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Keywords:

  • Salmonella enterica serovar Typhi;
  • Salmonella enterica serovar Typhimurium;
  • mutiplex PCR;
  • comparative genomics

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

This study was designed to develop a multiplex PCR method with five specific primer pairs for the detection of Salmonella spp., Salmonella subspecies I, Salmonella enterica serovars Typhimurium, Typhi and Enteritidis. A multiplex PCR was constructed with five primer pairs for the detection of Salmonella and pathogenic Salmonella serovars, including a specific primer pair for Salmonella Typhi, based on the sequence comparison between genomic DNA sequences of 12 Salmonella strains. Each primer pair was specifically targeted to Salmonella spp., Salmonella subspecies I, Salmonella Typhimurium, Typhi and Enteritidis. This multiplex PCR was evaluated with various DNAs of Salmonella serovars that yielded high specificity for amplifying the expected PCR products of Salmonella serovars. Using this primer pair, a set of multiplex PCR was performed for the rapid identification of salmonellae and major pathogenic Salmonella serovars. Although this multiplex PCR method will need to be evaluated for a wide range of Salmonella serovars among multilaboratories, it should be useful for identifying clinically significant strains of Salmonella serovars rapidly and accurately without the need for serological testing.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Salmonella is classified into >2500 serovars based on the Kauffmann–White scheme (Popoff et al., 2003). Among the >2500 Salmonella serovars, several serovars have been identified as major pathogens to humans and domestic animals, including Salmonella Typhimurium, Enteritidis, Typhi, Newport, Heidelberg and Paratyphi A. Salmonellae are divided taxonomically into two species: Salmonella enterica and Salmonella bongori (subspecies V). Salmonella enterica is comprised of six subspecies, which include S. enterica ssp. enterica (I), S. enterica ssp. salamae (II), S. enterica ssp. arizonae (IIIa), S. enterica ssp. diarizonae (IIIb), S. enterica ssp. houtenae (IV) and S. enterica ssp. indica (VI). Salmonella subspecies I, which causes most infections in warm-blooded animals, consists of almost 1500 serovars (Popoff, 2001). Among subspecies I, Salmonella serovars Typhimurium, Enteritidis, Newport, Typhi, Paratyphi A, Paratyphi C and Choleraesuis account for most human and domestic animal Salmonella infections (Porwollik et al., 2002, 2004; Chan et al., 2003), and different serovars belonging to subspecies I have different host ranges, diseases and virulence potentials (Bäumler et al., 1998; Edwards et al., 2002). These distinguishable characteristics are caused by genetic differences in each of the Salmonella serovars.

To date, numerous identification methods have been suggested to replace or complement traditional serotyping. These methods comprise ribotyping (Esteban et et al., 1993), random-amplified polymorphic DNA (RAPD)-PCR, real-time PCR (Hoorfar et al., 2000), PCR-single-strand conformation polymorphism (SSCP) analysis (Nair et al., 2002), amplified fragment length polymorphism (AFLP) (Torpdahl & Ahrens, 2004), DNA sequence analysis (Mortimer et al., 2004), DNA arrays (Chan et al., 2003) and protein arrays (Cai et al., 2005). The problems associated with these methods include reproducibility of results between different laboratories for AFLP, RAPD-PCR and PCR-SSCP analysis, the requirement of specialized equipment, high analysis costs per sample and the need for well-trained personnel for DNA sequencing, real-time PCR, and DNA and protein microarrays. Therefore, the evaluation of specific primers with various Salmonella serovars is necessary for rapid and accurate detection of Salmonella spp. using multiplex PCR in the food industry and epidemiology.

Salmonella Typhi causes typhoid fever in humans and thus remains a serious public health problem in many developing countries (Parkhill et al., 2001; Edwards et al., 2002; Hirose et al., 2002; McClelland et al., 2004). Many studies have reported on the identification of S. Typhi using PCR targeted for the fliC gene (Song et al., 1993), the ViaB region (Hashimoto et al., 1995). Hirose et al. (2002) have also demonstrated the identification of S. Typhi using PCR targeted for the fliC gene, the ViaB region, rfbE and rfbS. However, these studies showed that each primer pair was not specific only to S. Typhi and that multiplex PCR using several targeted genes was needed for the specific detection of S. Typhi.

In this present study, the multiplex PCR method was developed for the specific detection of Salmonella spp., Salmonella subspecies I, S. Typhimurium, Typhi and Enteritidis in a single reaction. An S. Typhi-specific primer pair was prepared using comparative genomics along with other primer pairs used in previous studies (Wang & Yeh, 2002; Kim et al., 2006a). This multiplex PCR was evaluated with various genomic DNAs of Salmonella serovars for the rapid identification of Salmonella spp. and major pathogenic Salmonella serovars of Salmonella subspecies I.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Bacterial strains

Salmonella strains used in this study were collected from Korea KCPB, KCDC, Germany (Malorny et al., 2003) and the United States (Seo et al., 2004) and are listed in Table 1. Non-Salmonella strains including foodborne pathogens and Enterobacteriaceae were collected from the American Type Culture Collection (ATCC, Rockville, MD).

Table 1. Salmonella strains used in this study and multiplex PCR results
Salmonella subspecies and serovars (no.)SerogroupSourceMultiplex PCR results
STM3098-f2, r2STM4057-f, rSTM4497M2-f, rSTY1599-f, rIE 2L, 3RIAC
  1. No. number of isolates; +, positive result; −, negative result; w+, weak positive result; BFR, Federal Institute for Risk Assessment (Malorny et al., 2003); KCPB, Korea Consumer Protection Board (Chung et al., 2003); FDA, US Food and Drug Administration (CFSAN/OPDFB) (Seo et al., 2004); KCDC, Korea Centers for Diseases Control and Prevention.

S. enterica subspecies I
 Agona (3)BBFR, KCPB+++
 Agona B FDA+++
 AnatumE1FDA+++
 Barcilly FDA+++
 BlockleyC2–C3BFR+++
 BovismorbificansC2–C3BFR+++
 BraenderupC1FDA+++
 BrandenburgBBFR+++
 Bredeney (2)BBFR, FDA+++
 CaliforniaBFDA+++
 CerroKFDA+++
 CholeraesuisC1ATCC 13312+++
 Derby (2)BBFR, FDA+++
 DublinD1BFR++++
 EdinburgC1KCPB+++
 EnteritidisD1ATCC 4931++++
 Enteritidis (25)D1BFR, FDA, KCPB++++
 Georgia (2)C1KCPB+++
 Give E1E1FDA+++
 Haardt (5)C2–C3KCPB+++
 Hadar (2)C2–C3BFR, KCPB+++
 Heidelberg (3)BBFR, FDA+++
 Illinois FDA+++
 Infantis (3)C1BFR, FDA, KCPB+++
 IstanbulC2–C3KCPB+++
 Java B FDA+++
 JavianaD1FDA+++
 JoalE1KCPB+++
 KentuckyC2–C3FDA+++
 Litchfield (2)C2–C3BFR, FDA+++
 LivingstoneC1BFR+++
 MadeliaHFDA+++
 ManhatanC2–C3FDA+++
 MbandlakaC1FDA+++
 MeleagridisE1FDA+++
 Mhenohen FDA+++
 MississippiGFDA+++
 Montevideo (2)C1BFR, FDA+++
 MuensterE1FDA+++
 Newington FDA+++
 NewportC2–C3BFR+++
 OhioC1FDA+++
 OranienburgC1FDA+++
 Paratyphi AAKCPB+++
 Paratyphi BBATCC 10719+++
 Paratyphi CC1ATCC 13428+++
 PoonaGFDA+++
 SaintpaulBBFR+++
 SandowC2–C3KCPB+++
 SenftenbergE4BFR+++
 Schwarzenground (2)BKCPB+++
 TennesseeC1KCPB+++
 TyphiD1ATCC 33459++++
 Typhi (30)D1KCDC++++
 TyphimuriumBATCC 19585++++
 TyphimuriumBATCC 13311++++
 TyphimuriumBATCC 14028++++
 Typhimurium (6)BBFR, FDA, KCPB++++
 VirchowC1BFR+++
 Virginia (5)C2–C3KCPB+++
S. enterica subspecies IIIa
 S. enterica ssp. arizonae ATCC 13314++
 18:z4,z32:-KBFR++
 21:g,z51:-LBFR++
 47:r:-XBFR++
S. enterica subspecies IIIb
 S. enterica ssp. diarizonae ATCC 43973++
 18:i,v:zKBFR++
 47 : l,v:zXBFR++
 50:z:z52ZBFR++
S. enterica subspecies IV
 S. enterica ssp. houtenae ATCC 43974++
 11:z4,z23:-FBFR++
 16:z4,z32:-IBFR++
 48:g,z51:-YBFR++
S. enterica subspecies VI
 S. enterica ssp. indica ATCC 43976++
 1,6,14,25:a:e,n,xHBFR++
 41:b:1,7SBFR++
 45:a:e,n,xWBFR++
S. bongori (V)
 S. bongori ATCC 43975++
 44:r:-VBFRw++
 48:z35:-YBFRw++
 66:z65:- BFR++
Bacillus anthracis ATCC 14578 
Bacillus cereus (3) ATCC 10876, 11778, 14579 
Bacillus mycoides ATCC 6462 
Bacillus subtilis (2) ATCC 6051, 6633 
Bacillus thuringiensis (2) ATCC 10792, 35646 
Campylobacter jejuni ATCC 33560 
Citrobacter freundii ATCC 8090 
Clostrdium perfringens ATCC 13124 
Escherichia coli (4) ATCC 11775, 23736, 25922, 27325 
Escherichia coli O157:H7 ATCC 43894 
Hafnia alvei ATCC 9760 
Listeria monocytogenes (2) ATCC 19111, 19113 
Listeria ivanovii ATCC 19119 
Listeria innocua ATCC 33090 
Listeria grayi (2) ATCC 19120, 25401 
Proteus mirabilis ATCC 9921 
Proteus vulgaris ATCC 6380 
Shigella flexneri ATCC 12022 
Shigella boydii ATCC 8700 
Shigella sonnei ATCC 25931 
Staphylococcus aureus (3) ATCC 6538, 25923, 29737 
Staphylococcus epidermidis ATCC 12228 
Staphylococcus haemolyticus ATCC 29970 
Vibrio parahaemolyticus (2) ATCC 17802, 33844 
Yersinia enterocolitica ATCC 23715 

Genomic DNA extraction

The Salmonella strains were inoculated in Luria–Bertani broth and cultured at 37 °C, with vigorous shaking at 230 r.p.m. Genomic DNAs of the Salmonella strains were extracted using the DNeasy Tissue Kit (Qiagen, Hilden, Germany) according to the manufacturer's instruction. DNA concentrations were measured using a UV-spectrophotometer (Model UV-1700, Shimadzu, Tokyo, Japan), and genomic DNA exhibiting a spectrophotometric ratio of 1.8–2 (A260 nm/A280 nm) was used. Genomic DNA of Salmonella strains were diluted in distilled water to 25 ng μL−1.

Genome sequences of Salmonella species

Genome sequences and the sources of the Salmonella strains used in this study have been listed in previous reports (Kim et al., 2006a), and a total of 29 Salmonella genome sequences were used for the current study. Genome-sequencing projects for S. Typhi CT18 (AL513382), S. Typhimurium LT2 (AE006468), S. Typhi Ty2 (AE014613), S. Gallinarum 287/91 (AM933173) and S. Paratyphi A ATCC 9150 (CP000026) have been completed (McClelland et al., 2001, 2004; Parkhill et al., 2001; Deng et al., 2003) and their genomic sequences were obtained from the National Center for Biotechnology Information (NCBI, http://www.ncbi.nlm.nih.gov/). Additional 24 genome-sequencing projects of Salmonella strains were not yet completed when the study was conducted, but raw sequence data were obtained from the Sanger Institute (http://www.sanger.ac.uk/Projects/Salmonella/), Washington University (St. Louis, MO) and the University of Illinois (Champaign, IL). Genome sequences for S. Typhimurium DT104, Typhimurium SL1344, Typhi 17 isolates (Holt et al., 2008), Enteritidis PT4 and S. Bongori 12419 were obtained from the Sanger Institute (Hinxton, Cambridge, UK), Salmonella Dublin, Pullorum were obtained from the University of Illinois (http://www.salmonella.org/genomics/) and Salmonella Diarizonae serovar 61:1,v:1,5,(7) was obtained from the Genome Sequencing Center of the Washington University (http://genome.wustl.edu/genomes/p170).

Comparative genomics of S. Typhi CT18 among Salmonella serovars

A total of 4395 gene sequences (NC_003198.ffn) of S. Typhi CT18 were submitted to the nonredundant(nr) DNA sequence database of the NCBI using the blast program (version 2.2.5) (Altschul et al., 1997). blast outputs that matched the Salmonella genus were eliminated and the highest scored output for each of the 4395 gene sequences was selected. Based on the blast outputs, Salmonella specific-expected genes that had an nr database match score of <40.14 and a matched length of <21 bp were selected and compared with the genomic sequences of 11 Salmonella strains using the blastn program. Specific-expected genes of Typhi were selected based on the comparison pattern.

Primer construction and PCR conditions

A total of 14 primer pairs (Bionics, Seoul, Korea) expected to be specific for S. Typhi were constructed. These primer pairs were used for each genomic DNA from the various Salmonella serovars and 25-μL PCR mixtures were reacted with one sample containing 1 × Ex Taq buffer (Mg2+ plus), 0.4 μmol L−1 of each primer, 200 μmol L−1 of dNTP, 0.5 U of Ex Taq DNA polymerase (TaKaRa, Shiga, Japan) and 25 ng μL−1 of template DNA. PCR amplification was performed in a thermocycler (Model PC 808, ASTEC, Fukuoka, Japan) with an initial denaturation at 94 °C for 3 min, followed by 30 cycles of 94 °C for 45 s, annealing temperature according to each primer pair for 30 s, 72 °C for 30 s and finishing with a final extension at 72 °C for 3 min. Amplified products were run on a 1.5% agarose electrophoresis gel in 0.5 × Tris-acetate–EDTA buffer, stained with 0.5 μg mL−1 of ethidium bromide, visualized under UV-irradiation and photographed using a digital camera (COOLPIX 4300, Nikon, Tokyo, Japan).

Construction of the internal amplification control (IAC)

The IAC was constructed using the STM3098 gene for the primer pair STM3098-f2, r2 which has been used to detect Salmonella species. The 100-bp artificial oligonucleotide including STM3098-f2, r2 primer sequences was synthesized, and then amplified using PCR for subsequent cloning in the pGEM-T easy vector (Promega, Mannheim, Germany). The sequence of cloned IAC, comprised of the STM3098 gene for specific Salmonella spp., was confirmed by a blast program.

Multiplex PCR of Salmonella

Multiplex PCR was performed using five pairs of primers, which were targeted for Salmonella spp., Salmonella subspecies I, S. Typhimurium, Typhi and Enteritidis, and the design and sequences of the primer pairs are shown in Table 2. The concentrations of reagents in the multiplex PCR for one sample were the same as those mentioned in the previous section, except for the Ex Taq DNA polymerase and primer concentrations: Ex Taq DNA polymerase (1 U), STM3098-f2, r2 (0.1 μmol L−1), STM4057-f, r (0.04 μmol L−1), STM4497M2-f, r (0.6 μmol L−1), STY1599-f, r (0.064 μmol L−1) and IE 2L, 3R (0.32 μmol L−1). PCR mixtures were heated at 94 °C for 3 min and subsequently amplified for 30 cycles, each cycle consisting of 94 °C for 45 s, 67 °C for 30 s and 72 °C for 30 s; the 3-min final extension step at 72 °C was followed by holding the mixture at 4 °C using a thermocycler (Model PC 808, ASTEC).

Table 2.   Primer pairs of multiplex PCR used in this study and their sources
PrimerTarget gene (synonym)TargetPCR product size (bp)Primer concentration (μmol L−1)SequencesReference (source)
STY1599-fSTY1599Salmonella Typhi2030.0645′-TTACC CCACA GGAAG CACGC-3′In this study
STY1599-r    5′-CTCGT TCTCT GCCGT GTGGG-3′ 
STM4497M2-fSTM4497Salmonella Typhimurium3100.65′-AACAA CGGCT CCGGT AATGA GATTG-3′In this study
STM4497M2-r    5′-ATGAC AAACT CTTGA TTCTG AAGAT CG-3′ 
IE 2L Salmonella Enteritidis5590.325′-GGATA AGGGA TCGAT AATTG CTCAC-3′Wang & Yeh (2002)
IE 3R    5′-GGACT TCCAG TTATA GTAGG TGGCC-3′ 
STM4057-fSTM4057Salmonella subspecies I1370.045′-GGTGG CCTCG ATGAT TCCCG-3′Kim et al. (2006a)
STM4057-r    5′-CCCAC TTGTA GCGAG CGCCG-3′ 
STM3098-f2STM3098Salmonella spp.4230.15′-TTTGG CGGCG CAGGC GATTC-3′Kim et al. (2006a)
STM3098-r2    5′-GCCTC CGCCT CATCA ATCCG-3′ 

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Specific gene screening of S. Typhi CT18 using genomic sequence comparison

Among the 4395 genes of serovar Typhi CT18, 195 genes matching a score of <40.14 and a length <21 bp of nucleotides using the nr database based on blast results were selected to screen for the genes that were present only in Salmonella (data not shown). These 195 genes were compared with the previously reported list of S. Typhi genes, which are not present in Escherichia coli (Parkhill et al., 2001), and 172 of the 195 genes (88%) were overlapping.

The 195 selected genes were compared with each genome sequence from 28 Salmonella strains using the blast program, and 35 genes that were expected to be specific to S. Typhi were selected. These 35 genes have been included in previously reported results, and were considered unique to S. Typhi CT18 (Parkhill et al., 2001; Porwollik et al., 2004). Among the 35 genes in this study, 15 genes were included in the Typhi-specific region as reported previously. Among the 195 genes, the STY2042 and STY2046 genes exhibited a Typhi CT18-specific pattern when the Salmonella genomic DNA sequences were compared.

Specific PCR results with S. Typhi

Among the 35 selected genes, 14 primer pairs were constructed and evaluated with various genomic DNAs of Salmonella serovars. The constructed primer pairs did not amplify with non-Salmonella strains, with the exception of the STY4675 primer pairs (data not shown). Most of the constructed primer pairs revealed positive results for S. Typhi, including several Salmonella serovars. In particular, primer pairs for STY1594, STY1599 and STY2020 yielded highly specific amplified results with S. Typhi (Fig. 1; PCR results of STY1594 and STY2020 are not shown). Therefore, in this study, S. Typhi-specific primer pairs were chosen based on their potential utility for the detection and identification of S. Typhi. The results of selected primer pairs showed a relatively high specificity for S. Typhi, Paratyphi C, Paratyphi A and Choleraesuis. These results further confirmed the close genetic proximity between Typhi and Paratyphi A, as reported previously (Chan et al., 2003; McClelland et al., 2004; Porwollik et al., 2004; Liu et al., 2009).

image

Figure 1.  Specificity of PCR for detection of Salmonella Typhi using the primer pair for STY1599. M, 100-bp ladder DNA marker; lane 1, S. Typhimurium ATCC 19585; lane 2, S. Typhimurium ATCC 13311; lane 3, S. Typhimurium ATCC 14028; lane 4, S. Typhi ATCC 33459; lane 5, S. Paratyphi B ATCC 10719; lane 6, S. Paratyphi C ATCC 13428; lane 7, S. Enteritidis ATCC 4931; lane 8, S. Choleraesuis ATCC 13312; lane 9, S. Salamae ATCC 15793; lane 10, S. Arizonae ATCC 13314; lane 11, S. Diarizonae ATCC 43973; lane 12, S. Houtenae ATCC 43974; lane 13, S. Bongori ATCC 43975; lane 14, S. Indica ATCC 43976; and lane 15, nontemplate.

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IAC

To verify the false-negative PCR results, an IAC was constructed with the primer pair of STM3098-f2, r2. The IAC was coamplified with the target gene using STM3098-f2, r2 primers, resulting in 100-bp (IAC) and 423-bp (target gene) products. We performed the PCR using anywhere from 30 to 300 000 copy numbers of IAC with 25 ng of the target gene to determine the detection limit. The detection limit was achieved at 300 000 copies of IAC for 25 ng of Salmonella genomic DNA (30 cycles of amplification).

Multiplex PCR

Multiplex PCR for Salmonella identification was designed using five primer pairs for Salmonella spp., Salmonella subspecies I, S. Typhimurium, Typhi and Enteritidis as described in Table 2, which includes the primer sequences and products' size. Each primer pair for STM3098-f2, r2, STM4057-f, r, and STM4497M2-f, r, was independently specific to the Salmonella genus, Salmonella subspecies I and S. Typhimurium, respectively, as modified from previous reports (Kim et al., 2006a, b). The primer pair for STY1599 was used as a specific primer for S. Typhi. Sequences of specific primer pairs for S. Enteritidis were obtained from a previous report (Wang & Yeh, 2002). However, this primer pair was also positive for S. Dublin, as shown in Table 1, and consequently might be nonspecific to S. Dublin.

We performed a singlet PCR to confirm the specificity of five primers using various foodborne microorganisms including Listeria monocytogenes, Staphylococcus aureus, Bacillus cereus and others as listed in Table 1. None of these microorganisms yielded a positive response. Before performing the multiplex PCR, single PCRs of each primer pair with S. Typhimurium, Typhi and Enteritidis were conducted (Fig. 2; lanes 1–9) and specific PCR products were obtained. Also, multiplex PCR was reacted with the mixed genomic DNA of Salmonella serovars to evaluate the amplification of five PCR products (Fig. 2; lanes 10–12). Six amplified products are shown with the mixed genomic DNA of S. Typhimurium, Typhi and Enteritidis (Fig. 2; lane 13). The amplified PCR product sizes were 423 bp for Salmonella spp.-specific, 137 bp for Salmonella subspecies I-specific, 310 bp for S. Typhimurium-specific, 203 bp for Typhi-specific, 559 bp for Enteritidis-specific and 100 bp for IAC products.

image

Figure 2.  Specificity of each primer pair and multiplex PCR result pattern with the genomic DNA combination of Salmonella Typhimurium, Salmonella Enteritidis and Salmonella Typhi. Primer (lanes 1–3, STM3098-f2, r2; lanes 4–6, STM4057-f, r; lane 7, STM4497M2-f, r; lane 8, STY1599-f, r; lane 9, IE 2L-3R; lanes 10–13, multiplex PCR with five primer pairs). M, 100-bp ladder DNA marker; lanes 1, 4 and 7, S. Typhimurium ATCC 19585; lanes 2, 5 and 8, S. Typhi ATCC 33459; lanes 3, 6 and 9, S. Enteritidis ATCC 4931; lane 10, S. Typhimurium ATCC 19585, S. Enteritidis ATCC 4931; lane 11, S. Typhimurium ATCC 19585, S. Typhi ATCC 33459; lane 12, S. Typhi ATCC 33459, S. Enteritidis ATCC 4931; lane 13, S. Typhimurium ATCC 19585, S. Typhi ATCC 33459, S. Enteritidis ATCC 4931; and lane 14, nontemplate.

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Multiplex PCR was evaluated with various genomic DNAs from Salmonella including Salmonella subspecies I–VI (Fig. 3 and Table 1). Amplification with the STM3098-f2, r2 primer pair yielded a PCR product of 423 bp size with all Salmonella spp. including subspecies I–VI in lanes 1–14 (Fig. 3). The primer pair for STM4057-f, r yielded only an amplified PCR product of 137 bp with Salmonella subspecies I in lanes 1–8. The primer pairs for STM4497M2-f, r and STY1599-f, r yielded amplified specific PCR products of 310 and 203 bp with Typhimurium and Typhi, respectively (Fig. 3; lanes 1–4). The primer pair for IE 2L, 3R produced an amplified PCR product of 559 bp with Enteritidis (Fig. 3; lane 7).

image

Figure 3.  Multiplex PCR results with genomic DNA of various Salmonella type strains. M, 100-bp ladder DNA marker; lane 1, S. Typhimurium ATCC 19585; lane 2, S. Typhimurium ATCC 13311; lane 3, S. Typhimurium ATCC 14028; lane 4, S. Typhi ATCC 33459; lane 5, S. Paratyphi B ATCC 10719; lane 6, S. Paratyphi C ATCC 13428; lane 7, S. Enteritidis ATCC 4931; lane 8, S. Choleraesuis ATCC 13312; lane 9, S. Salamae ATCC 15793; lane 10, S. Arizonae ATCC 13314; lane 11, S. Diarizonae ATCC150 43973; lane 12, S. Houtenae ATCC 43974; lane 13, S. Bongori ATCC 43975; lane 14, S. Indica ATCC 43976; and lane 15, nontemplate.

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Identification of S. Typhi using PCR has been reported previously (Song et al., 1993; Hashimoto et al., 1995; Hirose et al., 2002). Primer pairs used in these studies were constructed based on the flagellin gene and the Vi antigen gene of S. Typhi. However, these primers are limited in their ability for the specific detection of S. Typhi because of their sequence similarity between Salmonella serovars. In a previous report, a specific primer pair for S. Typhimurium was obtained using a genomic DNA comparison approach (Kim et al., 2006b). Also, in this study, specific-expected genes of S. Typhi were suggested using genomic sequence comparisons. Using this information, 14 primer pairs were constructed and three primer pairs (STY1594, STY1599 and STY2020) were suggested as specific primers for S. Typhi. The three primer pairs yielded positive reactions for S. Typhi and negative results with other serovars.

A multiplex PCR-based method with three or more pairs of primers has been developed for the simultaneous detection and identification of S. Typhimurium (Lim et al., 2003) and Typhi (Hirose et al., 2002). Until now, these methods were designed to identify a single Salmonella serovar using multiplex PCR. However, in these studies, each primer pair of multiplex PCR was not specific enough to target a Salmonella serovar. Therefore, target Salmonella serovars could only be distinguished based on the pattern of positive and negative results from all primer pairs with multiplex PCR. Also, only relatively few serovars of Salmonella have been used to test the specificity of the primers. In a study by Kim et al. (2006a), a multiplex PCR method including five primer pairs was designed and each of the primer pairs displayed specificity to its own target for Salmonella spp., Salmonella subspecies I, S. Typhimurium, Typhi and Enteritidis (Kim et al., 2006a). These five primer pairs identified Salmonella in stages. First, two primer pairs (STM3098-f2, r2 and STM4057-f, r) yielded a high specificity for Salmonella spp. and Salmonella subspecies I. Next, the other three primer pairs (STM4497M2-f, r, STY1599-f, r and IE 2L, 3R) identified the major pathogenic Salmonella serovars, including Typhimurium, Typhi and Enteritidis. If an unknown sample resulted in two bands at 423 bp (STM3098-f2, r2) and 137 bp (STM4057-f, r) after multiplex PCR, the unknown sample would be identified as Salmonella subspecies I. Therefore, an unknown sample identified by this approach has the potential of being a pathogen for humans and warm-blooded animals. If an unknown sample resulted in three bands at 423, 137 and 203 bp (STY1599-f, r) after multiplex PCR, the unknown sample would be identified as S. Typhi. However, this multiplex PCR method for Salmonella identification needs to be further evaluated with a greater variety of Salmonella serovars in conjunction with other laboratories to demonstrate the accuracy of Salmonella identification in epidemiological and taxonomical studies.

In previous reports, specific primer pairs for S. Typhimurium were suggested and evaluated using a genome sequence comparison approach (Kim et al., 2006b). These results demonstrated that the genome sequence comparison approach is a highly useful tool for identifying for target genes and characterizing the genomic contents of bacteria among closely related bacterial species. Genotyping results and a new identification scheme for Salmonella based on PCR have been suggested (Kim et al., 2006a). Recently, virulotyping and serotype array have also been applied to the identification of Salmonella. Virulotyping can be performed with a 20 virulence-associated genes profile in S. enterica that enables rapid monitoring of emerging pathogenic Salmonella spp. (Khoo et al., 2009). An antibody microarray-based assay that allows parallel analysis of multiple antigens has also been investigated for Salmonella serotyping (Cai et al., 2005). Both methods would contribute to the more rapid and comprehensive identification of Salmonella serovars.

In this study, new target genes specific to S. Typhi were sought using the genomic DNA sequence comparison of Salmonella, and the specificity of these selected target genes was demonstrated using the PCR methodology. Also, a Salmonella identification system including major Salmonella pathogens was designed using a multiplex PCR approach with five primer pairs and evaluated with various Salmonella serovars for the identification of Salmonella spp., Salmonella subspecies I, S. Typhimurium, Typhi and Enteritidis as suggested in identification schemes from previous reports. Salmonella spp. is regarded as a human pathogen and also in domestic animals in both the public and the food industry sectors, and this multiplex PCR method would allow for a rapid and convenient alternative for the identification of Salmonella spp. and major Salmonella pathogens, in these settings, as it can be conducted by nonspecialized laboratories. Also, these results suggest the possibility for a new screening method for specific marker genes and probes using comparative genomic analysis for the detection of pathogens.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

We thank Dr Reiner Helmuth and Dr Burkhard Malorny of the Federal Institute for Risk Assessment (BFR, Molecular Biology, National Salmonella Reference Laboratory, Germany) and Dr K.H. Seo of US Food and Drug of Administration (FDA, CFSAN/OPDFB) for the kind donation of Salmonella strains.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
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