We describe a multiplex PCR for the rapid detection of Salmonella enterica serovars Enteritidis, Hadar, Heidelberg, and Typhimurium. Specific Salmonella serovars can be identified by targeting a multiplex PCR to genes and sequences unique to the O-antigen biosynthesis cluster and flagellin of a given serovar. Serovar is assigned then to a Salmonella isolate based on the appearance of specific, size amplicons (PCR product) corresponding to the target allele.
Total time
~3-4 hours per sample (including PCR cycling and gel electrophoresis)
Steps
1
Prepare whole cell Salmonella template
Prepare whole cell lysate from Salmonella isolates to serve as PCR template. This step eliminates the need for DNA extraction.
▶ 02:00
2
Set up multiplex PCR reaction
Assemble multiplex PCR reactions targeting O-antigen biosynthesis cluster and flagellin genes specific to each serovar. Include appropriate controls and cycling parameters.
▶ 03:29
3
Perform gel electrophoresis on PCR products
Run PCR amplicons on agarose gel to separate products by size. Visualize and document bands for serovar assignment.
▶ 06:45
4
Identify serovars from amplicon patterns
Assign Salmonella serovar (Enteritidis, Hadar, Heidelberg, or Typhimurium) based on the specific combination of PCR product sizes present for each isolate.
▶ 09:20