Development of a simultaneous amplification and testing (SAT) system based fluorescence real-time isothermal RNA amplification for rapid detection of Cronobacter spp. in powdered infant formula

Abstract

Contamination of Cronobacter spp. in powdered infant formulas is a severe food safety problem. The present study developed a rapid and sensitive Simultaneous Amplification and Testing (SAT) system for the detection of Cronobacter spp. in powdered infant formula. SAT detection system is based on fluorescence real-time isothermal RNA amplification and mainly includes pre-enrichment, RNA isolation and detection by fluorescence real-time RNA isothermal amplification. The amplification targets 16 s/23 s rRNA for the specific detection and rapid identification of Cronobacter spp. and can accurately detect viable strains in infant formulas and other food products. Here, six C. sakazakii strains and 25 references strains were examined using one pair of primers, having the accuracy of 100% in reference to conventional methods like ISO-IDF 22964 and real-time PCR. The SAT assay was proved to be highly sensitive with a detection limit of 102 CFU/mL without pre-enrichment for powdered infant formula. After 3 h, 4 h and 8 h enrichment, the sensitivity was increased up to 100, 10− 1 and 10− 3 CFU/mL of Cronobacter spp., respectively. The SAT system including pre-enrichment performed for Cronobacter spp. detection was less than 4 h, dramatically shortened, in comparison to several days using standard culturing method and overnight using pre-enrichment real-time PCR method. And more importantly, the SAT assay can accurately distinguish viable strains from the dead one. Taken together, the SAT assay combined with pre-enrichment established in the present study should provide a rapid, sensitive, efficient and specific method for direct detection of Cronobacter spp. in powdered infant formula. Moreover, a full automatic food-borne pathogenic bacteria detector was developed based on the SAT assay.