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Rapid Detection of Salmonella in Pet Food: Design and Evaluationof Integrated Methods Based on Real-Time PCR Detection
PRIYA BALACHANDRAN,1* MARIA FRIBERG,2 V. VANLANDINGHAM,2 K. KOZAK,2 AMANDA MANOLIS,1
MAXIM BREVNOV,1 ERIN CROWLEY,3 PATRICK BIRD,3 DAVID GOINS,3 MANOHAR R. FURTADO,1
OLGA V. PETRAUSKENE,1 ROBERT S. TEBBS,1 AND DUANE CHARBONNEAU2
1Life Technologies, 850 Lincoln Centre Drive, Foster City, California 94404; 2Procter & Gamble, 8700 Mason-Montgomery Road, Mason, Ohio 45040; and3Q Laboratories, Inc., 1400 Harrison Avenue, Cincinnati, Ohio 45214, USA
Rapid Detection of Salmonella in Pet Food: Design and Evaluationof Integrated Methods Based on Real-Time PCR Detection
PRIYA BALACHANDRAN,1* MARIA FRIBERG,2 V. VANLANDINGHAM,2 K. KOZAK,2 AMANDA MANOLIS,1
MAXIM BREVNOV,1 ERIN CROWLEY,3 PATRICK BIRD,3 DAVID GOINS,3 MANOHAR R. FURTADO,1
OLGA V. PETRAUSKENE,1 ROBERT S. TEBBS,1 AND DUANE CHARBONNEAU2
1Life Technologies, 850 Lincoln Centre Drive, Foster City, California 94404; 2Procter & Gamble, 8700 Mason-Montgomery Road, Mason, Ohio 45040; and3Q Laboratories, Inc., 1400 Harrison Avenue, Cincinnati, Ohio 45214, USA
MS 11-210: Received 29 April 2011/Accepted 9 October 2011
ABSTRACT
Reducing the risk of Salmonella contamination in pet food is critical for both companion animals and humans, and its
importance is reflected by the substantial increase in the demand for pathogen testing. Accurate and rapid detection of foodborne
pathogens improves food safety, protects the public health, and benefits food producers by assuring product quality while
facilitating product release in a timely manner. Traditional culture-based methods for Salmonella screening are laborious and can
take 5 to 7 days to obtain definitive results. In this study, we developed two methods for the detection of low levels of Salmonellain pet food using real-time PCR: (i) detection of Salmonella in 25 g of dried pet food in less than 14 h with an automated
magnetic bead–based nucleic acid extraction method and (ii) detection of Salmonella in 375 g of composite dry pet food matrix in
less than 24 h with a manual centrifugation-based nucleic acid preparation method. Both methods included a preclarification step
using a novel protocol that removes food matrix–associated debris and PCR inhibitors and improves the sensitivity of detection.
Validation studies revealed no significant differences between the two real-time PCR methods and the standard U.S. Food and
Drug Administration Bacteriological Analytical Manual (chapter 5) culture confirmation method.
According to the U.S. Pet Ownership and DemographicSourcebook (3), 43 million households in the United States
own dogs, and about 37.5 million own cats. The recent 2009/2010 National Pet Owners Survey indicates that 62% of U.S.
households currently own a pet (2). Many pet owners feed
their companion animals dry pet food for ease of use and
nutritional benefit. Because pet foods and treats often contain
raw materials derived from avian and animal origin and each
ingredient may be processed under different conditions, these
items are at risk for contamination by various pathogens,
especially Salmonella, as has been demonstrated in previous
studies (7, 10, 11, 22). Pets that consume contaminated pet
treats may become colonized with Salmonella without
exhibiting clinical signs (12, 13). Pet food has been
implicated in some cases of human salmonellosis that,
although rare, occur mainly as a result of handling and/or
direct consumption of such contaminated food products (12,13). These findings highlight the importance of having robust
process controls, including pathogen screening methods, in
place during pet food production. Standard culture-based
microbiological methods for detection of Salmonella in food
may take as many as 5 days to obtain a presumptive result (4).Molecular methods such as real-time PCR are more sensitive
than the culture methods (21, 23) and are increasingly being
used to rapidly screen raw materials and finished products for
specific and early detection of foodborne pathogens (9, 25,29, 30).
With the increasing demands for pathogen testing,
methods that enable simultaneous screening of large number
of samples quickly are becoming more important for routine
screening of food. The aim of this study was to evaluate a
real-time PCR method (9, 14) for the rapid detection of
Salmonella in pet food and to compare this method with the
standard U.S. Food and Drug Administration Bacteriolog-ical Analytical Manual (FDA BAM) culture confirmation
method (4). Two sample amounts were tested, 25 and 375 g,
using two nucleic acid extraction protocols. The first series
of experiments evaluated a high-throughput process for a
25-g sample size in which individual samples were preen-
riched for 12 h. The second series of experiments was
conducted with composite or pooled samples of 375 g that
were enriched for 18 h. Implementation of such rapid
methods for routine screening can greatly enhance the safety
inspection process, allowing for more frequent proactive
inspections and reducing product hold time.
MATERIALS AND METHODS
Strains and medium. The Salmonella strains used in this
study are listed in Table 1. For inoculum preparation, an isolated
colony was added to 10 ml of brain heart infusion (BHI) broth
(Teknova, Hollister, CA) and incubated at 35uC for 22 to 24 h.* Author for correspondence. Tel: 650-638-6471; Fax: 650-638-6795;
E-mail: [email protected].
347
Journal of Food Protection, Vol. 75, No. 2, 2012, Pages 347–352doi:10.4315/0362-028X.JFP-11-210Copyright G, International Association for Food Protection
Preparation of artificially inoculated food samples. For
preliminary evaluation, 25-g pet food samples were inoculated
with 1 to 10 CFU of Salmonella. Control samples were not
inoculated.
For method comparison, 45 samples were analyzed in each
experiment: 5 uninoculated control samples, 20 samples inoculated
with a low level (0.2 to 2 CFU/25 or 375 g), and 20 samples
inoculated at a high level (2 to 10 CFU/25 or 375 g). The test
material for all experiments was ground dry pet food. A
background screen of the test material was conducted using the
FDA BAM method (4), and the target analyte (Salmonella) was not
detected. All test material was spiked within 72 h of the
background screen. Different serovars of Salmonella were used
in the independent experiments (Table 1). For each experiment, a
24-h broth culture inoculum was added to bulk samples of ground
dry pet food, mixed to obtain homogeneity, and maintained for
2 weeks at room temperature (25uC) as per AOAC guidelines (17).A three-tube most-probable-number (MPN) analysis of each
inoculated food was performed to determine the two target
inoculum levels. For the MPN analysis, triplicate samples of
100, 10, 1, and 0.1 g from each inoculum level were prepared from
each matrix, enriched with the appropriate medium, and confirmed
according to the FDA BAM reference method (4).
Method for 25-g high-throughput reduced enrichmentprotocol. A 25-g sample of ground dry pet food from an individual
inoculated sample was enriched in 225 ml of prewarmed BHI broth
at 37uC and agitated by shaking at 225 rpm overnight. After a 12-h
incubation step, samples were prepared for testing by PCR assay.
Nucleic acids were extracted using the PrepSEQ nucleic acid
extraction kit (Applied Biosystems, Foster City, CA) automated on
the MagMAX Express-96 magnetic particle processor (Applied
Biosystems) according to the manufacturer’s instructions, with
slight modifications to include the preclarification step. A 150-ml
aliquot of proteinase K (PK) containing digestion buffer (10 ml of
20 mg/ml PK in 140 ml of PK digestion buffer) was added to each
well of a 96-well deep-well plate. A 96-well preclarification tray
(Applied Biosystems) was inserted into the 96-well deep-well
plate, and 250 ml of each enriched sample was added to separate
wells of the clarification tray. Samples were spun through the
clarification tray directly into the PK buffer by centrifugation at
400 to 600 | g for 5 min on a tabletop plate centrifuge. The lysis
plate containing sample and PK buffer was processed according to
the manufacturer’s instructions.
At the end of the run, the elution plate containing the
extracted nucleic acid samples was recovered from the instrument,
and 30 ml was added directly to the real-time PCR assay as
described below.
Method for 375-g composite sample protocol. For the 375-
g compositing protocol, 15 25-g samples of inoculated ground dry
pet food from an inoculated sample were combined. Each 375-g
composite sample was enriched in 3.375 liters of buffered peptone
water (BPW) broth that had been prewarmed overnight at 37 ¡
1uC. Samples were incubated statically at 37 ¡ 1uC for 20 and 24 h
before proceeding to sample preparation and real-time PCR.
Sample preparation was performed with the PrepSEQ rapid
spin sample preparation kit (Applied Biosystems), which uses
individual preclarification tubes (9), according to the manufactur-
er’s instructions. A 30-ml volume of the final eluted sample was
added directly to the real-time PCR assay as described below.
Real-time PCR. A 30-ml volume of extracted DNA was added
to the MicroSEQ Salmonella lyophilized reagent mix, according to
the manufacturer’s instructions. The real-time PCR was performed
on the 7500 Fast instrument (Applied Biosystems). The RapidFinder
Express software (Applied Biosystems) differentiates positive from
negative samples based on a well-characterized threshold (9). For
the pet food matrix, the threshold settings were further tested based
on a study with 2,000 additional dry pet food samples.
Methods comparison. For both the 25-g short enrichment
protocol and the 375-g composite protocol, the real-time PCR
method was compared with the FDA BAM reference method as an
unpaired study.
For the FDA BAM reference method, 45 25-g or 375-g test
portions for each of the four Salmonella strains were sampled from
an inoculated sample and enriched in lactose broth for 24 ¡ 2 h at
37 ¡ 1uC. Secondary enrichment (in both Rappaport-Vasilliadis
medium and tetrathionate broth), plating on bismuth sulfate (BS),
xylose lysine desoxycholate (XLD), and Hektoen enteric (HE)
selective agars, and presumptive confirmation by biochemical and
serological methods were all performed as described in the FDA
BAM protocol for identification of Salmonella (4). Final
confirmations were made by VITEK GNIz using the AOAC
official method 991.13 (AOAC International, Wayne, PA).
Samples enriched according to the MicroSEQ method using
BHI (25-g samples) or BPW (375-g samples) were analyzed by
PCR (real-time PCR presumptive) and confirmed as positive or
negative by plating (real-time PCR confirmed) onto BS, XLD, and
HE selective agars, and suspect colonies were characterized as
described with the FDA BAM protocol.
Data analyses. Data were analyzed as outlined by the AOAC
Research Institute for an unpaired study (8) according to the
Mantel-Haenszel x2 formula. A x2 value of less than 3.84 is
indicative of no significant difference (i.e., failed to reject the null
hypothesis that there is no difference; P , 0.05). The equation for
Mantel-Haenszel x2 is
x2 ~ n{1ð Þ AF{ BzCzDð ÞEð Þ2= AzBzCzDð Þ AzEð Þ
| BzCzDzFð Þ EzFð Þ
TABLE 1. Pet food samples and test organisms used in this study
Ground dry pet food matrix Analytical unit (g)
Salmonella serovar
used for inoculation Source Origin
Dog food 25 Typhimurium ATCC 14028 ATCCa
Dog food 25 Senftenberg ATCC 43845 ATCC
Dog food 25 Tennessee Pet food Procter and Gamble
Dog food 25 Othmarschen Pet food Procter and Gamble
Dog food 375 Typhimurium ATCC 14028 ATCC
Cat food 375 Newport ATCC 6962 ATCC
a ATCC, American Type Culture Collection, Manassas, VA.
348 BALACHANDRAN ET AL. J. Food Prot., Vol. 75, No. 2
where n equals A z B z C z D z E z F. Each letter describes
the type of result: A is test positive, confirmed positive; B is test
positive, confirmed negative; C is test negative, confirmed
positive; D is test negative, confirmed negative; E is reference
positive; and F is reference negative. The following equations also
were used to determine relative sensitivity, false-negative rate, and
false-positive rate:
Relative sensitivity ~ A=E
False negative rate ~ C= AzCð Þ
False positive rate ~ B= BzDð Þ
RESULTS AND DISCUSSION
Sample preclarification eliminates PCR inhibitors.Food samples present unique challenges, particularly with
respect to sample preparation for microbial detection by
PCR (20). A reliable method will allow for separation of
bacteria from food debris and matrix-associated PCR
inhibitors (1, 20) while concentrating the bacteria for
detection with maximum sensitivity. Several methods,
including slow-speed centrifugation and filtration through
cheesecloth or filter paper, have been previously described
for separating bacteria from food debris (16, 20, 27). Other
methods rely on bacterial concentration by filter capture or
immunomagnetic capture as a way to remove food matrix-
associated inhibitors (20, 28).We observed carryover of fine food residue through all
steps of sample preparation and into the eluted sample when
nucleic acids were extracted with the magnetic bead capture
method. Transfer of the residual food matrix into the PCR
often resulted in PCR inhibition (data not shown). Although
it was possible to carefully avoid residual food material
during the transfer of eluted sample to the PCR, consider-
able challenges were associated with the ease of use of the
method and the reproducibility of the results obtained.
Therefore, we evaluated a novel preclarification step for
filtration of the enriched samples before bacterial lysis and
the subsequent nucleic acid extraction steps. This step
retained pet food debris while allowing the bacteria to filter
through into the lysis buffer.
We observed complete elimination of carryover food
particles during elution of nucleic acids from the magnetic
particles when samples were preclarified compared with
samples that did not undergo preclarification (Fig. 1A). This
step greatly simplified transfer of the eluted sample into the
real-time PCR assay beads. We also observed an improve-
ment in detection by up to 2 Ct (cycle threshold) values due
to preclarification of samples, indicating full recovery of
Salmonella and elimination of the PCR inhibitory effect
(Fig. 1B). This improvement in sensitivity can be particularly
important for samples containing low levels of pathogens
where PCR inhibitory effects can be more pronounced.
Evaluation of a less than 14-h method for detectionof Salmonella from 25-g pet food samples. One of the
major bottlenecks in routine food safety testing is time to
result, which directly impacts product hold time before
release. Reducing the time needed to arrive at a reliable
negative or presumptive-positive result for the presence of
pathogens in food can be of great benefit for any food safety
program. Several studies have been conducted on the
detection of Salmonella from food samples using short
preenrichment times in combination with molecular detec-
tion methods (18, 24, 26).We evaluated a real-time PCR method in combination
with a reduced preenrichment time to enable routine
screening within 14 h after collection of pet food samples.
The method included a reduced preenrichment step of 12 h
under shaking conditions to maximize growth of Salmonella(compared with the routine overnight growth typically
recommended for Salmonella) followed by sample prepa-
ration using an automated magnetic bead–based total
nucleic acid capture method and detection by real-time
PCR assay.
No significant differences were observed between the
real-time PCR–based detection method and the FDA BAM
method, with x2 values of less than 3.84 for all test groups
(Table 2). Overall, close agreement was obtained for low-
level and high-level inoculated samples when compared
with the reference culture method. The real-time PCR
method enabled detection of Salmonella from the spiked
sample in 14 h compared with the 5 days required for
presumptive detection of Salmonella by the traditional
culture method. All samples in the nonspiked group were
negative by both real-time PCR and the culture method,
indicating absence of false-positive results in this group.
One unconfirmed positive sample was found in each of the
groups spiked with Salmonella Typhimurium and Salmo-
FIGURE 1. (A) Clarified samples resulted in no food matrixcarryover into the eluted samples as observed with the unclarifiedsamples. (B) Sample preclarification improved the overallsensitivity of the nucleic acid extraction protocol when assayedby real-time PCR. Ct, cycle threshold or the fractional PCR cyclenumber at which the reporter fluorescence is greater than thethreshold (19).
J. Food Prot., Vol. 75, No. 2 RAPID DETECTION OF SALMONELLA IN PET FOOD 349
nella Senftenberg. One possible explanation for this result is
that overgrowth by competitor organisms masked the
growth of Salmonella colonies on agar plates in the culture
confirmation step yielding a negative result by culture, as
has been observed in previous studies (15). Higher detection
rates of positive samples by only one of the methods tested
(in this study, the PCR method) can occur when the levels of
contaminating target organism are low and the targets are in
mixed populations with other microorganisms. PCR assay
can have higher sensitivity than culture-based methods (21,23). Alternatively, cross-contamination from a positive
sample into a negative sample could result in a false-
positive result. However, false-positive results with the PCR
method are easily mitigated by additional testing that
requires all positive samples to be confirmed by culture.
Of the samples spiked with Salmonella Senftenberg,
one sample that was cultured positive was not detected by
the real-time PCR assay. This sample had an extremely low
level of Salmonella as indicated by culture, and the
combination of low level and some PCR inhibition may
have precluded detection, as reported by others (1, 20).Another possibility is that the Salmonella in this sample did
not reach the minimum cell density required for detection by
real-time PCR assay after the short 12-h enrichment step.
However, x2 analyses indicated that results of the two
methods were not significantly different.
Evaluation of 375-g composite sample. Small an-
alytical units can be composited into a single larger sample
to reduce the number of samples handled during sample
preparation and screening. This combining step also
increases sample throughput without the need for a high-
throughput sample preparation step, resulting in a substan-
tial reduction in time, cost, and labor compared with routine
testing.
We evaluated a composite sample protocol that was set
up according to the FDA BAM, in which 15 25-g units were
combined into a single 375-g composite sample (5). To
evaluate the minimum time to result, we sampled at two
times, 20 and 24 h, and prepared nucleic acids using a
centrifugation concentration method, which included a
preclarification step (9).No significant differences were found between the real-
time PCR method, after both 20 and 24 h of enrichment, and
the FDA BAM method, with x2 values of less than 3.84 for
all test groups (Table 3). All spiked samples that were
positive by real-time PCR assay were confirmed to be
positive by the culture method. All Salmonella-positive
samples were detected at both 20 and 24 h. These studies
revealed that even with a large sample size of 375 g, low
levels of inocula can be reliably detected with only a 20-h
enrichment period in a nonselective broth when coupled
with a highly sensitive method such as real-time PCR assay.
All nonspiked samples were negative by both the real-time
PCR method and the culture method, indicating the absence
of false-positive results.
In conclusion, we designed and tested two integrated
methods for detection of low levels of Salmonella in dry pet
food using a highly sensitive real-time PCR–based ap-
proach. These rapid methods offer a great advantage over
existing culture confirmation methods (4, 6) by reducing
time to result to less than 3 h after preenrichment, which can
be as little as 12 h for 25-g samples and 20 h for pooled 375-
g samples without loss of sensitivity versus the standard
culture protocol. These new methods can be effective tools
for monitoring the microbial safety of dry pet food.
TABLE 2. Comparison study of 25-g samples enriched for 12 h, processed with a magnetic bead–based nucleic acid capture method, andanalyzed with a real-time PCR assay
Inoculation level MPN/25 g
Total no. of
samples
Real-time
PCR
presumptive
Real-time
PCR
confirmed
FDA BAM
confirmed
x2 (real-time
PCR vs FDA
BAM)
Relative
sensitivity
(%)
False-
negative rate
False-
positive rate
Salmonella Typhimurium ATCC 14028
Control 0 5 0 0 0
0.2–2 CFU/25 g 0.1 20 0 0 0
2–10 CFU/25 g 2.3 20 6 5 6 0.12 83 0 6.6
Salmonella Senftenberg ATCC 43845
Control 0 5 0 0 0
0.2–2 CFU/25 g 0.1 20 4 3 3 100 0 5.8
2–10 CFU/25 g 0.4 20 12 13 14 0.43 85 7.6 0
Salmonella Tennessee
Control 0 5 0 0 0
0.2–2 CFU/25 g 2.3 20 2 2 6 2.44 33 0 0
2–10 CFU/25 g 11.5 20 18 18 19 0.35 95 0 0
Salmonella Othmarchen
Control 0 5 0 0 0
0.2–2 CFU/25 g 0.2 20 6 6 7 0.11 86 0 0
2–10 CFU/25 g 2.3 20 16 16 18 0.76 89 0 0
350 BALACHANDRAN ET AL. J. Food Prot., Vol. 75, No. 2
ACKNOWLEDGMENTS
We thank Allan Minn, Bruce DeSimas, Fujun Zhang, Ravi Gupta,
Mary Baltazar, Justin Liao, Peyman Fatemi, and Bob Kays (Life
Technologies) for their significant contributions.
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Inoculation level MPN/25 g
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Real-time
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FDA BAM
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False-
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False-
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24 h of preenrichment
Control 0 5 0 0 0
0.2–2 CFU/25 g 0.1 20 5 5 3 0.39 167 0 0
2–10 CFU/25 g 0.4 20 14 14 16 0.39 87.5 0 0
J. Food Prot., Vol. 75, No. 2 RAPID DETECTION OF SALMONELLA IN PET FOOD 351
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