Sensitive and visual detection of Cronobacter spp. in powdered infant formula by saltatory rolling circle amplification method

Cronobacter species are opportunistic foodborne pathogens involved in neonatal infections from powdered infant formula (PIF). In this study, a novel saltatory rolling circle amplification (SRCA) method has been developed for detecting Cronobacter spp. in PIF. 16 of Cronobacter spp. strains and 32 of non-Cronobacter strains were examined by using the DNA-directed RNA polymerase subunit beta (rpoB) gene. All Cronobacter strains were identified in pure cultures, whereas no SRCA products were found in non-Cronobacter strains. The detection limits were 3.4 x 10(2) CFU/mL through observing the white precipitate by naked eye, while 3.4 x 10(1) CFU/mL by fluorescence visualization. After enrichment, the detection limits of SRCA method in PIF were 8.7 x 10(0) CFU/g and 8.7 x 10(-1) CFU/g by visualization with white precipitate and fluorescence, respectively. Compared with the conventional PCR method, the SRCA has at least 100-fold higher sensitivity and 100-fold lower detection limit. In addition, 85 of PIF samples were investigated for the Cronobacter contamination, and the results were 100% sensitivity, 97.62% specificity and 97.65% accuracy compared with the ISO method. Therefore, the results show that SRCA is a sensitive and visual detection method for the detection of Cronobacter spp. in PIF with great potential.