Development of a rapid Salmonella detection method via phage-conjugated magnetic bead separation coupled with real-time PCR quantification

摘要

In this study, we used the lytic Siphovirus phage LPST10 to develop a rapid and accurate detection method for Salmonella. Salmonella cells were separated from complex food matrices through magnetic separation, then subjected to SYBR green-based real-time quantitative PCR detection. Bacterial concentrations of 10(2)-10(6) (Colony forming unit per milliliter, CFU/mL), with 200 of mu g LPST10 phage-MBs, and 15 min reactions at 37 degrees C were found to be optimal to allow the phage-magnetic beads to capture the Salmonella cells. The phage-magnetic beads specifically recognized Salmonella cells of different serotypes. Under optimal conditions, DNA was extracted from the resulting bacteria phage-magnetic bead complexes through either alkaline lysis with sodium dodecyl sulfate (SDS) or boiling. The detection assay was subsequently assessed in food matrices (Tryptic soy broth medium, milk, and lettuce). The detection limit reached <30 CFU/mL in the food matrices. The entire assay, including bacterial capture, DNA extraction, and real-time polymerase chain reaction (RT-PCR) detection, could be completed within 1.5 h. This approach using a lytic Siphovirus phage has potential for rapid, specific, and accurate detection of Salmonella enterica subsp.enterica in different food matrices.