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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: priya.balachandran@lifetech.com.

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.

REFERENCES

1. Abu Al-Soud, W., and P. Radstrom. 2000. Effects of amplification

facilitators on diagnostic PCR in the presence of blood, feces, and

meat. J. Clin. Microbiol. 38:4463–4470.

2. American Pet Products Association. 2010. 2009/2010 national pet

owners survey. American Pet Products Association, Greenwich, CT.

3. American Veterinary Medical Association. 2007. U.S. pet ownership

and demographics sourcebook. American Veterinary Medical Asso-

ciation, Shaumburg, IL.

4. Andrews, W. H., and T. Hammack. 2007. Salmonella, chap. 5. In

Bacteriological analytical manual online. U.S. Food and

Drug Administration, Silver Spring, MD. Available at: http://

www.fda.gov/Food/ScienceResearch/LaboratoryMethods/Bacteri

ologicalAnalyticalManualBAM/ucm070149.htm. Accessed 18 July

2011.

5. Andrews, W. H., and T. S. Hammack. 2003. Food sampling and

preparation of sample homogenate, chap. 1. In Bacteriological

analytical manual online. U.S. Food and Drug Administration, Silver

Spring, MD. Available at: http://www.fda.gov/Food/ScienceResearch/

LaboratoryMethods/BacteriologicalAnalyticalManualBAM/ucm063335.

htm. Accessed 18 July 2011.

6. Anonymous. 2002. Microbiology of food and animal feeding stuffs—

horizontal method for the detection of Salmonella spp. ISO 6579:2002

(E). International Organization for Standardization, Geneva.

7. Anonymous. 2008. Mars recalls pet food because of potential link

with human illness. J. Am. Vet. Med. Assoc. 233:1198.

8. AOAC International. 2009. Research institute performance tested

methods program. Validation of rapid microbiological methods.

Available at: http://www.aoac.org/testkits/PTM_MicroWorkshop_

8-12-09.pdf. Accessed 28 July 2011.

9. Balachandran, P., Y. Cao, L. Wong, M. R. Furtado, O. V.

Petrauskene, and R. S. Tebbs. 2011. Evaluation of Applied

Biosystems MicroSEQH real-time PCR system for dection of

Salmonella spp. in food. J. AOAC Int. 94:1106–1116.

10. Behravesh, C. B., A. Ferraro, M. Deasy III, V. Dato, M. Moll, C. Sandt,

N. K. Rea, R. Rickert, C. Marriott, K. Warren, V. Urdaneta, E. Salehi,

E. Villamil, T. Ayers, R. M. Hoekstra, J. L. Austin, S. Ostroff, I. T.

Williams, and the Salmonella Schwarzengrund Outbreak Investigation

Team. 2010. Human Salmonella infections linked to contaminated dry

dog and cat food, 2006–2008. Pediatrics 126:477–483.

11. Centers for Disease Control and Prevention. 2008. Multistate

outbreak of human Salmonella infections caused by contaminated

dry dog food—United States, 2006–2007. Morb. Mortal. Wkly. Rep.557:521–524.

12. Centers for Disease Control and Prevention. 2009. Multistate

outbreaks of Salmonella infections associated with live poultry:

United States, 2007. Morb. Mortal. Wkly. Rep. 58:25–29.

13. Centers for Disease Control and Prevention. 2009. Compendium of

measures to prevent disease associated with animals in public

settings, 2009. National Association of State Public Health

Veterinarians, Inc. (NASPHV). Morb. Mortal. Wkly. Rep. Recomm.

Rep. 58:1–21.

14. Corbitt, N. 2007. Food safety testing: the U.S. market. BCC

Research, Wellesley, MA. Available at: http://www.bccresearch.

com/report/food-safety-testing-us-fod011e.html. Accessed 18 July

2011.

15. Cudjoe, K. S., and R. Krona. 1997. Detection of Salmonella from raw

food samples using Dynabeads anti-Salmonella and a conventional

reference method. Int. J. Food Microbiol. 37:55–62.

16. Dwivedi, H. P., and L. Jaykus. 2011. Detection of pathogens in food:

the current state-of-the-art and future directions. Crit. Rev. Microbiol.37:40–63.

17. Feldsine, P., C. Abeyta, and W. H. Andrews. 2002. AOAC

International methods committee guidelines for validation of

qualitative and quantitative food microbiological official methods

of analysis. J. AOAC Int. 85:1187–1200.

18. Ferretti, R., I. Mannazzu, L. Cocolin, G. Comi, and F. Clementi.

2001. Twelve-hour PCR-based method for detection of Salmonella

spp. in food. Appl. Environ. Microbiol. 67:977–978.

19. Heid, C. A., J. Stevens, K. J. Livak, and P. M. Williams. 1996. Real

time quantitative PCR. Genome Res. 6:986–994.

20. Jaykus, L.-A. 2003. Challenges to developing real-time methods to

detect pathogens in foods. ASM News 69:341–347.

21. Johansson, A., L. Berglund, U. Eriksson, I. Goransson, R. Wollin, M.

Forsman, A. Tarnvik, and A. Sjostedt. 2000. Comparative analysis of

TABLE 3. Comparison study of 375-g composite samples of ground dry dog food or ground dry cat food enriched for 20 or 24 h,processed with a centrifugation concentration nucleic acid preparation method, and analyzed 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

Dry dog food, Salmonella Typhimurium ATCC 14028

20 h of preenrichment

Control 0 5 0 0 0

0.2–2 CFU/25 g 0.1 20 6 6 3 1.26 200 0 0

2–10 CFU/25 g 2.3 20 14 14 10 1.62 140 0 0

24 h of preenrichment

Control 0 5 0 0 0

0.2–2 CFU/25 g 0.1 20 6 6 3 1.26 200 0 0

2–10 CFU/25 g 0.4 20 14 14 10 1.62 140 0 0

Dry cat food, Salmonella Newport ATCC 6962

20 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 2.3 20 14 14 16 0.39 87.5 0 0

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

PCR versus culture for diagnosis of ulceroglandular tularemia. J.

Clin. Microbiol. 38:22–26.

22. Kukanich, K. S. 2011. Update on Salmonella spp contamination of

pet food, treats, and nutritional products and safe feeding recom-

mendations. J. Am. Vet. Med. Assoc. 238:1430–1434.

23. Maddox, C. W. 2003. Detection/diagnosis of Salmonella, p. 83–88. In

M. E. Torrence and R. E. Isaacson (ed.), Microbial food safety in animal

agriculture: current topics, sect. 3. Wiley-Blackwell, Hoboken, NJ.

24. Malorny, B., E. Paccassoni, P. Fach, C. Bunge, A. Martin, and R.

Helmuth. 2004. Diagnostic real-time PCR for detection of Salmonella

in food. Appl. Environ. Microbiol. 70:7046–7052.

25. McCarthy, N., F. J. Reen, J. F. Buckley, J. G. Frye, E. F. Boyd, and

D. Gilroy. 2009. Sensitive and rapid molecular detection assays for

Salmonella enterica serovars Typhimurium and Heidelberg. J. Food

Prot. 72:2350–2357.

26. Mozola, M. A., X. Peng, and M. Wendorf. 2007. Evaluation of the

GeneQuence DNA hybridization method in conjunction with 24-hour

enrichment protocols for detection of Salmonella spp. in select foods:

collaborative study. J. AOAC Int. 90:738–755.

27. Stevens, K. A., and L. Jaykus. 2004. Bacterial separation and

concentration from complex matrices: a review. Crit. Rev. Microbiol.30:7–24.

28. Tatavarthy, A., K. Peak, W. Veguilla, T. Cutting, V. J. Harwood, J.

Roberts, P. Amuso, J. Cattani, and A. Cannons. 2009. An accelerated

method for isolation of Salmonella enterica serotype Typhimurium

from artificially contaminated foods, using a short preenrichment,

immunomagnetic separation, and xylose-lysine-desoxycholate agar

(6IX method). J. Food Prot. 72:583–590.

29. Tebbs, R. S., Y. Y. Cao, P. Balachandran, and O. Petrauskene. 2009.

TaqManH Salmonella enterica detection kit. Performance tested

method 020803. J. AOAC Int. 92:1895–1901.

30. Techathuvanan, C., F. A. Draughon, and D. H. D’Souza. 2010. Real-

time reverse transcriptase PCR for the rapid and sensitive detection of

Salmonella Typhimurium from pork. J. Food Prot. 73:507–514.

352 BALACHANDRAN ET AL. J. Food Prot., Vol. 75, No. 2