Rapid detection of Shigella flexneri on egg samples by the real time polymerase chain reaction method

Abstract

Shigella flexneri is one of the foodborne pathogen bacteria that caused food poisoning. Detection methods that are sensitive, specific and fast are very necessary in dealing with food poisoning, among these methods is Real Time PCR (RT-PCR). This study aims to apply the RT-PCR method to detect and quantify the Shigella flexneri bacteria in egg samples with the ipaH target gene. The amplicon of the target gene from the amplification process is 188 base pairs. Confirmation test of the ipaH primers pair with DNA template in concentration of Shigella flexneri culture of ±50 ng/μL gave the value of Cycle threshold (Ct) ±12. Primers sensitivity evaluation in detecting target bacteria gives the results that up to the smallest concentration of 8.05 pg/μL with a Ct value of 24.939. Specificity testing shows that the ipaH primers pair can differentiate Shigella flexneri bacteria significantly with some non-target bacteria as negative controls. Quantification of the number of bacteria found in egg samples using the flow of line equations by the RT-PCR method of 15.85 × 10-5CFU/mL. These results provide more sufficiently information compared to the culture method. Based on the results, it can be concluded that the RT-PCR method was successfully applied in detecting Shigella flexneri bacteria with the target ipaH gene in egg samples quickly, sensitive and specific as well as can determine the number of bacteria accurately.