Nucleic acid lateral flow immunoassay (NALFIA) with integrated DNA probe degradation for the rapid detection of Cronobacter sakazakii and Cronobacter malonaticus in powdered infant formula

Abstract

A novel nucleic acid lateral flow immunoassay (NALFIA) system for Cronobacter (C.) sakazakii and C. malonaticus is based on amplification of the rpoB gene in the presence of a heterobifunctional, Cronobacter-specific DNA probe. Partial degradation of this probe during amplification is then detected by lateral flow immunoassay. The NALFIA detected all of the 22 C. sakazakii and 8 C. malonaticus isolates under study, but showed no reaction with other Cronobacter species or non-Cronobacter species. The minimal visual and instrumental detection limits were 104 cfu/ml for both species in buffered peptone water and in reconstituted infant formula. C. sakazakii or C. malonaticus at levels of 100-101 cfu/g in powdered infant formula were reliably detected by NALFI after reconstitution and 24 h incubation at 37 °C. A major advantage of this approach is that the use of an integrated DNA probe provides additional test specificity. Since C. sakazakii and C. malonaticus are by far the most important Cronobacter species occurring in PIF, the novel NALFIA is a promising tool to enhance rapid control of infant formula.