RESEARCH LETTER

Identi¢cation of Salmonella enterica subspecies I, Salmonella

enterica serovars Typhimurium, Enteritidis and Typhi using
multiplex PCR
Si Hong Park1, Hyun Joong Kim1, Woo Hee Cho1, Jae Hwan Kim1, Mi Hwa Oh1, Sung Hun Kim2,
Bok Kwon Lee2, Steven C. Ricke3 & Hae Yeong Kim1
1
Institute of Life Sciences and Resources, Graduate School of Biotechnology, Kyung Hee University, Yongin, Korea; 2Centers for Infectious Diseases,
Division of Enteric Bacterial Infection, National Institute of Health, Seoul, Korea; and 3Department of Food Science, Center for Food Safety, University of
Arkansas, Fayetteville, AR, USA

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Correspondence: Hae Yeong Kim, Institute Abstract
of Life Sciences and Resources, Graduate
School of Biotechnology, Kyung Hee
This study was designed to develop a multiplex PCR method with five specific
University, Yongin 446-701, Korea. Tel.: 182 primer pairs for the detection of Salmonella spp., Salmonella subspecies I,
31 201 2660; fax: 182 31 204 8116; e-mail: Salmonella enterica serovars Typhimurium, Typhi and Enteritidis. A multiplex
[email protected] PCR was constructed with five primer pairs for the detection of Salmonella and
pathogenic Salmonella serovars, including a specific primer pair for Salmonella
Present address: Si Hong Park, Department Typhi, based on the sequence comparison between genomic DNA sequences of 12
of Food Science, Center for Food Safety, Salmonella strains. Each primer pair was specifically targeted to Salmonella spp.,
University of Arkansas, Fayetteville, AR
Salmonella subspecies I, Salmonella Typhimurium, Typhi and Enteritidis. This
72704-5690, USA.
multiplex PCR was evaluated with various DNAs of Salmonella serovars that
Received 13 May 2009; accepted 24 September
yielded high specificity for amplifying the expected PCR products of Salmonella
2009. serovars. Using this primer pair, a set of multiplex PCR was performed for the
Final version published online 16 October 2009. rapid identification of salmonellae and major pathogenic Salmonella serovars.
Although this multiplex PCR method will need to be evaluated for a wide range of
MICROBIOLOGY LETTERS

DOI:10.1111/j.1574-6968.2009.01809.x Salmonella serovars among multilaboratories, it should be useful for identifying

clinically significant strains of Salmonella serovars rapidly and accurately without
Editor: Craig Winstanley the need for serological testing.

Keywords
Salmonella enterica serovar Typhi; Salmonella
enterica serovar Typhimurium; mutiplex PCR;
comparative genomics.

indica (VI). Salmonella subspecies I, which causes most

Introduction infections in warm-blooded animals, consists of almost
Salmonella is classified into 4 2500 serovars based on the 1500 serovars (Popoff, 2001). Among subspecies I, Salmo-
Kauffmann–White scheme (Popoff et al., 2003). Among the nella serovars Typhimurium, Enteritidis, Newport, Typhi,
4 2500 Salmonella serovars, several serovars have been Paratyphi A, Paratyphi C and Choleraesuis account for most
identified as major pathogens to humans and domestic human and domestic animal Salmonella infections (Porwol-
animals, including Salmonella Typhimurium, Enteritidis, lik et al., 2002, 2004; Chan et al., 2003), and different
Typhi, Newport, Heidelberg and Paratyphi A. Salmonellae serovars belonging to subspecies I have different host ranges,
are divided taxonomically into two species: Salmonella diseases and virulence potentials (Bäumler et al., 1998;
enterica and Salmonella bongori (subspecies V). Salmonella Edwards et al., 2002). These distinguishable characteristics
enterica is comprised of six subspecies, which include are caused by genetic differences in each of the Salmonella
S. enterica ssp. enterica (I), S. enterica ssp. salamae (II), serovars.
S. enterica ssp. arizonae (IIIa), S. enterica ssp. diarizonae To date, numerous identification methods have been
(IIIb), S. enterica ssp. houtenae (IV) and S. enterica ssp. suggested to replace or complement traditional serotyping.

FEMS Microbiol Lett 301 (2009) 137–146 

c2009 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
138 S.H. Park et al.

These methods comprise ribotyping (Esteban et al., 1993), Genomic DNA extraction
random-amplified polymorphic DNA (RAPD)-PCR,
The Salmonella strains were inoculated in Luria–Bertani
real-time PCR (Hoorfar et al., 2000), PCR-single-strand
broth and cultured at 37 1C, with vigorous shaking at
conformation polymorphism (SSCP) analysis (Nair et al.,
230 r.p.m. Genomic DNAs of the Salmonella strains were
2002), amplified fragment length polymorphism
extracted using the DNeasy Tissue Kit (Qiagen, Hilden,
(AFLP) (Torpdahl & Ahrens, 2004), DNA sequence analysis
Germany) according to the manufacturer’s instruction.
(Mortimer et al., 2004), DNA arrays (Chan et al., 2003)
DNA concentrations were measured using a UV-spectro-
and protein arrays (Cai et al., 2005). The problems
photometer (Model UV-1700, Shimadzu, Tokyo, Japan),
associated with these methods include reproducibility of
and genomic DNA exhibiting a spectrophotometric ratio
results between different laboratories for AFLP, RAPD-PCR
of 1.8–2 (A260 nm/A280 nm) was used. Genomic DNA of
and PCR-SSCP analysis, the requirement of specialized
Salmonella strains were diluted in distilled water to
equipment, high analysis costs per sample and the need

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25 ng mL1.
for well-trained personnel for DNA sequencing, real-time
PCR, and DNA and protein microarrays. Therefore, the
evaluation of specific primers with various Salmonella Genome sequences of Salmonella species
serovars is necessary for rapid and accurate detection of Genome sequences and the sources of the Salmonella strains
Salmonella spp. using multiplex PCR in the food industry used in this study have been listed in previous reports (Kim
and epidemiology. et al., 2006a), and a total of 29 Salmonella genome sequences
Salmonella Typhi causes typhoid fever in humans and were used for the current study. Genome-sequencing pro-
thus remains a serious public health problem in many jects for S. Typhi CT18 (AL513382), S. Typhimurium LT2
developing countries (Parkhill et al., 2001; Edwards et al., (AE006468), S. Typhi Ty2 (AE014613), S. Gallinarum 287/91
2002; Hirose et al., 2002; McClelland et al., 2004). Many (AM933173) and S. Paratyphi A ATCC 9150 (CP000026)
studies have reported on the identification of S. Typhi using have been completed (McClelland et al., 2001, 2004; Parkhill
PCR targeted for the fliC gene (Song et al., 1993), the ViaB et al., 2001; Deng et al., 2003) and their genomic sequences
region (Hashimoto et al., 1995). Hirose et al. (2002) have were obtained from the National Center for Biotechnology
also demonstrated the identification of S. Typhi using PCR Information (NCBI, http://www.ncbi.nlm.nih.gov/). Addi-
targeted for the fliC gene, the ViaB region, rfbE and rfbS. tional 24 genome-sequencing projects of Salmonella strains
However, these studies showed that each primer pair was were not yet completed when the study was conducted, but
not specific only to S. Typhi and that multiplex PCR using raw sequence data were obtained from the Sanger Institute
several targeted genes was needed for the specific detection (http://www.sanger.ac.uk/Projects/Salmonella/), Washing-
of S. Typhi. ton University (St. Louis, MO) and the University of Illinois
In this present study, the multiplex PCR method (Champaign, IL). Genome sequences for S. Typhimurium
was developed for the specific detection of Salmonella DT104, Typhimurium SL1344, Typhi 17 isolates (Holt et al.,
spp., Salmonella subspecies I, S. Typhimurium, Typhi 2008), Enteritidis PT4 and S. Bongori 12419 were obtained
and Enteritidis in a single reaction. An S. Typhi-specific from the Sanger Institute (Hinxton, Cambridge, UK),
primer pair was prepared using comparative genomics along Salmonella Dublin, Pullorum were obtained from the Uni-
with other primer pairs used in previous studies (Wang & versity of Illinois (http://www.salmonella.org/genomics/)
Yeh, 2002; Kim et al., 2006a). This multiplex PCR was and Salmonella Diarizonae serovar 61:1,v:1,5,(7) was ob-
evaluated with various genomic DNAs of Salmonella tained from the Genome Sequencing Center of the Washing-
serovars for the rapid identification of Salmonella spp. ton University (http://genome.wustl.edu/genomes/p170).
and major pathogenic Salmonella serovars of Salmonella
subspecies I.
Comparative genomics of S. Typhi CT18 among
Salmonella serovars
Materials and methods A total of 4395 gene sequences (NC_003198.ffn) of S. Typhi
CT18 were submitted to the nonredundant(nr) DNA se-
Bacterial strains
quence database of the NCBI using the BLAST program
Salmonella strains used in this study were collected from (version 2.2.5) (Altschul et al., 1997). BLAST outputs that
Korea KCPB, KCDC, Germany (Malorny et al., 2003) and matched the Salmonella genus were eliminated and the
the United States (Seo et al., 2004) and are listed in Table 1. highest scored output for each of the 4395 gene sequences
Non-Salmonella strains including foodborne pathogens and was selected. Based on the BLAST outputs, Salmonella specific-
Enterobacteriaceae were collected from the American Type expected genes that had an nr database match score of
Culture Collection (ATCC, Rockville, MD). o 40.14 and a matched length of o 21 bp were selected and


c 2009 Federation of European Microbiological Societies FEMS Microbiol Lett 301 (2009) 137–146
Published by Blackwell Publishing Ltd. All rights reserved
 Table 1. Salmonella strains used in this study and multiplex PCR results
Multiplex PCR results

Salmonella subspecies and serovars (no.) Serogroup Source STM3098-f2, r2 STM4057-f, r STM4497M2-f, r STY1599-f, r IE 2L, 3R IAC
S. enterica subspecies I
Agona (3) B BFR, KCPB 1 1    1
Agona B FDA 1 1    1
Anatum E1 FDA 1 1    1
Barcilly FDA 1 1    1
Blockley C2–C3 BFR 1 1    1

FEMS Microbiol Lett 301 (2009) 137–146

Bovismorbificans C2–C3 BFR 1 1    1
Braenderup C1 FDA 1 1    1
Brandenburg B BFR 1 1    1
Bredeney (2) B BFR, FDA 1 1    1
California B FDA 1 1    1
Cerro K FDA 1 1    1
Identification of Salmonella serovars using multiplex PCR

Choleraesuis C1 ATCC 13312 1 1    1

Derby (2) B BFR, FDA 1 1    1
Dublin D1 BFR 1 1   1 1
Edinburg C1 KCPB 1 1    1
Enteritidis D1 ATCC 4931 1 1   1 1
Enteritidis (25) D1 BFR, FDA, KCPB 1 1   1 1
Georgia (2) C1 KCPB 1 1    1
Give E1 E1 FDA 1 1    1
Haardt (5) C2–C3 KCPB 1 1    1
Hadar (2) C2–C3 BFR, KCPB 1 1    1
Heidelberg (3) B BFR, FDA 1 1    1
Illinois FDA 1 1    1
Infantis (3) C1 BFR, FDA, KCPB 1 1    1

c
Istanbul C2–C3 KCPB 1 1    1


Java B FDA 1 1    1
Javiana D1 FDA 1 1    1
Joal E1 KCPB 1 1    1
Kentucky C2–C3 FDA 1 1    1
Litchfield (2) C2–C3 BFR, FDA 1 1    1
Livingstone C1 BFR 1 1    1
Madelia H FDA 1 1    1
Manhatan C2–C3 FDA 1 1    1
Mbandlaka C1 FDA 1 1    1
Meleagridis E1 FDA 1 1    1
Mhenohen FDA 1 1    1
Mississippi G FDA 1 1    1
Montevideo (2) C1 BFR, FDA 1 1    1
Muenster E1 FDA 1 1    1
139

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 c

140

Table 1. Continued.
Multiplex PCR results

Salmonella subspecies and serovars (no.) Serogroup Source STM3098-f2, r2 STM4057-f, r STM4497M2-f, r STY1599-f, r IE 2L, 3R IAC
Newington FDA 1 1    1
Newport C2–C3 BFR 1 1    1
Ohio C1 FDA 1 1    1
Oranienburg C1 FDA 1 1    1
Paratyphi A A KCPB 1 1    1
Paratyphi B B ATCC 10719 1 1    1
Paratyphi C C1 ATCC 13428 1 1    1
Poona G FDA 1 1    1
Saintpaul B BFR 1 1    1

Published by Blackwell Publishing Ltd. All rights reserved

Sandow C2–C3 KCPB 1 1    1

2009 Federation of European Microbiological Societies

Senftenberg E4 BFR 1 1    1
Schwarzenground (2) B KCPB 1 1    1
Tennessee C1 KCPB 1 1    1
Typhi D1 ATCC 33459 1 1  1  1
Typhi (30) D1 KCDC 1 1  1  1
Typhimurium B ATCC 19585 1 1 1   1
Typhimurium B ATCC 13311 1 1 1   1
Typhimurium B ATCC 14028 1 1 1   1
Typhimurium (6) B BFR, FDA, KCPB 1 1 1   1
Virchow C1 BFR 1 1    1
Virginia (5) C2–C3 KCPB 1 1    1
S. enterica subspecies IIIa
S. enterica ssp. arizonae ATCC 13314 1     1
18:z4,z32:- K BFR 1     1
21:g,z51:- L BFR 1     1
47:r:- X BFR 1     1
S. enterica subspecies IIIb
S. enterica ssp. diarizonae ATCC 43973 1     1
18:i,v:z K BFR 1     1
47 : l,v:z X BFR 1     1
50:z:z52 Z BFR 1     1
S. enterica subspecies IV
S. enterica ssp. houtenae ATCC 43974 1     1
11:z4,z23:- F BFR 1     1
16:z4,z32:- I BFR 1     1
48:g,z51:- Y BFR 1     1
S. enterica subspecies VI
S. enterica ssp. indica ATCC 43976 1     1
1,6,14,25:a:e,n,x H BFR 1     1
41:b:1,7 S BFR 1     1
S.H. Park et al.

FEMS Microbiol Lett 301 (2009) 137–146

45:a:e,n,x W BFR 1     1

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 S. bongori (V)
S. bongori ATCC 43975 1     1
44:r:- V BFR w1     1
48:z35:- Y BFR w1     1
66:z65:- BFR 1     1
Bacillus anthracis ATCC 14578     
Bacillus cereus (3) ATCC 10876, 11778, 14579     
Bacillus mycoides ATCC 6462     
Bacillus subtilis (2) ATCC 6051, 6633     

FEMS Microbiol Lett 301 (2009) 137–146

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     
Identification of Salmonella serovars using multiplex PCR

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     

c

Yersinia enterocolitica ATCC 23715     

No. number of isolates; 1, positive result;  , negative result; w1, 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.
141

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142 S.H. Park et al.

Table 2. Primer pairs of multiplex PCR used in this study and their sources
Primer
Target gene PCR product concentration
Primer (synonym) Target size (bp) (mmol L1) Sequences Reference (source)
STY1599-f STY1599 Salmonella Typhi 203 0.064 5 0 -TTACC CCACA GGAAG CACGC-3 0 In this study
STY1599-r 5 0 -CTCGT TCTCT GCCGT GTGGG-3 0
STM4497M2-f STM4497 Salmonella 310 0.6 5 0 -AACAA CGGCT CCGGT AATGA In this study
Typhimurium GATTG-3 0
STM4497M2-r 5 0 -ATGAC AAACT CTTGA TTCTG
AAGAT CG-3 0
IE 2L Salmonella 559 0.32 5 0 -GGATA AGGGA TCGAT AATTG Wang & Yeh (2002)
Enteritidis CTCAC-3 0

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IE 3R 5 0 -GGACT TCCAG TTATA GTAGG
TGGCC-3 0
STM4057-f STM4057 Salmonella 137 0.04 5 0 -GGTGG CCTCG ATGAT TCCCG-3 0 Kim et al. (2006a)
subspecies I
STM4057-r 5 0 -CCCAC TTGTA GCGAG CGCCG-3 0
STM3098-f2 STM3098 Salmonella spp. 423 0.1 5 0 -TTTGG CGGCG CAGGC GATTC-3 0 Kim et al. (2006a)
STM3098-r2 5 0 -GCCTC CGCCT CATCA ATCCG-3 0

compared with the genomic sequences of 11 Salmonella in the pGEM-T easy vector (Promega, Mannheim, Ger-
strains using the BLASTN program. Specific-expected genes of many). The sequence of cloned IAC, comprised of the
Typhi were selected based on the comparison pattern. STM3098 gene for specific Salmonella spp., was confirmed
by a BLAST program.
Primer construction and PCR conditions
Multiplex PCR of Salmonella
A total of 14 primer pairs (Bionics, Seoul, Korea) expected
to be specific for S. Typhi were constructed. These primer Multiplex PCR was performed using five pairs of primers,
pairs were used for each genomic DNA from the various which were targeted for Salmonella spp., Salmonella sub-
Salmonella serovars and 25-mL PCR mixtures were reacted species I, S. Typhimurium, Typhi and Enteritidis, and
with one sample containing 1  Ex TaqTM buffer (Mg21 the design and sequences of the primer pairs are shown in
plus), 0.4 mmol L1 of each primer, 200 mmol L1 of dNTP, Table 2. The concentrations of reagents in the multiplex
0.5 U of Ex Taq DNA polymerase (TaKaRa, Shiga, Japan) PCR for one sample were the same as those mentioned
and 25 ng mL1 of template DNA. PCR amplification was in the previous section, except for the Ex Taq DNA
performed in a thermocycler (Model PC 808, ASTEC, polymerase and primer concentrations: Ex Taq DNA poly-
Fukuoka, Japan) with an initial denaturation at 94 1C for merase (1 U), STM3098-f2, r2 (0.1 mmol L1), STM4057-f, r
3 min, followed by 30 cycles of 94 1C for 45 s, annealing (0.04 mmol L1), STM4497M2-f, r (0.6 mmol L1), STY1599-
temperature according to each primer pair for 30 s, 72 1C for f, r (0.064 mmol L1) and IE 2L, 3R (0.32 mmol L1). PCR
30 s and finishing with a final extension at 72 1C for 3 min. mixtures were heated at 94 1C for 3 min and subsequently
Amplified products were run on a 1.5% agarose electro- amplified for 30 cycles, each cycle consisting of 94 1C for
phoresis gel in 0.5  Tris-acetate–EDTA buffer, stained with 45 s, 67 1C for 30 s and 72 1C for 30 s; the 3-min final
0.5 mg mL1 of ethidium bromide, visualized under UV- extension step at 72 1C was followed by holding the mixture
irradiation and photographed using a digital camera at 4 1C using a thermocycler (Model PC 808, ASTEC).
(COOLPIX 4300, Nikon, Tokyo, Japan).
Results
Construction of the internal amplification
control (IAC) Specific gene screening of S. Typhi CT18 using
genomic sequence comparison
The IAC was constructed using the STM3098 gene for the
primer pair STM3098-f2, r2 which has been used to detect Among the 4395 genes of serovar Typhi CT18, 195 genes
Salmonella species. The 100-bp artificial oligonucleotide matching a score of o 40.14 and a length o 21 bp of
including STM3098-f2, r2 primer sequences was synthe- nucleotides using the nr database based on BLAST results
sized, and then amplified using PCR for subsequent cloning were selected to screen for the genes that were present only


c 2009 Federation of European Microbiological Societies FEMS Microbiol Lett 301 (2009) 137–146
Published by Blackwell Publishing Ltd. All rights reserved
Identification of Salmonella serovars using multiplex PCR 143

in Salmonella (data not shown). These 195 genes IAC

were compared with the previously reported list of S. Typhi
To verify the false-negative PCR results, an IAC was con-
genes, which are not present in Escherichia coli (Parkhill
structed with the primer pair of STM3098-f2, r2. The IAC
et al., 2001), and 172 of the 195 genes (88%) were
was coamplified with the target gene using STM3098-f2, r2
overlapping.
primers, resulting in 100-bp (IAC) and 423-bp (target gene)
The 195 selected genes were compared with each genome
products. We performed the PCR using anywhere from 30 to
sequence from 28 Salmonella strains using the BLAST pro-
300 000 copy numbers of IAC with 25 ng of the target gene
gram, and 35 genes that were expected to be specific to S.
to determine the detection limit. The detection limit was
Typhi were selected. These 35 genes have been included in
achieved at 300 000 copies of IAC for 25 ng of Salmonella
previously reported results, and were considered unique to
genomic DNA (30 cycles of amplification).
S. Typhi CT18 (Parkhill et al., 2001; Porwollik et al., 2004).
Among the 35 genes in this study, 15 genes were included in

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the Typhi-specific region as reported previously. Among the Multiplex PCR
195 genes, the STY2042 and STY2046 genes exhibited a
Multiplex PCR for Salmonella identification was designed
Typhi CT18-specific pattern when the Salmonella genomic
using five primer pairs for Salmonella spp., Salmonella
DNA sequences were compared.
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,
Specific PCR results with S. Typhi
STM4057-f, r, and STM4497M2-f, r, was independently
Among the 35 selected genes, 14 primer pairs were con- specific to the Salmonella genus, Salmonella subspecies I
structed and evaluated with various genomic DNAs of and S. Typhimurium, respectively, as modified from pre-
Salmonella serovars. The constructed primer pairs did not vious reports (Kim et al., 2006a, b). The primer pair for
amplify with non-Salmonella strains, with the exception of STY1599 was used as a specific primer for S. Typhi.
the STY4675 primer pairs (data not shown). Most of the Sequences of specific primer pairs for S. Enteritidis were
constructed primer pairs revealed positive results for S. obtained from a previous report (Wang & Yeh, 2002).
Typhi, including several Salmonella serovars. In particular, However, this primer pair was also positive for S. Dublin,
primer pairs for STY1594, STY1599 and STY2020 yielded as shown in Table 1, and consequently might be nonspecific
highly specific amplified results with S. Typhi (Fig. 1; PCR to S. Dublin.
results of STY1594 and STY2020 are not shown). Therefore, We performed a singlet PCR to confirm the specificity of
in this study, S. Typhi-specific primer pairs were chosen five primers using various foodborne microorganisms in-
based on their potential utility for the detection and cluding Listeria monocytogenes, Staphylococcus aureus, Bacillus
identification of S. Typhi. The results of selected primer cereus and others as listed in Table 1. None of these micro-
pairs showed a relatively high specificity for S. Typhi, organisms yielded a positive response. Before performing the
Paratyphi C, Paratyphi A and Choleraesuis. These results multiplex PCR, single PCRs of each primer pair with S.
further confirmed the close genetic proximity between Typhimurium, Typhi and Enteritidis were conducted (Fig. 2;
Typhi and Paratyphi A, as reported previously (Chan et al., lanes 1–9) and specific PCR products were obtained. Also,
2003; McClelland et al., 2004; Porwollik et al., 2004; Liu multiplex PCR was reacted with the mixed genomic DNA of
et al., 2009). Salmonella serovars to evaluate the amplification of five PCR

Fig. 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.

FEMS Microbiol Lett 301 (2009) 137–146 

c 2009 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
144 S.H. Park et al.

Fig. 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.

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Typhi ATCC 33459, S. Enteritidis ATCC 4931; and lane 14, nontemplate.

Fig. 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.

products (Fig. 2; lanes 10–12). Six amplified products are constructed based on the flagellin gene and the Vi antigen
shown with the mixed genomic DNA of S. Typhimurium, gene of S. Typhi. However, these primers are limited in their
Typhi and Enteritidis (Fig. 2; lane 13). The amplified PCR ability for the specific detection of S. Typhi because of their
product sizes were 423 bp for Salmonella spp.-specific, 137 bp sequence similarity between Salmonella serovars. In a pre-
for Salmonella subspecies I-specific, 310 bp for S. Typhimur- vious report, a specific primer pair for S. Typhimurium was
ium-specific, 203 bp for Typhi-specific, 559 bp for Enteritidis- obtained using a genomic DNA comparison approach (Kim
specific and 100 bp for IAC products. et al., 2006b). Also, in this study, specific-expected genes of
Multiplex PCR was evaluated with various genomic DNAs S. Typhi were suggested using genomic sequence compar-
from Salmonella including Salmonella subspecies I–VI (Fig. 3 isons. Using this information, 14 primer pairs were con-
and Table 1). Amplification with the STM3098-f2, r2 primer structed and three primer pairs (STY1594, STY1599 and
pair yielded a PCR product of 423 bp size with all Salmonella STY2020) were suggested as specific primers for S. Typhi.
spp. including subspecies I–VI in lanes 1–14 (Fig. 3). The The three primer pairs yielded positive reactions for S. Typhi
primer pair for STM4057-f, r yielded only an amplified PCR and negative results with other serovars.
product of 137 bp with Salmonella subspecies I in lanes 1–8. A multiplex PCR-based method with three or more pairs
The primer pairs for STM4497M2-f, r and STY1599-f, r of primers has been developed for the simultaneous detec-
yielded amplified specific PCR products of 310 and 203 bp tion and identification of S. Typhimurium (Lim et al., 2003)
with Typhimurium and Typhi, respectively (Fig. 3; lanes and Typhi (Hirose et al., 2002). Until now, these methods
1–4). The primer pair for IE 2L, 3R produced an amplified were designed to identify a single Salmonella serovar using
PCR product of 559 bp with Enteritidis (Fig. 3; lane 7). 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
Discussion could only be distinguished based on the pattern of positive
Identification of S. Typhi using PCR has been reported and negative results from all primer pairs with multiplex
previously (Song et al., 1993; Hashimoto et al., 1995; Hirose PCR. Also, only relatively few serovars of Salmonella have
et al., 2002). Primer pairs used in these studies were been used to test the specificity of the primers. In a study by


c 2009 Federation of European Microbiological Societies FEMS Microbiol Lett 301 (2009) 137–146
Published by Blackwell Publishing Ltd. All rights reserved
Identification of Salmonella serovars using multiplex PCR 145

Kim et al. (2006a), a multiplex PCR method including five Salmonella spp. is regarded as a human pathogen and also in
primer pairs was designed and each of the primer pairs domestic animals in both the public and the food industry
displayed specificity to its own target for Salmonella spp., sectors, and this multiplex PCR method would allow for a
Salmonella subspecies I, S. Typhimurium, Typhi and Enter- rapid and convenient alternative for the identification of
itidis (Kim et al., 2006a). These five primer pairs identified Salmonella spp. and major Salmonella pathogens, in these
Salmonella in stages. First, two primer pairs (STM3098-f2, settings, as it can be conducted by nonspecialized labora-
r2 and STM4057-f, r) yielded a high specificity for Salmo- tories. Also, these results suggest the possibility for a new
nella spp. and Salmonella subspecies I. Next, the other three screening method for specific marker genes and probes
primer pairs (STM4497M2-f, r, STY1599-f, r and IE 2L, 3R) using comparative genomic analysis for the detection of
identified the major pathogenic Salmonella serovars, includ- pathogens.
ing Typhimurium, Typhi and Enteritidis. If an unknown
sample resulted in two bands at 423 bp (STM3098-f2, r2)

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and 137 bp (STM4057-f, r) after multiplex PCR, the un-
Acknowledgements
known sample would be identified as Salmonella subspecies
I. Therefore, an unknown sample identified by this approach We thank Dr Reiner Helmuth and Dr Burkhard Malorny of
has the potential of being a pathogen for humans and warm- the Federal Institute for Risk Assessment (BFR, Molecular
blooded animals. If an unknown sample resulted in three Biology, National Salmonella Reference Laboratory, Ger-
bands at 423, 137 and 203 bp (STY1599-f, r) after multiplex many) and Dr K.H. Seo of US Food and Drug of Adminis-
PCR, the unknown sample would be identified as S. Typhi. tration (FDA, CFSAN/OPDFB) for the kind donation of
However, this multiplex PCR method for Salmonella identi- Salmonella strains.
fication needs to be further evaluated with a greater variety
of Salmonella serovars in conjunction with other labora-
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c 2009 Federation of European Microbiological Societies FEMS Microbiol Lett 301 (2009) 137–146
Published by Blackwell Publishing Ltd. All rights reserved