Sensitive dual readout assays based on rolling circle amplification for fluorescent and colorimetric detection of Cronobacter spp. in powdered infant formula

Abstract

Cronobacter spp. are important opportunistic foodborne pathogens in powdered infant formula (PIF). In this study, a sensitive and specific assay was developed for fluorescent and colorimetric detection of Cronobacter spp. in PIF samples by using ligase chain reaction and rolling circle amplification (LCR-RCA) dual-amplification strategy. A pair of gap-padlock probes was designed to induce specific genomic DNA recognition and initiate the LCR. Hyperbranched rolling circle amplification (HRCA) combined with fluorescent dye and linear rolling circle amplification (LRCA) with AuNP probes were performed to achieve fluorescent or colorimetric detection of Cronobacter spp. The limits of detection (LODs) of the proposed assays were 8.6 × 101 CFU/mL (fluorescence) and 7.5 × 102 CFU/mL (colorimetric) in pure culture and 9.2 × 102 CFU/mL (fluorescence) and 8.4 × 103 CFU/mL (colorimetric) in spiked PIF samples, respectively. After 5–7 h enrichment, the proposed methods could detect as low as 100 CFU/mL bacteria in pure culture and PIF sample. The recovery of both methods was in the range of 89.4%–103.8% in pure culture or spiked PIF samples. The proposed assay has the potential to be a rapid and efficient method for detection of Cronobacter spp. in PIF.