concentration of 50 ng/µL. The results of the Real Time PCR confirmation test of Salmonella typhi bacteria at a pre-denaturation of 3 minutes with a concentration of 10 pmol obtained the Ct value according to thestandard with previous studies. The Ct value obtained was 13.96 for S. typhi bacteria. Based on the results obtained, it can be conclude that the condition validation stage for pure cultures was successfully carried out by producing consistentand reproducible data

The Potential of

Cronobacter sakazakii is one of the bacteria that causes food poisoning that contaminates infant formula. This pathogen causes necrotizing enterocolitis, sepsis, and meningitis in infants or neonates with reported case fatality rates ranging from 40% to 80%. Therefore, it is necessary to develop fast and accurate detection of C. sakazakii in infant formula milk. This research aims to develop a method for detecting C. sakazakii bacteria using real-time PCR with high sensitivity, specificity, and accuracy. A rapid detection method using real-time PCR with the target gene grxB successfully detects the presence of C. sakazakii DNA in artificially contaminated formula milk. The results of the real-time PCR test showed that C. sakazakii DNA with a concentration of 53 ng/µL could be amplified by the grxB gene primer pair with a Ct value of 12 and a Tm value of 85.8ºC. The specificity test showed that the grxB primer could differentiate between target and some non-target bacteria. The sensitivity test showed the ability of the grxB primer to detect the smallest concentration of 3,392 pg/µL with a Ct of 24,06. Based on the results obtained, it can be concluded that the grxB primer has the potential to be used as rapid detection method for C. sakazakii bacteria in infant formula using real-time PCR

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

Validation of the Detection Kit for Pathogenic Bacteria

Salmonella typhi is a gram-negative bacteria that causes food poisoning. Salmonella typhi bacteria can cause typhus, which can produce lipopolysaccharide complex endotoxin, which plays an important role in the pathogenesis oftyphoid fever. Cases of food poisoning are still common and are one of the causes of death and death in Indonesia. In 2019, there were 77 cases of food poisoning in Indonesia. A fast, accurate, and specific detection method is needed to detect poisoning. In previous studies, the optimum conditions and formulas for detecting these bacteria have been obtained using Real Time PCR. The results of previous studies are used as the basis for the development of a prototypedetection kit. In this study, validation was carried out, which aimed to confirm the results of previous studies so that a reproducible and accurate product for the detection of Salmonella typhi bacteria could be obtained. The results of this study showed that fim-C primers for Salmonella typhi were amplified at 95 base pairs (bp) with an annealing temperature of 60ºC and a standard DNA concentration of 50 ng/µL. The results of the Real Time PCR confirmation test of Salmonellatyphi bacteria at a pre-denaturation of 3 minutes with a concentration of 10 pmol obtained the Ct value according to thestandard with previous studies. The Ct value obtained was 13.96 for S. typhi bacteria. Based on the results obtained, it can be conclude that the condition validation stage for pure cultures was successfully carried out by producing consistentand reproducible data

Validation of the Detection Kit for Pathogenic Bacteria

Salmonella typhi is a gram-negative bacteria that causes food poisoning. Salmonella typhi bacteria can cause typhus, which can produce lipopolysaccharide complex endotoxin, which plays an important role in the pathogenesis of typhoid fever. Cases of food poisoning are still common and are one of the causes of death and death in Indonesia. In 2019, there were 77 cases of food poisoning in Indonesia. A fast, accurate, and specific detection method is needed to detect poisoning. In previous studies, the optimum conditions and formulas for detecting these bacteria have been obtained using Real Time PCR. The results of previous studies are used as the basis for the development of a prototype detection kit. In this study, validation was carried out, which aimed to confirm the results of previous studies so that a reproducible and accurate product for the detection of Salmonella typhi bacteria could be obtained. The results of this study showed that fim-C primers for Salmonella typhi were amplified at 95 base pairs (bp) with an annealing temperature of 60ºC and a standard DNA concentration of 50 ng/µL. The results of the Real Time PCR confirmation test of Salmonella typhi bacteria at a pre-denaturation of 3 minutes with a concentration of 10 pmol obtained the Ct value according to thestandard with previous studies. The Ct value obtained was 13.96 for S. typhi bacteria. Based on the results obtained, it can be conclude that the condition validation stage for pure cultures was successfully carried out by producing consistentand reproducible data