Minimum Detectable Level of Salmonellae Using a Binomial-Ice Based Bacterial Ice Nucleation Detection Assay (BIND®)

Abstract

A modified ice nucleation detection (BIND®) assay was used for rapid and sensitive detection of several Salmonella species. For the BIND assay, Salmonella cells are Infected with bacteriophage genetically modified to contain DNA encoding an ice nucleation protein (INP). After infection, de novo protein synthesis occurs and INPs are incorporated into the outer membrane of the organism. After supercoding (-9.3°C), only buffer solutins containing transfected salmonellae freeze, causing a phase-sensitive dye to change color. This technique, and a probability-based protocol modification, provided quantitative detection with a minimum detectable level (MDL) of 2.0 ± 0.3 S. enteritidis cells/mL in buffer (about 3 h). The MDLs for S. typhlmurium DT104and S. abaetetuba were 4.2 ± 0.2 and 11.1 ± 0.4 cells.mL, respectively. Using salmonellae-specific immunomagnetic bead separation technology in conjunction with the modified BIND protocol, we achieved an MDL of about 4.5 S. enteritidis cells/mL with an apparent capture efficiency of 56%.