Development of a test for the real-time fluorescence RNA targeted isothermal amplification to quickly identify Cronobacter species in powdered infant formula.

Foodborne bacteria such as Cronobacter are opportunistic and have been connected to potentially fatal infections. The current work created a fast and sensitive RNA targeting amplification and detection system for Cronobacter spp., including enrichment, RNA isolation and detection by real-time RNA isothermal amplification, capable of detecting viable Cronobacter spp. in powdered infant formula (PIF) and other food products. Total RNA was extracted to optimize Simultaneous Amplification and Testing (SAT) reaction conditions including concentrations of primers, molecular beacon, Mg²⁺, dNTP, and NTP. The optimized SAT assay targeting 16s/23s rRNA was used to demonstrate the specificity, sensitivity of the detection assay. Seven Cronobacter sakazakii strains and 24 control strains were examined in comparison that of real time PCR (SN/T 1632.3) and ISO 22964.The SAT assay showed sensitivity with detection limit of 2 log10 CFU/ml without pre-enrichment, 2 log CFU/10ml with 4 hours enrichment and 2 log CFU/1000ml with 7 hours pre-enrichment (The sensitivity of real-time PCR are 3 log CFU/ml without pre-enrichment, log CFU/ml with 4 hours pre-enrichment and 2 log CFU/10ml with 8 hours pre-enrichment) The new developed assay could provide results in 4 hours including enrichment, which has been significantly shortened compare with real time PCR method with overnight enrichment. Moreover, SAT assay did not give false positive results when detecting dead C. sakazakii (7-2 log CFU/ml). In contrast, the real-time PCR assay exhibited a detection limit equivalent to that for detecting viable bacteria.The developed SAT assay, combined with enrichment, provide a rapid, sensitive and simple approach, has great potential in the detection of Cronobacter species in baby formula and other food goods.