Development and evaluation of a PCR-ELISA assay for the detection and quantification of Cronobacter spp.

Abstract

In this study, a PCR-enzyme-linked immunosorbent assay (PCR-ELISA) was developed for the detection and quantification of Cronobacter spp. in powdered infant formula (PIF). The method uses digoxigenin- and biotin-labelled primers targeting the rpoB gene, and PCR products were detected using ELISA with antidigoxigenin-detecting antibodies. The specificity of the PCR-ELISA assay was evaluated with 39 strains, including 19 strains of Cronobacter spp. After 25 cycles of amplification, the PCR-ELISA assay showed a good linear relationship between cell numbers and PCR-ELISA values. The sensitivity of PCR-ELISA assay was 1.06×102 and 1.06×103cfumL-1 for pure cultures and PIF without enrichment, respectively. When the PCR-ELISA system was applied to artificially contaminated PIF, as few as 100cfu mL-1of Cronobacter sakazakii could be detected after 10h enrichment. The PCR-ELISA assay is thus a sensitive and accurate method for the rapid detection and quantification of Cronobacter spp. in PIF. © 2013 Elsevier Ltd.