grxB gene as a potential target for the development of Cronobacter sakazakii detection method in infant formula milk using real-time PCR

Cronobacter sakazakii is a pathogen that causes necrotizing enterocolitis, sepsis, and meningitis in infants or neonates, with reported case-fatality rates varying from 40 to 80 percent. Therefore, it is necessary to develop fast and accurate detection of C. sakazakii. This study aims to get the primer pairs and optimal annealing temperature for the Polymerase Chain Reaction (PCR) method to detect C. sakazakii. The glutaredoxin B (grxB) gene was found suitable for detecting Cronobacter sakazakii, and a primer pair based on the grxB gene sequence was designed and synthesized. The genomic DNA of Cronobacter sakazakii strain ATCC 29544 was isolated and then used as a template for PCR assay. Optimization of running PCR conditions was carried out by varying the annealing temperature at 53-62°C, and the template DNA concentration of C. sakazakii is 53 ng/µL with a purity (A260/280) of 1,853. The result showed that the primer grxB gave optimal results at the annealing temperature range between 57-61°C and could amplify the grxB C. sakazakii fragment to produce an amplicon with a size of 151 base pairs (bp), and only a single band was formed. Furthermore, the primer design results and the optimum temperature obtained will be used to develop a sensitive and specific rapid detection method of C. sakazakii in food samples using real-time PCR.