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Updated: Jun 16, 2026
An Allelotyping PCR for Identifying Salmonella enterica serovars Enteritidis, Hadar, Heidelberg, and Typhimurium
Published on: July 22, 2011
Development and evaluation of a multiplex real-time polymerase chain reaction procedure to clinically type prevalent
Nélida Muñoz1, Miguel Diaz-Osorio, Jaime Moreno
1Grupo de Microbiología, Instituto Nacional de Salud, Calle 26 51-20 CAN, Bogotá, Colombia. nmunoz@ins.gov.co
Abstract:
A multiplex real-time polymerase chain reaction procedure was developed to identify the most prevalent clinical isolates of Salmonella enterica subsp. enterica. Genes from the rfb, fliC, fljB, and viaB groups that encode the O, H, and Vi antigens were used to design 15 primer pairs and TaqMan probes specific for the genes rfbJ, wzx, fliC, fljB, wcdB, the sdf-l sequence, and invA, which was used as an internal amplification control. The primers and probes were variously combined into six sets. The first round of reactions used two of these sets to detect Salmonella O:4, O:9, O:7, O:8, and O:3,10 serogroups. Once the serogroups were identified, the results of a second round of reactions that used primers and probes for the flagellar antigen l genes, 1,2; e,h; g,m; d; e,n,x; and z(10), and the Vi gene were used to identify individual serovars. The procedure was standardized using 18 Salmonella reference strains and other enterobacteria. The procedure's reliability and sensitivity was evaluated using 267 randomly chosen serotyped Salmonella clinical isolates. The procedure had a sensitivity of 95.5% and was 100% specific. Thus, our technique is a quick, sensitive, reliable, and specific means of identifying S. enterica serovars and can be used in conjunction with traditional serotyping. Other primer and probe combinations could be used to increase the number of identifiable serovars.
Insights
A new multiplex real-time PCR method accurately identifies Salmonella enterica serovars. This rapid, sensitive, and specific technique enhances Salmonella detection in clinical isolates.
Area of Science:
- Microbiology
- Molecular Biology
- Immunology
Background:
- Salmonella enterica is a significant foodborne pathogen.
- Accurate serovar identification is crucial for public health surveillance and outbreak investigations.
- Traditional serotyping methods can be time-consuming and labor-intensive.
Purpose of the Study:
- To develop a multiplex real-time polymerase chain reaction (PCR) assay for rapid and accurate identification of prevalent Salmonella enterica serovars.
- To design specific primers and probes targeting genes encoding O, H, and Vi antigens.
- To evaluate the sensitivity and specificity of the developed PCR assay.
Main Methods:
- Developed a multiplex real-time PCR assay using 15 primer pairs and TaqMan probes.
- Targeted genes included rfbJ, wzx, fliC, fljB, wcdB, sdf-1, and invA (internal control).
- Assay involved two rounds of reactions to identify serogroups and then individual serovars.
Main Results:
- The assay demonstrated high sensitivity (95.5%) and 100% specificity.
- Successfully identified Salmonella O:4, O:9, O:7, O:8, and O:3,10 serogroups in the first round.
- Further identified specific serovars based on flagellar and Vi antigens in the second round.
Conclusions:
- The developed multiplex real-time PCR procedure is a quick, sensitive, reliable, and specific method for identifying S. enterica serovars.
- This molecular technique can complement traditional serotyping methods.
- The assay has the potential for broader application with additional primer and probe combinations.
