Name: an allelotyping pcr for identifying salmonella enterica serovars enteritidis, hadar, heidelberg, and typhimurium
Uploaded: 2011-07-22T13:00:00
Duration: 12 min 18 s
Description: Watch this Scientific Journal Video about An Allelotyping PCR for Identifying Salmonella enterica serovars Enteritidis, Hadar, Heidelberg, and Typhimurium at JoVE.com

This work has been supported by USDA Grants 1999-35212-8680, 2005-01378, and 2010-04288-01, USDA Formula Funds and the State of Georgia&#x2019;s Veterinary Medicine Agricultural Research Grant. The protocol described in this publication was developed for use by undergraduates as part of directed research courses: MIBO 4900L, GENE 4960H, BCMB 4960L, and BIOL 4960H at the University of Georgia and used by the students on several molecular epidemiology research projects in the Maurer laboratory. We wish to thank the many undergraduate students who have performed research in Maurer and Lee laboratories, and our department chair, John Glisson for supporting our efforts to integrate undergraduate instruction with research.

<ol>
	<li>Dauga, C., Zabrovskaia, A., Grimont, P. A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Restriction+fragment+length+polymorphism+analysis+of+some+flagellin+genes+of+Salmonella+enterica.">Restriction fragment length polymorphism analysis of some flagellin genes of Salmonella enterica.</a> J. Clin. Microbiol. 36, 2835-2843 .</li><li>Hong, Y., Liu, T., Lee, M. D., Hofacre, C. L., Maier, M., White, D. G., Ayers, S., Wang, L., Berghaus, R., Maurer, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+rapid+screen+of+broth+enrichments+for+Salmonella+enterica+serovars+Enteritidis,+Hadar,+Heidelberg,+and+Typhimurium+using+an+allelotyping+multiplex+PCR+that+targets+O+and+H+antigen+alleles.">A rapid screen of broth enrichments for Salmonella enterica serovars Enteritidis, Hadar, Heidelberg, and Typhimurium using an allelotyping multiplex PCR that targets O and H antigen alleles.</a> J. Food Prot. 72, 2198-2201 (2009).</li><li>Hong, Y., Liu, T., Lee, H. o. f. a. c. r. e., Maier, C. L., White, M., Ayers, D. G., Wang, S., Berghaus, L., R, M. a. u. r. e. r. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=J.Rapid+screening+of+Salmonella+enterica+serovars+Enteritidis,+Hadar,+Heidelberg+and+Typhimurium+using+a+serologically-correlative+allelotyping+PCR+targeting+the+O+and+H+antigen+alleles.">J.Rapid screening of Salmonella enterica serovars Enteritidis, Hadar, Heidelberg and Typhimurium using a serologically-correlative allelotyping PCR targeting the O and H antigen alleles.</a> BMC Microbiol. , (2008).</li><li>Liu, T., Liljebjelke, K., Bartlett, E., Hofacre, C. L., Sanchez, S., Maurer, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Application+of+nested+PCR+to+detection+of+Salmonella+in+poultry+environments.">Application of nested PCR to detection of Salmonella in poultry environments.</a> J. Food Prot. 65, 1227-1232 (2002).</li><li>Maurer, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+detection+and+limitation+of+molecular+techniques.">Rapid detection and limitation of molecular techniques.</a> Ann. Rev. Food Sci. Tech. 2, 259-279 (2011).</li><li>Moscoso, H., Alvarado, I., Hofacre, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+analysis+of+infectious+bursal+disease+virus+from+bursal+tissues+collected+on+FTA+filter+paper.">Molecular analysis of infectious bursal disease virus from bursal tissues collected on FTA filter paper.</a> Avian Dis. 50, 391-396 (2006).</li><li>Sambrook, J., Fritsch, E. F., Maniatis, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Molecular cloning: a laboratory. , (1989).</li><li>Wittwer, C. T., Fillmore, G. C., Hillyard, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Automated+polymerase+chain+reaction+capillary+tubes+with+hot+air.">Automated polymerase chain reaction capillary tubes with hot air.</a> Nucleic Acids Res. 17, 4353-4357 (1989).</li></ol>

No conflicts of interest declared.

Most commercially available PCR tests for the detection of Salmonella targets genes unique to the genus (ex. invA). However, not all Salmonella are equal in their ability to cause disease in humans or prevalence in animal populations. Early recognition of antigenic differences in Salmonella LPS O-antigen and flagellinH1 and H2 antigens has lead to the differentiation of Salmonella into over 2,500 serovars based on these antigenic differences. There are 46 distinct O antigens and 86 distinct flagellin (H) antigens identified in the genus Salmonella. Salmonella may possess one (H1) or two different (H1&amp;H2) flagellins. The different combination of O, H1, and H2 antigens account for the 2,500 Salmonella serovars recognized today. A serovar is assigned based on the antigenic formula derived from the identification of the individual O and H antigens. The antigenic formula is written as O: H1: H2; and where "-", refers to the absence of H1 or H2 flagellin and "NT" (non-typeable) for Salmonella negative for the O-antigen. For example, the serovar Typhimurium is assigned to a S. enterica isolate with the antigenic formula B: i: 1,2 while Enteritidis is given to an isolate with the formula D1: g,m: -. This antigenic variation in the Salmonella population is the outward manifestation of differences at the nucleotide level that can therefore be easily exploited by PCR. 
We have identified the genetic differences associated with specific O and H alleles to develop an allelotyping PCR for identifying S. enterica serovars: Enteritidis, Hadar, Heidelberg, and Typhimurium (3). The correct assignment and reporting of Salmonella serovar requires testing for all alleles associated with O: H1: H2 antigenic formulas for Enteritidis (D1: g,m: -), Hadar (C2: z10: e,n,x), Heidelberg (B: r: 1,2), and Typhimurium (B: i: 1,2). Without knowledge of O allele or antigen, the finding of the H1 g,m allele alone does not necessarily identify the Salmonella isolate as Enteritidis as other Salmonella serovars: Agona, California, and Montevideo, also possess this same allele. While the allelotyping PCR was originally designed to detect the fore mentioned Salmonella serovars, this test can identify an additional 19 serovars (Table 3). Our allelotyping PCR was originally designed to detect O and H alleles. In practice, it has become more practical and cost effective for us to identify the O-antigen by slide agglutination test, using O-specific antisera, rather than performing the O allelotyping PCR when working with isolated Salmonella cultures. However, we do recommend using the O allelotyping PCR if your lab uses this PCR as a screen (2) or for typing Salmonella isolates submitted on FTA cards (6), and include a Salmonella-specific PCR with the first screen of samples (4). Limit the allelotyping PCRs to only those samples that are identified as Salmonella by PCR or biochemical/antigenic tests.
The protocol described in this article has been optimized for use with the Idaho Technology Rapidcycler, a hot-air thermocycler that allows one to scale down reaction volumes and runs reaction conditions in less time than many heating block thermocyclers. To adapt this protocol for use with a heating block thermocycler may require optimizing reaction conditions empirically by lowering/raising annealing temperatures; adjusting primer concentrations; or adjusting MgCl2 concentrations. For example, we have had to adapt the protocol for the MJ Research PTC-200 thermocycler as follows. Working with the same reaction volumes described in Table 1, the working stock concentrationof the i primer set was diluted to 2.5 &mu;M, the annealing temperature was lowered from 55&deg;C to 45&deg;C, and incubation times at the denaturation, annealing and extension steps was lengthened to 20 s for the i/g, m allelotyping PCR. Having the appropriate positive and negative controls are essential in the initial start up and optimization for any PCR, including published protocols. We recommend acquiring known Salmonella serovars, specifically Anatum (E1: e,h: 1,6), Enteritidis (D1: g,m: -), Hadar (C2: z10: e,n,x), Heidelberg (B: r: 1,2), Montevideo (C1: g,m,s: 1,2,7) and Typhimurium (B: i: 1,2); and a laboratory Escherichia coli K12 strain (DH5&alpha;, LE393, JM109, etc.) as positive and negative controls respectively for the allelotyping PCR tests described in this article. Several of these Salmonella serovars will serve as either positive or negative controls, depending on which allele is tested.
Certain precautions and guidelines need to be followed while performing this and any other diagnostic PCR. First, the physical separation of template preparation, PCR set-up, and gel electrophoresis is critical in preventing possible PCR carry-over contamination and erroneous reporting of false-positives. In addition, the inclusion of appropriate controls is necessary in any routine PCR as these controls identify problems as they arise and assist in the interpretation of results (5). Most importantly, consistency is essential, especially with regards to the electrophoretic conditions for amplicon (PCR product) detection andestimation of amplicon size. To illustrate this point, we purposely suspended electrophoresis earlier than intended in Figure 1A. The molecular weight standards (lanes 1 and 13) and positive control (lane 2) are not sufficiently separated to accurately estimate sizes or observe separation of DNA fragments where there are small differences (&tilde;50 bp) in sizes. Adequate separation of DNA fragments, especially the molecular weight standards, is essential as reporting positives is dependent on the accurate estimation of amplicon size and distinguishing "true positive" from non-specific amplicons that are sometimes observed in the daily running of any PCR (5). For example, there is a non-specific amplicon in Figure 1B, lane 7 (&tilde;700 bp in size). Had electrophoresis been discontinued too early, when the dye front was only at the half way point in the agarose gel, there would be little differentiation between 500 bp and the 700 bp DNA fragments. Therefore, sample in lane 7 would have been erroneously reported as positive for both i and g,m alleles. 
Finally, one needs to recognize the limitations associated with any test. Several of the H1/H2 allelotyping PCR do not have the discriminatory power to discern subtle genetic differences in alleles belonging to the H2 1,2; 1,5; 1,6; 1,7 antigen complex; e,n,x/e,n,z15 antigen complex and the H1 G antigen complex, which includes the g,m allele. One or two amino acid changes account for the different epitopes present in these flagellar antigens. It was therefore difficult for us to design primers that could exploit these sometimes single base pair differences in the flagellin gene sequence (3). For example, Salmonella serovars Dublin (D1: g,p: -) and Berta (D1: f,g,t: -) are also PCR positive with our g,mallelotyping primers. The H2 allelotyping primers cannot distinguish Typhimurium (B: i : 1,2) from Lagos (B: i: 1,5), Heidelberg (B: r: 1,2) from Bradford (B: r: 1,5), Winneba (B: r: 1,6), or Remo ( B: r: 1,7), or Hadar (C2: z10: e,n,x) from Glostrup (C2: z10: e,n,z15). While the likelihood of encountering some of these serovars: Lagos, Bradford, Winneba, Remo or Glustrup is remote, it is a possibility and requires either providing disclaimer on the tests' limitation or another confirmatory test. 
In conclusion, this PCR-based serotyping rapidly identifies S. enterica serovars Enteritidis, Hadar, Heidelberg and Typhimurium, and O, H1, and H2 alleles associated with 19 additional serovars. This assay is a rapid, cost-effective alternative to traditional microbiological approach to serotyping Salmonella.

<table border="1">
 <tbody>
 <tr>
 <th>Material Name</th>
 <th>Type</th>
 <th>Company</th>
 <th>Catalogue Number</th>
 <th>Comment</th>
 </tr>
 <tr>
 <td>Luria Bertani</td>
 <td>&nbsp;</td>
 <td>Fisher</td>
 <td>&nbsp;</td>
 <td>(or any comparable source)</td>
 </tr>
 <tr>
 <td>Microfuge tubes</td>
 <td>1.5 ml capacity</td>
 <td>Fisher</td>
 <td>&nbsp;</td>
 <td>(or any comparable source)</td>
 </tr>
 <tr>
 <td>Ethanol</td>
 <td>200 proof</td>
 <td>Fisher</td>
 <td>&nbsp;</td>
 <td>(or any comparable source)</td>
 </tr>
 <tr>
 <td>dH2O</td>
 <td>Molecular Biology Grade</td>
 <td>Sigma</td>
 <td>&nbsp;</td>
 <td>(or any comparable source; PCR only)</td>
 </tr>
 <tr>
 <td>10 x PCR Buffer:</td>
 <td>20 mM MgCl2</td>
 <td>Idaho Technology</td>
 <td>&nbsp;</td>
 <td>(buffer comes with BSA, Ficoll and Tartrazine)</td>
 </tr>
 <tr>
 <td>&nbsp;</td>
 <td>30 mM MgCl2</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>&nbsp;</td>
 <td>40 mM MgCl2</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>PCR primers</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>DMSO</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>dNTP</td>
 <td>&nbsp;</td>
 <td>Roche</td>
 <td>&nbsp;</td>
 <td>(or any comparable source)</td>
 </tr>
 <tr>
 <td>Taq DNA Polymerase</td>
 <td>&nbsp;</td>
 <td>Denville</td>
 <td>&nbsp;</td>
 <td>(or any comparable source)</td>
 </tr>
 <tr>
 <td>Capillary pipettes</td>
 <td>10 &mu;l volume</td>
 <td>Idaho Technology</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Rapid Cycler</td>
 <td>&nbsp;</td>
 <td>Idaho Technology</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Agarose</td>
 <td>Low EEO; Molecular Biology Grade</td>
 <td>BioRad</td>
 <td>&nbsp;</td>
 <td>(or any comparable source)</td>
 </tr>
 <tr>
 <td>50 X TAE Buffer or 5X TBE Buffer</td>
 <td>&nbsp;</td>
 <td>Fisher</td>
 <td>&nbsp;</td>
 <td>(or any comparable source)</td>
 </tr>
 <tr>
 <td>Ethidium bromide</td>
 <td>&nbsp;</td>
 <td>BioRad</td>
 <td>&nbsp;</td>
 <td>(or any comparable source)</td>
 </tr>
 <tr>
 <td>Horizontal, submersible gel electrophoresis chamber with gel mold</td>
 <td>&nbsp;</td>
 <td>BioRad</td>
 <td>&nbsp;</td>
 <td>(or any comparable source)</td>
 </tr>
 <tr>
 <td>Power supply</td>
 <td>&nbsp;</td>
 <td>BioRad</td>
 <td>&nbsp;</td>
 <td>(or any comparable source)</td>
 </tr>
 <tr>
 <td>Molecular Imager Gel Doc System</td>
 <td>&nbsp;</td>
 <td>BioRad</td>
 <td>&nbsp;</td>
 <td>(or any comparable source)</td>
 </tr>
 </tbody>
</table>
Table 4. Materials

an allelotyping pcr for identifying salmonella enterica serovars enteritidis, hadar, heidelberg, and typhimurium

Current commercial PCRs tests for identifying Salmonella target genes unique to this genus. However, there are two species, six subspecies, and over 2,500 different Salmonella serovars, and not all are equal in their significance to public health. For example, finding S. enterica subspecies IIIa Arizona on a table egg layer farm is insignificant compared to the isolation of S. enterica subspecies I serovar Enteritidis, the leading cause of salmonellosis linked to the consumption of table eggs. Serovars are identified based on antigenic differences in lipopolysaccharide (LPS)(O antigen) and flagellin (H1 and H2 antigens). These antigenic differences are the outward appearance of the diversity of genes and gene alleles associated with this phenotype.
We have developed an allelotyping, multiplex PCR that keys on genetic differences between four major S. enterica subspecies I serovars found in poultry and associated with significant human disease in the US. The PCR primer pairs were targeted to key genes or sequences unique to a specific Salmonella serovar and designed to produce an amplicon with size specific for that gene or allele. Salmonella serovar is assigned to an isolate based on the combination of PCR test results for specific LPS and flagellin gene alleles. The multiplex PCRs described in this article are specific for the detection of S. enterica subspecies I serovars Enteritidis, Hadar, Heidelberg, and Typhimurium.
Here we demonstrate how to use the multiplex PCRs to identify serovar for a Salmonella isolate.

Watch this Scientific Journal Video about An Allelotyping PCR for Identifying Salmonella enterica serovars Enteritidis, Hadar, Heidelberg, and Typhimurium at JoVE.com

An Allelotyping PCR for Identifying Salmonella enterica serovars Enteritidis, Hadar, Heidelberg, and Typhimurium

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.

An Allelotyping PCR for Identifying Salmonella enterica serovars Enteritidis, Hadar, Heidelberg, and Typhimurium

Current commercial PCRs tests for identifying Salmonella target genes unique to this genus.  However, there are two species, six subspecies, and over ...

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