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Microbiology and Infectious Disease / SALMONELLA AND SALMONELLA SEROTYPE TYPHI–SPECIFIC PCR
Broad-Range (Pan) Salmonella and Salmonella Serotype
Typhi–Specific Real-Time PCR Assays
Potential Tools for the Clinical Microbiologist
John J. Farrell, MD,1,2 Laura J. Doyle, MT(ASCP),2 Rachel M. Addison, MPH, MT(ASCP),3
L. Barth Reller, MD,3 Geraldine S. Hall, PhD,2 and Gary W. Procop, MD2
Key Words: Pan-Salmonella; Salmonella Typhi; Real-time PCR
DOI: 10.1309/DP0HY5UT10HQW9YM
Abstract
We describe broad-range salmonellae (ie,
Salmonella) and Salmonella serotype Typhi–specific
LightCycler (Roche Diagnostics, Indianapolis, IN) realtime polymerase chain reaction assays. We validated
these with a battery of 280 bacteria, 108 of which were
salmonellae representing 20 serotypes. In addition, 298
isolates from 170 clinical specimens that were
suspected to possibly represent Salmonella were tested
with the pan-Salmonella assay. Finally, the panSalmonella assay also was used to test DNA extracts
from 101 archived, frozen stool specimens, 55 of which
were culture-positive for salmonellae. Both assays were
100% sensitive and specific when cultured isolates of
the battery were tested. The pan-Salmonella assay also
characterized correctly all salmonellae on the primary
isolation agar and was 96% sensitive (53/55) and 96%
specific (49/51) when nucleic acid extracts from direct
stool specimens were tested. These assays represent
potential tools the clinical microbiologist could use to
screen suspect isolates or stool specimens for
Salmonella.
Salmonella species are important causes of enteritis
throughout the world. In addition, Salmonella enterica
serotypes Typhi and Paratyphi are important causes of
enteric fever in underdeveloped countries that lack adequate
sewage disposal and water treatment facilities.1,2 It is standard practice in many clinical microbiology laboratories to
culture for Salmonella and Shigella species by using primary
isolation media, such as MacConkey and Hektoen Enteric
agars.3,4 Isolates suspected to possibly represent salmonellae
(ie, lactose non-fermenting colonies with or without hydrogen sulfide production) are submitted for additional screening or identification, depending on individual laboratory
practices.5-7 These practices, although effective, are timeconsuming and costly.
The identification of Salmonella directly from the primary isolation plates or, ideally, directly from stool samples is
attractive.8 This has been achieved using polymerase chain
reaction (PCR) for the invA gene, a gene associated with the
invasive nature of Salmonella.9 We hypothesized that the prgK
gene, which is part of a genetic complex that also is thought to
be important for enterocyte invasion, might serve as a useful
target for a real-time PCR-based assay for the detection of
Salmonella enterica from cultures and clinical stool specimens.10-12 In addition, we developed a real-time PCR assay
directed against the vexC gene, which encodes for the Vi antigen, as a means of detecting the Salmonella serotype Typhi.
The usefulness of the gene that encodes for the Vi antigen as
a specific target for the identification of Salmonella Typhi has
been demonstrated previously.13-15 The real-time PCR assays
described were validated against a large battery of lysates
from well-characterized bacteria, including 108 strains of
Salmonella, representing 20 serotypes. In addition, we used
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the pan-Salmonella real-time PCR assay to test culture isolates from the primary inoculation media of routinely obtained
stool cultures, as well as DNA extracts from stool specimens
that had been cultured.
Materials and Methods
Primer and Probe Design
The primers and fluorescent resonance energy transfer
(FRET) probes, for use with LightCycler system (Roche
Diagnostics, Indianapolis, IN), for the broad-range or panSalmonella assay were designed based on the prgK gene of
Salmonella serotype Typhimurium (GenBank accession number, AE008831).10,16 This primer set generated a 193-basepair nucleotide product that demonstrated high predicted
specificity for Salmonella when submitted to a National
Center for Biotechnology Information (NCBI) Blast, with perfect matches obtained only for Salmonella enterica. The
GenBank accession numbers of these perfect matches were
AE008831, L33855, AC117230, and AL627276.
The PanSalm FRET hybridization probe set was
designed to be 100% homologous for Salmonella
Typhimurium. The 3' end of the donor FRET hybridization
probe (PanSalmHP1) was labeled with fluorescein (fam).
The 3' end of the acceptor FRET hybridization probe
(PanSalmHP2) was phosphorylated (p) to prevent probe
extension during PCR, and the 5' end was labeled with 705N-hydroxysuccinimide ester (LC Red 705). The sequences
for the pan-Salmonella primers and probes were as follows:
Pan-SalmF, 5'-CCTTTCTTATTGCGGGCA-3' (forward primer;
position 4179-4196); PanSalmR, 5'-GCCGATGTGGATTATGAC-3' (reverse primer; position 4372-4355); PanSalmHP1, HP1
5'-GGATTGTTTTGATTATTTTGTTATCCGTGATG-[fam]-3'
(donor FRET probe; position 4266-4235); and PanSalmHP2, 5'[LC Red 705]-AGCAGGCTTTGGCGTCTGGGTGG-p-3'
(acceptor FRET probe; position 4232-4210).
The primers and FRET hybridization probes used for the
Salmonella Typhi–specific real-time PCR assay were
designed based on the vexC gene of Salmonella Typhi
(GenBank accession number, AL627283), which encodes for
the Vi antigen. This primer set generated a 215-base-pair
nucleotide product that demonstrated high predicted specificity for Salmonella Typhi when submitted to an NCBI
Blast, with perfect matches obtained only for Salmonella
Typhi. The Salmonella Typhi GenBank accession numbers
that demonstrated perfect matches were AL627283,
AE016848, and D14156.1.
The 3' end of the donor FRET hybridization probe
(STyphiHP1) was labeled with fluorescein (fam). The 3' end
of the acceptor FRET hybridization probe (STyphiHP2) was
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phosphorylated (p) to prevent probe extension during PCR,
and the 5' end was labeled with LC Red 640. The sequences
for the Salmonella Typhi–specific primers and probes were as
follows: STyphiF, 5'-ACCCCGTAGCCCAATA-3' (forward
primer; position 42395-42410); STyphiR, 5'-AGGAGAGACGCATTCG-3' (reverse primer; position 42610-42595);
STyphiHP1, 5'-GCATATCGGTATTCTGGCGGC-[fam]-3'
(donor FRET probe; position 42500-42480); and STyphiHP2,
5'-[LC Red 640]-CTGGTTCAGGCAAAACGACG-p-3'
(acceptor FRET probe; position 42477-42458).
PCR Reagents and Thermocycling Parameters
LightCycler FastStart DNA Master Hybridization Probe
kit reagents (Roche Diagnostics), primers, and probes were
combined in a master mix. The PCR reaction was carried out
in LightCycler capillary tubes (Roche Diagnostics) with 15 µL
of master mix and 5 µL of sample. For the pan-Salmonella
PCR assay, the following concentrations were used: 1.0
µmol/L of both forward and reverse primers, 0.4 µmol/L of
both PanSalm FRET hybridization probes, and 4.0 mmol/L of
magnesium chloride. The same concentrations were used for
the Salmonella Typhi–specific assay, except a 3.0-mmol/L
concentration of magnesium chloride was used. The thermocycling protocol of the pan-Salmonella PCR consisted of 10
minutes at 95°C for DNA polymerase activation, 45 cycles of
PCR amplification (95°C for 10 seconds, 50°C for 10 seconds, and 72°C for 20 seconds), postamplification melting
(35°C to 95°C at 0.1°C/second), and a cooling step (35°C for
30 seconds). The protocol for the Salmonella Typhi–specific
assay was the same, except primer annealing occurred at 55°C
for 10 seconds and the postamplification melting ranged from
45°C to 75°C.
Validation Battery
The sensitivity and specificity of both assays were
assessed using lysates of 280 well-characterized bacterial
strains obtained from Duke University Medical Center
(Durham, NC) and the Cleveland Clinic Foundation
(Cleveland, OH) stock collections of bacteria. These bacteria
included numerous American Type Culture Collection
(Manassas, VA) strains. The other non-Salmonella stock isolates were identified by traditional biochemical methods by
experienced microbiologists and were characterized unambiguously. Of the 280 isolates tested, 108 were Salmonella
species, which included 23 Salmonella Typhi and 24
Salmonella Paratyphi. The 108 Salmonella isolates tested consisted of 20 serotypes, including those routinely identified (ie,
commonly occurring) in these 2 clinical microbiology laboratories. The serotyping of these isolates was performed in the
local public health laboratories according to routine. A variety
of commonly encountered bacteria was included in the
remaining 172 non-Salmonella bacteria tested and included
© American Society for Clinical Pathology
Microbiology and Infectious Disease / ORIGINAL ARTICLE
other enteric pathogens such as Shigella species, Yersinia
enterocolitica, and Escherichia coli O157:H7 ❚Table 1❚.
DNA lysates of the cultured bacteria were prepared by
placing a 1.0-µL loop of each cultured isolate into 500 µL of
lysis buffer, which has been described previously and contains
1× tris(hydroxymethyl)aminomethane-EDTA buffer supplemented with 1% Triton X-100 (Sigma, St Louis, MO) and
0.5% polysorbate 20.17 This solution was heated to 100°C for
10 minutes, centrifuged at 10,000g for 30 seconds, and stored
at –20°C. The criterion for a positive test result was a cycle
threshold (CT) (ie, crossing point) of 35 cycles or fewer.
Limits of Detection
The limit of detection was determined only for the panSalmonella assay because it was not our intention to use the
Salmonella Typhi–specific assay on direct specimens, but
rather on cultured isolates wherein the target copy number is
excessive. To determine the analytic sensitivity of this assay
for lysates of Salmonella in pure culture, duplicate serial dilutions of a 0.5-McFarland suspension of Salmonella
Typhimurium (American Type Culture Collection, 23854)
were prepared in 0.85% saline. Lysates of each serial dilution
were tested by the LightCycler prgK real-time PCR protocol
as described. One hundred microliters from each of the serial
dilutions was inoculated and spread onto sheep blood agar
plates (Becton Dickinson Microbiology Systems, Cockeysville,
MD) and then incubated in atmospheric oxygen at 35°C for 24
hours to determine colony counts for each dilution. The lowest limit of detection was determined by averaging the values
of the duplicate quantitative cultures that were correlated with
the lowest limit of detection by PCR.
A stool specimen, proven by culture to be negative for all
bacterial enteric pathogens, was used to determine the analytic sensitivity of the pan-Salmonella assay for stool specimen
extracts. Aliquots of this stool specimen were spiked with serial 10-fold dilutions of a 0.5-McFarland preparation of
Salmonella Typhimurium, as described in the preceding text.
DNA extracts were prepared, as described subsequently, for
the clinical stool specimens, and real-time PCR was performed on these extracts and compared with the results of the
quantitative cultures, which were performed as described in an
earlier section.
Testing Suspect Colonies From Routine Stool Cultures
With the Pan-Salmonella Assay
A prospective validation of the pan-Salmonella assay was
conducted during a 5-month period on all isolates from stool
cultures that were suspected to possibly represent Salmonella
because of morphologic features of their colonies (ie,
green/green or black colonies on Hektoen enteric agar). Cell
lysates were prepared as described in the preceding text from
298 suspect isolates from the stool cultures of 170 patients. The
lysates were batch tested once a week by the protocol described
herein, with the same positive test criterion (ie, CT ≤35 cycles).
❚Table 1❚
Validation Battery of Cultured Bacteria*
PCR Results
Bacteria Tested
Pan-Salmonella
Gram-positive bacteria tested (n = 40)
Staphylococcus aureus (5), Staphylococcus epidermidis (3), Staphylococcus saprophyticus (2),
All negative
Micrococcus species (2), Stomatococcus species (2), Lactobacillus species (2), Enterococcus
species (3), Viridans streptococci (3), Streptococcus pneumoniae (3), group A streptococcus (3),
group B streptococcus (3), Aerococcus species (3), Listeria species (3), Bacillus species (3)
Non–Salmonella gram-negative bacteria tested (n = 132)
Providencia species (2), Shigella sonnei (10), Shigella flexneri (17), Shigella boydii (6), Shigella
All negative
dysenteriae (2), Burkholderia species (2), Yersinia kristensenii (1), Yersinia enterocolitica (27),
Citrobacter species (3), Escherichia coli non-O157:H7 (2), E coli O157:H7 (12), Proteus species (3),
Klebsiella species (3), Enterobacter species (3), Pseudomonas species (3), Acinetobacter
species (3), Haemophilus species (3), Neisseria meningitidis (3), Neisseria gonorrhoeae (3),
Non-gonococcal Neisseria species (3), Moraxella species (3), Bacteroides species (3), Serratia
species (3), Corynebacterium species (3), 1 each of Afipia felis, Vibrio cholerae, Eikenella corrodens,
Pasteurella multocida, Campylobacter jejuni, Mesorhizobium species, Rhizobium species,
Bartonella henselae, and Bartonella quintana
Salmonella isolates tested (n = 108)
Salmonella Typhimurium (15), Salmonella Enteritidis (6), Salmonella Choleraesuis (5), Salmonella
All positive
Paratyphi (24) Salmonella Infantis (8), Salmonella Heidelberg (5), Salmonella Arizonae (8), Salmonella
Java (2), Salmonella Newport (2), and single isolates of Salmonella Agona, Salmonella Oslo, Salmonella
Poona, Salmonella Senfrenberg, Salmonella Havana, Salmonella Javiana, Salmonella Anatum,
Salmonella Saint Paul, Salmonella Berta, Salmonella Braenderup
Salmonella Typhi (23)
All positive
Serotype Typhi–
Specific
All negative
All negative
All negative
All positive
PCR, polymerase chain reaction.
* Numbers in parentheses following names of bacteria indicate the number of strains tested.
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DNA Extracts From Clinical Stool Specimens
DNA extracts of 101 stool samples identified by culture or
Campylobacter enzyme immunoassay to be positive for a bacterial enteric pathogen and 5 samples that were negative for enteric
pathogens were tested. The pathogens detected by culture were
Salmonella (55), Shigella (20), E coli O157:H7 (9),
Campylobacter jejuni (4), Y enterocolitica (3), and Plesiomonas
shigelloides (1). Nine stool samples were positive for
Campylobacter by the ProSpecT (Alexon-Trend, Ramsey, MN)
Campylobacter enzyme immunoassay. All stool samples were
stored at –20°C before extraction. Nucleic acid extracts were
prepared from thawed stool specimens with the Qiagen Stool
Extraction Kit (Qiagen, Valencia, CA). The pan-Salmonella realtime PCR assay was performed with the master mix and thermocycling protocol described in preceding text and with the same
criteria for test positivity (ie, CT ≤35 cycles). The Salmonella
Typhi–specific PCR also was not performed on the direct stool
extracts because of the low incidence of Salmonella Typhi in the
United States. An evaluation of the Salmonella Typhi real-time
Fluorescence (F3/F1)
1.8
1.6
1.4
1.2
1.0
0.8
0.6
0.4
0.2
0.0
–0.2
Positive control
Negative control
Non-Salmonella
Salmonella
0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44 46
Cycle Number
❚Figure 1❚ Pan-Salmonella real-time polymerase chain
reaction. The positive control (Salmonella Typhimurium) and
all Salmonella isolates (16 strains, 12 serotypes) in this
experiment demonstrates unequivocal amplification
compared with the negative control sample (lysis buffer) and
the non-Salmonella isolates (Yersinia species [3 strains] and
Escherichia coli O157:H7 [11 strains]) tested. (If no number of
strains is given, 1 strain was assayed.)
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PCR on routinely occurring stool culture isolates and nucleic
acid extracts from direct stool specimens should be performed in
an area with a sufficient incidence of Salmonella Typhi infections to appropriately validate this assay.
Results
Validation Battery
All lysates from the 108 Salmonella strains included in
the validation battery were positive with the pan-Salmonella
PCR, whereas only the 23 strains of Salmonella Typhi were
positive with the Salmonella Typhi–specific PCR (Table 1)
❚Figure 1❚ and ❚Figure 2❚. The 85 strains of Salmonella that
were not serotype Typhi were negative with the Salmonella
Typhi–specific PCR. The 172 lysates from non-Salmonella
bacteria were negative with both the pan-Salmonella and the
Salmonella Typhi–specific assays (Table 1). These assays
both demonstrated 100% sensitivity and 100% specificity
when the bacterial lysates from the validation battery were
tested (Table 1). The approximate analytic sensitivity of the
pan-Salmonella assay for cultured bacteria was 8 colonyforming units per milliliter.
Routine Stool Culture Isolates
All suspect colony lysates from the stool samples proven
by culture to contain Salmonella in the prospective study were
positive with the pan-Salmonella PCR assay. All remaining
suspect colonies, determined to not represent salmonellae by
2.4
2.2
2.0
1.8
1.6
1.4
1.2
1.0
0.8
0.6
0.4
0.2
0.0
–0.2
Fluorescence (F2/F1)
Each batch included a DNA extract from Salmonella
Typhimurium as a positive control sample and lysis buffer as
a negative control sample. The assay results subsequently
were compared with the culture results to determine the sensitivity and specificity of the assay in this setting. The
Salmonella Typhi–specific PCR was not performed on these
colony extracts, primarily because of the low incidence of
Salmonella Typhi in the United States and because the sensitivity and specificity of the Salmonella Typhi–specific assay
was defined previously in the validation phase of this study.
Positive control
Negative control
E coli O157:H7
Non-Typhi Salmonella
0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40
Cycle Number
❚Figure 2❚ The Salmonella serotype Typhi–specific real-time
polymerase chain reaction demonstrated amplification of only
the Salmonella Typhi isolate (ie, the positive control). The
negative control sample (lysis buffer), non-Salmonella bacteria
(Escherichia coli O157:H7 [11 strains]), and non-Typhi
Salmonella isolates (19 strains, 15 serotypes) were all
negative.
© American Society for Clinical Pathology
Microbiology and Infectious Disease / ORIGINAL ARTICLE
routine bacteriologic methods, were negative with the panSalmonella PCR. During this study, there was 1 case of apparent misinoculation of lysis buffer or a mislabeling of the test
tube that resulted in an apparent false-positive result; reevaluation of the 2 colony types involved gave correct identification
with the pan-Salmonella assay. Therefore, as with the validation battery, we believe the sensitivity and specificity of the
pan-Salmonella PCR on colony lysates from colonies on the
primary recovery media were both 100%.
PCR Testing of Frozen, Archived Stool Specimens
Of the 55 stool specimens positive by culture for salmonellae, 53 were positive with the pan-Salmonella PCR (ie,
there were 2 false-negative PCR results) ❚Table 2❚. Of the
extracts from the 51 stool specimens that were culture negative for salmonellae, there were 2 that tested positive in the
pan-Salmonella assay (ie, 2 false-positive results). Both were
from stool samples from which E coli O157:H7 had been cultured. The sensitivity and specificity for the pan-Salmonella
assay when DNA extracts from stool specimens were tested
were 96% (53/55) and 96% (49/51), respectively (Table 2).
Low-level amplification (CT = 44) was detected in one of the
stool extracts, but this had to be categorized as a false-negative
result because of our predefined interpretation criteria.
The testing of DNA extracts of normal stool samples
spiked with serial dilutions of Salmonella Typhimurium to
determine the limit of detection of this assay demonstrated
that PCR was positive at a dilution of 10–5 but not at greater
dilutions. Duplicate quantitative cultures of 100 µL of this
dilution revealed 91 organisms. Therefore, the limit of detection of this assay for Salmonella in DNA extracts of stool
specimens is estimated at approximately 910 colony-forming
units per milliliter.
Discussion
The taxonomy of Salmonella has been reviewed extensively by Janda and Abbott.18 Two species of Salmonella currently are recognized, S enterica and Salmonella bongori.
Eight subgroups or subspecies have been defined using a variety of molecular methods.18-20 S bongori contains only a single
subspecies or subgroup (ie, subgroup V, bongori). S enterica
contains the remaining 7 subgroups, 2 of which contain the
clinically relevant salmonellae. Most of these are contained
within subgroup I, enterica, and are subcategorized further as
serotypes (eg, Typhi, Typhimurium, Enteritidis).21,22 Subgroup
IIIa, arizonae, contains the clinically important salmonellae
that are associated with reptiles and cause human infection.
We designed and tested and now report a sensitive and
specific LightCycler real-time PCR assay for the detection of
Salmonella and another for the detection of the Typhi serotype.
❚Table 2❚
PCR of Stool Specimen Extracts
Pan-Salmonella
PCR Result
Stool Culture/EIA Results
N
Positive Negative
Total Salmonella-positive stool samples
Salmonella serotype Typhi
O antigen serogroups
Group B
Group C
Group D non–Salmonella Typhi
Group E or G
Not typeable
Other enteric pathogens and negative
stool cultures
Campylobacter jejuni
Campylobacter by EIA
Shigella sonnei
Shigella flexneri
Yersinia enterocolitica
Escherichia coli O157:H7
Plesiomonas shigelloides
No pathogen isolated
55
1
53
1
2
0
20
15
14
2
3
19
14
14
2
3
1
1
0
0
0
51
4
9
19
1
3
9
1
5
2
0
0
0
0
0
2
0
0
49
4
9
19
1
3
7
1
5
EIA, enzyme immunoassay; PCR, polymerase chain reaction.
The pan-Salmonella assay was directed against a single-copy
gene, which is part of a complex that is thought to be important
for the entry of salmonellae into enterocytes.23,24 A similar
genetic target, the invA gene, previously has been shown to be
useful for the detection of salmonellae by PCR.25,26 We think
this assay potentially could be useful as a molecular means of
screening colonies from stool cultures that have characteristics
that suggest the possibility of salmonellae. We proved this
hypothesis, first by testing lysates from a large battery of wellcharacterized bacteria, many of which are isolated commonly
in a clinical microbiology laboratory. The validation battery
also included 108 strains of Salmonella, representing 20
serotypes, including Typhi, Paratyphi, and commonly occurring serotypes. The pan-Salmonella and the Salmonella
Typhi–specific assay correctly characterized all isolate lysates
as a Salmonella species or the Typhi serotype, respectively.
The additional studies performed examined only the panSalmonella assay, which was the assay that was of greatest
interest to us for implementation into our laboratory for routine testing. Furthermore, we thought additional studies of the
Salmonella Typhi–specific PCR assay on stool culture isolates
or stool nucleic acid extracts from patients in Cleveland would
not sufficiently validate this assay further, given the low incidence of Salmonella Typhi infections in this area. These studies, which are important before assay implementation, should
be performed in an area wherein Salmonella Typhi is endemic to validate the assay.
We further examined the pan-Salmonella assay in a
prospective study wherein we examined a large number of
lysates (n = 298) from colonies suspected to possibly represent
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Salmonella because of their features on the primary inoculation media. These 298 isolates were from the routine stool cultures of 170 patients. All colonies were categorized appropriately as Salmonella or non-Salmonella species based on the
results of the pan-Salmonella assay. These results largely were
expected, given the results of the testing of the validation battery. This aspect of the study, however, was done on lysates
prepared by routine medical technologists and done directly
from media used for primary inoculation. There was a
misinoculation or a mislabeling of a lysate, which reiterates
the need for good quality control with any laboratory test.
Although not performed in this study, the possibility exists
of testing multiple different colonies or “sweeps” from individual plates, rather than individual colonies. The feasibility of this
approach remains to be proven, but it likely is possible given the
low limit of detection of this assay for lysates derived from cultured isolates. Although the Salmonella Typhi–specific assay
was not tested on the colonies that grew on the primary inoculation media because of the low incidence of enteric fever in the
United States, it is highly likely that this assay could be used to
exclude the Typhi serotype for isolates that tested positive within
the pan-Salmonella assay; this, too, however, remains speculative. This prospective validation at the stool bench in our clinical laboratory demonstrated that real-time PCR was as accurate
for the differentiation of Salmonella from non-Salmonella isolates as conventional methods, but considerably faster.
Not surprisingly, the pan-Salmonella assay was considerably less sensitive (approximately 2 logs) when DNA extracts
from stool specimens were tested compared with colony
lysates. However, even with extracts from this complex specimen type, the assay was highly sensitive with 96% (53/55) of
Salmonella-containing stool samples detected. Two of the 51
stool samples that did not contain salmonellae by culture were
positive with the pan-Salmonella PCR. Both of the extracts
that generated false-positive results were from cultures that
grew E coli O157:H7. There was no cross-reactivity with this
assay, and the E coli O157:H7 isolates tested (Figure 1), nor is
there any expected based on GenBank searches. The possibility remains that salmonellae were present in these stool samples but not detected by routine culture (ie, false-negative cultures), but this remains speculative. The greater than 95% sensitivity and specificity of this assay for detecting Salmonella
in extracts of stool specimens suggests that, perhaps owing to
the large number of organisms typically present in stool samples from patients with Salmonella enterocolitis, this assay has
adequate sensitivity for a clinical application. The complete
Salmonella genome contains only 1 copy of the prgK gene,
which might limit the analytic sensitivity of this assay and,
thus, the applicability of this assay for the detection of
Salmonella directly in the blood of patients with enteric fever,
in whom the number of bacteria per milliliter would be considerably lower than in stool samples.
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Initial studies (data not shown) included the use of an alternative acceptor FRET probe, the sequence of which was: 5'-[LC
Red 705]-AGCAGGCTTTGGCGTCTGG-p-3' (acceptor
FRET probe; position 4232-4210). This probe, however, did not
detect Salmonella arizonae. This is not surprising, in retrospect,
because S arizonae is in a distinctly different subgroup (IIIa)
from the other clinically important salmonellae (subgroup I).
Unfortunately, the sequence of the prgK gene for S arizonae
was not available in GenBank for electronic analysis when this
assay was designed. When we discovered that S arizonae was
missed by the initial version of the pan-Salmonella assay, we
first demonstrated that the amplicon was generated by the
primers, through gel electrophoresis, and then sequenced the
amplicons of S arizonae, Salmonella Typhimurium, and
Salmonella Typhi for comparison. This comparison led to the
redesign of the acceptor FRET hybridization probe used in this
study. The extension of the 3' of the acceptor FRET probe by 4
nucleotides (GTGG) was necessary to detect S arizonae.
The use of PCR and, more recently, real-time PCR has
become commonplace in molecular microbiology and molecular pathology laboratories. This technology often is used to
detect fastidious microbes or microorganisms that cannot be
cultivated. Real-time applications, however, have been used
for the detection of commonly cultured bacteria and their
mechanisms of resistance. Examples of this include the detection of Staphylococcus aureus and the mecA gene and the
detection of vancomycin-resistant enterococci.27,28 In some
cases, real-time PCR has been found to be cost-competitive
compared with culture.29 The detection of Salmonella and the
differentiation of these organisms from the many mimics that
occur in stool cultures are now possible with real-time PCR of
lysates from cultured colonies or direct nucleic extracts of the
stool specimens. How such technologies will be incorporated
into the stool bench of the future, however, remains to be seen.
From the 1Department of Infectious Diseases, University of
Illinois/OSF Infectious Diseases, Peoria, IL; 2Section of Clinical
Microbiology, Department of Clinical Pathology, Cleveland Clinic
Foundation, Cleveland, OH; 3Clinical Microbiology Laboratory,
Duke University Medical Center, Durham, NC.
Address reprint requests to Dr Procop: Clinical
Microbiology, Dept of Microbiology L40, Cleveland Clinic
Foundation, 9500 Euclid Ave, Cleveland, OH 44195.
References
1. Bern C, Martines J, de Zoysa I, et al. The magnitude of the
global problem of diarrhoeal disease: a ten-year update. Bull
World Health Organ. 1992;70:705-714.
2. Parry CM, Hien TT, Dougan G, et al. Medical progress:
typhoid fever. N Engl J Med. 2002;347:1770-1782.
3. Bopp CA, Brenner FW, Fields PI, et al. Escherichia, Shigella,
and Salmonella. In: Manual of Clinical Microbiology. 8th ed.
Washington, DC: ASM Press; 2003:chap 42.
© American Society for Clinical Pathology
Microbiology and Infectious Disease / ORIGINAL ARTICLE
4. Procop G. Gastrointestinal infections. Infect Dis Clin North
Am. 2001;15:1073-1108.
5. Bonev S, Zakhariev Z, Gentchev P. Comparative study of
media for determination of lysine decarboxylase activity. Appl
Microbiol. 1974;27:464-468.
6. Kolmos HJ, Schmidt J. Failure to detect hydrogen-sulphide
production in lactose/sucrose-fermenting Enterobacteriaceae,
using triple sugar iron agar. Acta Pathol Microbiol Immunol
Scand. 1987;95:85-87.
7. Johnson JG, Kunz L, Barron W, et al. Biochemical
differentiation of the Enterobacteriaceae with the aid of
lysine-iron-agar. Appl Microbiol. 1966;14:212-217.
8. Kurowski PB, Traub-Dargatz JL, Morley PS, et al. Detection of
Salmonella spp in fecal specimens by use of real-time
polymerase chain reaction assay. Am J Vet Res. 2002;63:12651268.
9. Rahn K, De Grandis SA, Clarke RC, et al. Amplification of
an invA gene sequence of Salmonella typhimurium by
polymerase chain reaction as a specific method of detection of
Salmonella. Mol Cell Probes. 1992;6:271-279.
10. Pegues DA, Hantman MJ, Behlau I, et al. PhoP/PhoQ
transcriptional repression of Salmonella typhimurium invasion
genes: evidence for a role in protein secretion. Mol Microbiol.
1995;17:169-181.
11. Jones B, Falkow S. Identification and characterization of a
Salmonella typhimurium oxygen-related gene required for
bacterial internalization. Infect Immun. 1994;62:3745-3752.
12. Klein JR, Fahlen TF, Jones BD. Transcriptional organization
and function of invasion genes within Salmonella enterica
serovar Typhimurium pathogenicity island 1, including the
prgH, prgI, prgJ, prgK, orgA, orgB, and orgC genes. Infect
Immun. 2000;68:3368-3376.
13. Hashimoto Y, Itho Y, Fujinaga Y, et al. Development of nested
PCR based on the ViaB sequence to detect Salmonella typhi. J
Clin Microbiol. 1995;33:775-777.
14. Hirose K, Itoh K, Nakajima H, et al. Selective amplification of
tyv (rfbE), prt (rfbS), viaB, and fliC genes by multiplex PCR for
identification of Salmonella enterica serovars Typhi and
Paratyphi A. J Clin Microbiol. 2002;40:633-636.
15. Sharma K, Arya S. Detection of Salmonella typhi by nested
PCR based on the ViaB sequence [letter]. J Clin Microbiol.
1995;33:3361.
16. McClelland M, Sanderson KE, Spieth J, et al. Complete
genome sequence of Salmonella enterica serovar Typhimurium
LT2. Nature. 2001;413:852-856.
17. Reischl U, Linde HJ, Metz M, et al. Rapid identification of
methicillin-resistant Staphylococcus aureus and simultaneous
species confirmation using real-time fluorescence PCR. J Clin
Microbiol. 2000;38:2429-2433.
18. Janda J, Abbott S. The Enterobacteria. Philadelphia, PA:
Lippincott-Raven; 1998.
19. Brown EW, Kotewicz ML, Cebula TA. Detection of
recombination among Salmonella enterica strains using the
incongruence length difference test. Mol Phylogenet Evol.
2002;24:102-120.
20. Chan K, Baker S, Kim CC, et al. Genomic comparison of
Salmonella enterica serovars and Salmonella bongori by use of an
S enterica serovar Typhimurium DNA microarray. J Bacteriol.
2003;185:553-563.
21. Boyd EF, Wang F, Whittam TS, et al. Molecular genetic
relationships of the salmonellae. Appl Environ Microbiol.
1996;62:804-808.
22. Koneman EW, Allen SD, Janda WM, et al. Color Atlas and
Textbook of Diagnostic Microbiology. 5th ed. Philadelphia, PA:
Lippincott Williams & Wilkins; 1997.
23. Kubori T, Sukhan A, Aizawa SI, et al. Molecular
characterization and assembly of the needle complex of the
Salmonella typhimurium type III protein secretion system. Proc
Natl Acad Sci U S A. 2000;97:10225-10230.
24. Pucciarelli M, Garcia-del Portillo F. Protein-peptidoglycan
interactions modulate the assembly of the needle complex in
the Salmonella invasion–associated type III secretion system.
Mol Microbiol. 2003;48:573-585.
25. Isacsson J, Cao H, Ohlsson L, et al. Rapid and specific
detection of PCR products using light-up probes. Mol Cell
Probes. 2000;14:321-328.
26. Scholz HC, Arnold T, Marg H, et al. Improvement of an
invA-based PCR for the specific detection of Salmonella
typhimurium in organs of pigs. Berl Munch Tierarztl Wochenschr.
2001;114:401-403.
27. Shrestha N, Tuohy M, Hall G, et al. Rapid identification of
Staphylococcus aureus and the mecA gene from BacT/ALERT
blood culture bottles by using the LightCycler system. J Clin
Microbiol. 2002;40:2659-2661.
28. Sloan L, Uhl J, Vetter E, et al. Comparison of the Roche
LightCycler vanA/vanB detection assay and culture for
detection of vancomycin-resistant enterococci from perianal
swabs. J Clin Microbiol. 2004;42:2636-2643.
29. Shrestha N, Shermock K, Gordon S, et al. Predictive value
and cost-effectiveness analysis of a rapid polymerase chain
reaction for preoperative detection of nasal carriage of
Staphylococcus aureus. Infect Control Hosp Epidemiol.
2003;24:327-333.
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