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Quantitative Detection of Pork Contamination in Cooked Meat Products by

ELISA

Article  in  Journal of AOAC International · September 2017

DOI: 10.5740/jaoacint.17-0036

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810 THIENES ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 101, NO. 3, 2018
FOOD COMPOSITION AND ADDITIVES
Quantitative Detection of Pork Contamination in Cooked
Meat Products by ELISA
CORTLANDT P. THIENES and JONGKIT MASIRI
Molecular Epidemiology, Inc., 15300 Bothell Way NE, Lake Forest Park, WA 98155
LORA A. BENOIT
IEH Laboratories and Consulting Group, Inc., 15300 Bothell Way NE, Lake Forest Park, WA 98155
BRIANDA BARRIOS-LOPEZ, SANTOSH A. SAMUEL, DAVID P. COX, ANATOLY P. DOBRITSA, and CESAR NADALA
Molecular Epidemiology, Inc., 15300 Bothell Way NE, Lake Forest Park, WA 98155
MANSOUR SAMADPOUR1
Molecular Epidemiology, Inc., 15300 Bothell Way NE, Lake Forest Park, WA 98155; IEH Laboratories and Consulting Group, Inc.,
15300 Bothell Way NE, Lake Forest Park, WA 98155
Recent news of many cases of adulteration of
meats with pork has bolstered the need for a way
to detect and quantify the unwanted contamination
of pork in other meats. To address this need,
Microbiologique, Inc. has produced a sandwich
ELISA assay that can rapidly quantify the presence
of pork in cooked horse, beef, chicken, goat, and
lamb meats. We carried out a validation study and
showed that this assay has an analytical sensitivity
of 0.00014 and 0.00040% (w/v) for cooked and
autoclaved pork, respectively, and an analytical
range of quantitation of 0.05–3.2% (w/v) in the
absence of other meats. The assay can measure
pork contamination down to 0.1% (w/w) in the
presence of cooked horse, beef, chicken, goat, and
lamb meats. The assay is quick and can be
completed in 1 h and 10 min.
P
ork is the third most highly consumed meat in the United
States at 9409 metric tons per year compared with 15 233
metric tons of poultry and 11 671 metric tons of beef (1).
Pork is the most consumed meat in Asia, with China alone
consuming 57 140 metric tons (1). Pork, at an average retail
value of $3.78 USD per pound, is far less expensive than beef
at $6.12 per pound (2). Due to economic incentives and cross-
contamination on meat-processing equipment, the potential for
intentional and accidental adulteration is high.
Many high-profile cases of meat adulteration have occurred
recently, notably the 2013 horsemeat scandal (3), when the Food
Safety Authority of Ireland found traces of pig and horse DNA in
products labeled as beef. Of 27 beef burger products analyzed,
37% were positive for horse DNA and 85% were positive for pig
DNA (3). The evolving investigation eventually found horsemeat
contamination in the United Kingdom, France, Germany, Sweden,
Belgium, the Netherlands, and Switzerland (4). Because of the
scandal, the confidence of European customers in the meat-
processing industry was damaged.
Adulteration of pork into other meats is a global concern, and
even accidental contamination at low levels poses significant
Received February 3, 2017. Accepted by SG August 9, 2017.
1
Corresponding author’s e-mail: ms@iehinc.com
DOI: https://doi.org/10.5740/jaoacint.17-0036
concern among Muslim and Jewish consumers for religious
reasons. In the United States, no official acceptable level of
meat contamination has been adopted by the U.S. Department of
Agriculture (USDA). In the United Kingdom, however, a
threshold of 1% (w/w) was established (5), and the United
Kingdom Food Standards Agency (FSA) recommends
investigation of the causes of contamination between 0.1 and
1% (w/w).
Previous published methods for the detection of pork
meat have used qualitative ELISA (6–8), with LODs down to
0.05% (w/w) in cooked chicken and 0.1% (w/w) in cooked
beef. None of the commercially available kits are validated to
detect to a level of 0.1% (w/w), which is necessary to comply
with the recommendation of the FSA. The ELISA-TEK Cooked
Pork Species Kit (ELISA Technologies, Gainesville, FL) was
tested by the USDA and found to detect at least 4% (w/w)
pork when pork and background meat extracts were mixed
(9). Perestam et al. (10) found the ELISA-TEK kit sensitive
to 10% (w/w) using extracts made from pork meat mixed into
a beef background. A potential improvement, the Neogen
BIOKITS Cooked Species Identification Kit (Neogen,
Lansing, MI) is still only sensitive to 1% (w/w) according to
the manufacturer.
Lateral flow tests (11) are a quick alternative for pork
detection, but are semiquantitative and only sensitive to 1%
(w/w) cooked pork in other meats. Real-time PCR can potentially
quantitate the level of pork in meat backgrounds to 0.01% (w/w;
12), but reliable quantitation can be technically demanding (13).
An ELISA-based kit for the quantitation of meat contamination
can be performed quickly on site with limited equipment,
whereas PCR-based techniques require equipment and
expertise that are not available in most meat-processing
plants. To address the need for a quick, on-site method to
determine and quantify the adulteration of pork into beef or
other meats, Microbiologique (Seattle, WA) produced an ELISA
that can quantify cooked pork contamination at a concentration
as low as 0.1% (w/w) in only 70 min.
Materials and Methods
Chemicals and Reagents
Extraction buffer, dilution buffer, antibody-coated assay plates,
wash buffer, horse radish peroxidase-conjugated detection
 THIENES ET AL.: JOURNAL OF
Figure 1. LOD determination calculating the reliable detection limit
(RDL) as the interpolated intersection of the upper 95% confidence
interval (CI) horizontal asymptote with the lower 95% CI of the
standards data. Percent pork (w/v) is the final concentration of pork
meat in the assay after extraction and dilution. Absorbance values
are an average of eight replicates. (A) Cooked pork and
(B) autoclaved pork.
antibody, enzyme substrate, and stop buffer were supplied in
the Microbiologique Cooked Pork ELISA kit.
Meat Samples
Raw pork shoulder roast, boneless beef sirloin roast, breast
meat from whole chickens, turkey breast, leg of lamb roast, and
prepared meats were purchased from a Fred Meyer grocery store
(Seattle, WA). Goat leg meat was purchased from Better Meat
(Seattle, WA). Horse leg meat was a gift from Washington State
University, College of Veterinary Medicine (Pullman, WA).
Meat identities were confirmed with a species-specific
conventional multiplex PCR, followed by DNA lateral-flow
detection at IEH Laboratories, Inc. (Seattle, WA). Pet foods
were obtained from a PetSmart retailer (Seattle, WA).
Preparation of Meat Extracts
Preparation of meat extracts was performed exactly as described
in the Microbiologique Cooked Pork ELISA kit instructions-for-
use manual. Excess fat and connective tissue were trimmed from
AOAC INTERNATIONAL VOL. 101, NO. 3, 2018 811
Table 1. PRSDR values used in this study
Concn before extraction, %a Analytical concn, % PRSDR, %
3.20 0.08 5.8
1.60 0.04 6.5
0.80 0.02 7.2
0.40 0.010 8.0
0.20 0.0050 8.9
0.10 0.0025 9.9
0.050 0.0013 10.9
a
The concentration before extraction is the analytical concentration
multiplied by the 40-fold dilution factor.
the meat, which was ground twice in a Nesco electric meat grinder
(Metal Ware Corp., Two Rivers, WI). A 1% (w/w) mixed raw
meat sample was prepared by adding 0.5 g each of pork, horse,
beef, chicken, turkey, goat, and lamb to 46.5 g ground background
meat and homogenizing the sample 10 times for 5 s in a blender
(Model No. 31BL92; Waring, New Hartford, CT) with an MC2
cup and mixing with a tongue depressor between each cycle. Five
grams of the 1% mixed meat were added to 45 g background meat
and homogenized to produce the 0.1% (w/w) sample. For the
matrix-matched negative (0%) control, 50 g background meat
were homogenized alone.
For extract preparation, 20 g homogenized mixed meat
were added to a 55 oz Whirl-Pak stomacher bag (Nasco, Fort
Atkinson, WI) containing 200 mL extraction solution and
homogenized for 1 min at high speed in a Stomacher 400
circulator (Seward, West Sussex, United Kingdom), manually
agitated for 3 s, and then homogenized for another minute in the
circulator. After a 10 min incubation at room temperature (RT),
the extract was centrifuged in 5–50 mL polypropylene disposable
tubes (Corning, Corning, NY) at 2000 × g for 10 min at 4°C, and
the supernatant was transferred to a new 50 mL tube. Cooked
meat extracts were prepared by incubating 25 mL of the raw meat
extracts in a 50 mL tube in a boiling water bath 15 min after the
extract temperature reached 95°C. For autoclaved meat extracts,
20 g homogenized mixed meat samples were autoclaved at 121°C
for 35 min in a 100 mL Pyrex jar (Corning), added to a stomacher
bag, and extracted with extraction solution as above. Extracts
Figure 2. Reactivity of the pork ELISA. Means of triplicate samples
from serial dilutions of raw, cooked, and autoclaved pork (n = 3).
812 THIENES ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 101, NO. 3, 2018

Table 2. Analytical range of quantitation determination

Result Accuracy
a
Cooked pork concn (w/v), % Analyst 1, % Analyst 2, % Analyst 1, % Analyst 2, % CV, % RSDr/PRSDr

3.2 3.4 3.5 105 108 1.7 0.28

1.6 1.6 1.6 100 101 0.3 0.05
0.80 0.77 0.78 96 98 1.2 0.16
0.40 0.40 0.40 99 100 0.4 0.06
0.20 0.20 0.19 98 97 0.7 0.07
0.10 0.10 0.09 100 94 4.8 0.49
0.050 0.050 0.051 100 103 1.7 0.15
a
Comparison between known concentrations and values determined by Microbiologique Cooked Pork ELISA (n = 3).

were stored at –20°C and thawed samples spun at 16 000 × g for
10 min at 4°C before use. Commercially processed meat products
and pet food samples were extracted using the same protocol after
first homogenizing the 20 g sample without extraction buffer in
the blender (Waring) with an MC2 cup.
Extracts used in the LODs and range of quantitation (ROQ),
reproducibility, and ruggedness tests were made from 20 g of
2× ground 100% pork by the extraction method above.
Cooked Pork ELISA
The Cooked Pork ELISA kit was obtained from
Microbiologique, Inc. The assay protocol was as follows.
(1) Meat extracts were diluted 4-fold in dilution buffer.
Prepared meat and pet food extracts were diluted in a
10–0.01% (w/v) series in dilution buffer. Diluted extracts were
added at 100 µL per well (n = 3, unless otherwise noted) and
incubated for 30 min at RT on a titer plate shaker (Laboratory-Line
Instruments, Melrose Park, IL) on setting No. 2. (2) The
wells were washed four times with wash buffer, and 100 µL
horse radish peroxidase-conjugated detection antibody were
added per well, incubated at RT, and shaken for 30 min. (3)
The wells were washed four times with wash buffer, and
100 µL enzyme substrate were added per well and incubated
at RT and shaken for 10 min. (4) Stop solution was added at
50 µL per well, and the absorbance was read at a 450 nm
wavelength with a SpectraMax ELISA microplate reader
(Molecular Devices, Sunnyvale, CA). Standard curves were
Table 3. Cooked pork quantitation in the presence of
a meat matrix (n = 3)
Spiked samples OD450 SD Result, % Accuracy, %
generated in Excel using a five-parameter nonlinear curve-
fitting model (14).
Comparison Against a Reference Method
The USDA-tested Cooked Pork ELISA (ELISA-TEK,
Gainesville, FL; 9) was performed using the same 1% (w/w)
mixed meat and matrix-matched negative controls that
were used in the Microbiologique Cooked Pork ELISA
following the manufacturer’s instructions. The validity of
the assay, result interpretation, and classification of the
unknown samples were determined using the USDA
criteria detailed in the ELISA-TEK kit instructions-for-use
manual.
Determination of LOD
The analytical LOD for the Microbiologique Cooked Pork
ELISA was determined using eight replicates of 2-fold serial
dilutions of 100% cooked and autoclaved pork meat extracts in
dilution buffer in the absence of a meat matrix. For the
analytical LOD, the concentration of the initial extract of
100% pork meat diluted in 10 volumes of extraction buffer
was 10% (w/v). The reliable detection limit (RDL), i.e., the
“lowest concentration that has a high probability of producing
a response that is significantly greater than the response at
zero” (15), was used to calculate the LOD. Briefly, the upper
and lower 95% confidence intervals (CIs) of the average
absorbance readings for the diluted samples were graphed
using a five-parameter nonlinear curve-fitting model (14).
The RDL was the concentration of pork corresponding to the
interpolated intersection of the upper 95% CI horizontal
asymptote, with the lower 95% CI of the absorbance readings

1% pork in horse 0.80 0.0092 0.38 38 Table 4. Method concordance assay: ELISA-TEK and
0.1% pork in horse 0.13 0.0036 0.059 59 Microbiologique Cooked Pork ELISA kit results
1% pork in beef 0.83 0.0087 0.40 40
ELISA-TEK
0.1% pork in beef 0.13 0.0045 0.060 60 Microbiologique
1% pork in chicken 1.0 0.017 0.49 49 Spiked samples OD450 – 3× SD Result (n = 2) Result, % (n = 3)
0.1% pork in chicken 0.15 0.018 0.069 69
1% pork in horse 0.756 +a 0.57
1% pork in goat 1.8 0.025 1.0 102
1% pork in beef 0.696 + 0.49
0.1% pork in goat 0.25 0.0061 0.11 113
1% pork in chicken 0.749 + 0.61
1% pork in lamb 0.94 0.011 0.45 45
1% pork in goat 0.938 + 1.31
0.1% pork in lamb 0.11 0.0017 0.053 53
a
+ = Positive.
 THIENES ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 101, NO. 3, 2018 813

Table 5. Reproducibility within a single run (n = 9)

Run, %

Concn cooked pork (w/v), % 1 2 3 4 5 6 7 8 9 CV, % RSDr/PRSDr

3.20 3.43 3.37 3.55 3.2 3.3 3.4 3.4 3.6 3.5 3.2 0.55
1.60 1.62 1.58 1.64 1.6 1.6 1.7 1.6 1.7 1.8 4.5 0.69
0.80 0.78 0.78 0.79 0.80 0.79 0.80 0.80 0.80 0.85 2.5 0.34
0.40 0.40 0.39 0.40 0.40 0.39 0.40 0.39 0.40 0.40 1.1 0.14
0.20 0.19 0.20 0.19 0.20 0.19 0.19 0.19 0.20 0.19 2.9 0.32
0.10 0.09 0.09 0.10 0.10 0.10 0.10 0.09 0.10 0.10 2.9 0.29
0.05 0.05 0.05 0.06 0.05 0.06 0.05 0.05 0.06 0.06 7.5 0.68

for the diluted samples (Figure 1) calculated using the five-
parameter equation.
Analytical Range of Quantitation, Reproducibility, and
Ruggedness Testing
Analytical range of quantitation (ROQ), reproducibility, and
ruggedness testing were carried out using 2-fold serial
dilutions of 100% cooked pork that had been initially
extracted with 10 volumes of extraction solution and diluted
4-fold in dilution buffer. Cooked pork concentration values
for ROQ, reproducibility, and ruggedness testing are reported as
concentrations before extraction and dilution. The concentration of
pure pork extracted with 10 volumes of extraction buffer and diluted
4-fold in dilution buffer would be 100% (w/w) in the ROQ,
reproducibility, and ruggedness testing.
The effect of temperature was tested by assaying triplicate
samples at 5°C above and below the standard assay temperature
of 23°C (RT) at 18, 23, and 28°C.
The effect of incubation time on cooked pork quantitation was
investigated by varying the incubation periods ±25%. Three
experiments were set up in which the incubation times were
shortened to 22.5–22.5–7.5 min, kept at a standard of 30–30–10
min, and lengthened to 37.5–37–12.5 min for the sample
extract, detection antibody, and enzyme substrate incubations,
respectively.
The acceptability criterion for reproducibility performance
was assessed by the Horwitz ratio (HorRat; 16). HorRat is
calculated from the ratio of the RSD or the percent CV to the
predicted RSDR (PRSDR; Table 1). Acceptable HorRats are
between 0.3 and 1.3 (17) for repeatability conditions (by the
same analyst) or 0.5–2 under reproducibility conditions
(between-laboratories). PRSDR = 2C–0.1505, where C = percent
concentration of the analyte in this study.
Results and Discussion
The reactivity of the Microbiologique Cooked Pork
ELISA toward raw, cooked, and autoclaved pork was
evaluated using 2-fold serial dilutions of each extract in
the dilution buffer. Results show similar reactivity, with
raw and cooked pork being slightly better than autoclaved
pork (Figure 2).
Analytical Sensitivity
The analytical LOD for the Microbiologique Cooked Pork
ELISA was determined through calculation of the RDL
(Figure 1) for the final concentration of pork meat in the
assay after extraction and dilution. The LOD for the assay
was calculated as 0.00014% (w/v) for cooked pork
(Figure 1A) and 0.00040% (w/v) for autoclaved pork
(Figure 1B).
Analytical Range of Quantitation
The analytical ROQ for the Microbiologique Cooked Pork
ELISA was determined using 2-fold serial dilutions of cooked
pork extract, first diluted 4-fold in dilution buffer in the absence
of a meat background matrix. Assays were performed by two
analysts. The ROQ for pork ELISA was determined to be
between at least 0.05 and 3.2% (w/v) cooked pork (Table 2).
Pork concentrations above and below this range were not tested.
Accuracy was calculated to be within 10% of the known values,
analyst-to-analyst CV was below 5%, and the HorRat was
consistently on the low side (values of <0.5) for samples with
0.05–3.2% (w/v) cooked pork. This indicates excellent

Table 6. Reproducibility between runs (n = 3)

Concn cooked pork (w/v), % Day 1, % Day 2, % Day 3, % CV, % RSDr/PRSDr

3.2 3.4 3.5 3.2 3.7 0.63

1.6 1.6 1.6 1.6 1.7 0.27
0.80 0.82 0.78 0.79 2.7 0.38
0.40 0.41 0.40 0.39 2.2 0.27
0.20 0.20 0.19 0.20 1.8 0.20
0.10 0.10 0.09 0.10 5.1 0.51
0.050 0.047 0.051 0.052 5.6 0.51
814 THIENES ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 101, NO. 3, 2018

Table 7. Ruggedness: effect of temperature (n = 3)

Temperature, %

Concn cooked pork (w/v), % 18°C 23°C 28°C CV, % RSDr/PRSDr

3.2 3.7 3.4 3.4 4.3 0.73

1.6 1.6 1.6 1.7 1.3 0.21
0.80 0.80 0.82 0.80 1.7 0.24
0.40 0.40 0.41 0.40 1.2 0.14
0.20 0.20 0.20 0.20 1.4 0.16
0.10 0.10 0.10 0.10 1.3 0.14
0.050 0.048 0.047 0.050 3.7 0.34

performance by the analysts on a highly reproducible assay
within the ROQ.
Detection of Pork in Various Cooked Meat
Backgrounds
To estimate the LOD of pork in cooked meat backgrounds,
nonspiked meat extracts were tested with the Microbiologique
Cooked Pork ELISA kit. The ELISA absorbance reading above
which it can be reasonably affirmed that cooked pork is detected
in the presence of each background meat was estimated as the
average absorbance of the nonspiked background meat sample
plus 3.3 times the SD. This absorbance was highest with horse
meat, estimated at 0.075 (data not shown). The LOD of the
Cooked Pork ELISA kit in meat background is, therefore,
predicted to have an absorbance of 0.075.
The data in Table 3 show that the lowest absorbance reading
for all the meats spiked with 0.1% (w/w) cooked pork is 0.11,
which is 1.5 times higher than with horse meat. Therefore, 0.1%
(w/w) pork is above the LOD and clearly detectable in all meat
backgrounds. Although the pork ELISA has an analytical ROQ
as low as 0.05% in the absence of a meat matrix, it is calibrated to
measure pork contamination in the presence of a meat matrix at
0.1% or higher due to the background signal contributed from the
meat matrix.
To determine the accuracy of the results obtained with the
Microbiologique Cooked Pork ELISA when measuring cooked
samples containing a background meat spiked with pork, the
assay was performed on various meat samples spiked with 1 and
0.1% (w/w) pork. The accuracy of the pork ELISA result was
determined as the percent pork calculated from the assay
compared with the expected percent of pork spiked into
background meat.
The accuracy of the ELISA results ranged from 53 to 113% for
meat spiked with 0.1% (w/w) pork and 38–102% for meat spiked
with 1% (w/w) pork (Table 3). Recovery was higher in goat
background than in other meats.
Concordance
Concordance with the ELISA-TEK pork detection kit was
determined using the same spiked 1% (w/w) cooked mixed meat
extracts and nonspiked meat backgrounds as used for testing the
Microbiologique Cooked Pork ELISA kit. Both the ELISA-TEK
and Microbiologique ELISA kits detected pork in the spiked
meat samples (Table 4). Nonspiked meat backgrounds were
negative in both assays (data not shown). In the ELISA-TEK
kit result interpretation by the USDA criteria, a sample is positive
if its mean blank-corrected value minus 3 SDs is higher than
0.250. By this criterion, all the 1% (w/w) pork samples were
positive in the ELISA-TEK assay. Although the USDA found the
ELISA-TEK kit sensitive to at least 4% pork, the sensitivity in
this assay was at least 1%. In the Microbiologique kit results,
pork meat concentrations ranged from 0.49 to 1.31% (w/w)
depending on the meat background. The ELISA-TEK data
agrees with the Microbiologique results as pork was detected
by the ELISA-TEK kit, which has an LOD of at least 1% in this
assay. Interestingly, the values were higher in goat meat
background for both ELISA kits.
Precision of the Microbiologique Cooked Pork ELISA
Reproducibility of the Microbiologique ELISA kit was
measured using 2-fold serial dilutions of cooked pork meat
extract, first diluted 4-fold in dilution buffer. In each

Table 8. Ruggedness: effect of incubation time (n = 3)

Concn cooked pork (w/v), % Shorter(–25%), % Standard, % Longer (+25%), % CV, % RSDr/PRSDr

3.2 3.3 3.5 3.4 2.4 0.41

1.6 1.6 1.6 1.6 1.1 0.17
0.8 0.80 0.78 0.76 2.3 0.32
0.4 0.41 0.40 0.40 2.1 0.27
0.2 0.20 0.19 0.20 0.7 0.08
0.10 0.10 0.094 0.10 3.8 0.39
0.050 0.047 0.051 0.042 10 0.94
 THIENES ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 101, NO. 3, 2018 815

Ruggedness of the Microbiologique Cooked Pork

ELISA

The robustness of the Microbiologique ELISA kit was

Figure 3. Microbiologique Cooked Pork ELISA kit of prepared
meats (n = 3). P = pork, B = beef, C = chicken, and t = Turkey.
measured using 2-fold serial dilutions of cooked pork meat
extract, first diluted 4-fold in dilution buffer. In each
determination, CVs were determined and the HorRat calculated.
The effect of temperature was tested by assaying triplicate
samples at 5°C above and below the standard assay temperature
of 23°C (RT). CVs ranged from 1.2 to 4.3%, and the HorRat was
below 0.75 for cooked pork concentrations of 0.05–3.2% (w/v;
Table 7), indicating that temperature variations of ±5°C do not
significantly affect cooked pork quantitation in this ELISA.
The effect of incubation time on cooked pork quantitation was
investigated by varying the incubation periods ±25%. The results
show that the CVs between experiments are <4% (Table 8), and
the HorRat is below 0.95, indicating that the incubation times can
be shortened or extended by up to 25% for cooked pork
concentrations of 0.05–3.2% (w/v).

Detection of Pork in Commercially Processed Meat

Products
reproducibility determination, the CV and HorRat were
determined for the measured pork concentration.
Extracts from commercially prepared meats were tested using
Reproducibility within a single run was determined using nine
the Microbiologique Cooked Pork ELISA. Pork was detected in
replicates of each sample. CVs ranged between 1.1 and 7.5% for
pepperoni sticks, pork salami, Spam, pork and beef meat loaf,
cooked pork concentrations of 0.05–3.2% (w/v, Table 5). The
pork-containing polish sausage, and chicken and pork franks
HorRat was below 0.7 and on the low side for most samples tested,
(Figure 3). Note that estimates of percent pork reflect the
indicating that the assay is highly reproducible.
concentration of pork meat, not including fat and connective
Reproducibility between runs was determined on 3 separate
tissue. Beef franks and beef jerky were negative for pork as
days by the same analyst using triplicate 2-fold dilutions of each
expected (Figure 3). No undeclared pork was detected in any of
sample. CVs ranged between 1.7 and 5.6% for cooked pork
the processed meats tested.
concentrations of 0.05–3.2% (w/v; Table 6). The HorRat was
again on the low side (0.2–0.63), indicating that the assay is
highly reproducible between runs. Detection of Pork in Pet Foods
Reproducibility between analysts was determined by two
analysts on separate days using triplicate 2-fold dilutions of Pork was detected in the pet foods that listed pork in the
cooked pork extract. CVs ranged between 0.3 and 4.8% for ingredients (Table 9): Castor and Pollux dry dog food containing
cooked pork concentrations of 0.05–3.2% (w/v), and the HorRat pork meal (sample ID No. 50) and Royal Canin canned food
was below 0.5 (Table 2), indicating that the assay is reproducible containing pork by-products and pork liver (sample ID No. 29).
between analysts. In the four pet food products that contained natural flavors, meat

Table 9. Percent pork in pet foods

ID No. Product Dry/canned Meat content Pork, % (n = 3)

1 Royal Canin Mini Adult Dog Food Dry Chicken meal, chicken fat, natural flavor 0.62 ± 0.012
28 Max Cat Adult Chicken and Lamb Formula Canned Chicken broth, chicken, chicken liver, beef liver, 0
turkey, lamb, copper proteinate
29 Royal Canin Puppy in gel Canned Chicken, pork by-products, pork liver, zinc proteinate 1.3 ± 0.17
32 Eukanuba Adult Chicken and Rice Entrée Canned Chicken broth, chicken, beef by-products, chicken 0
by-products, beef liver, chicken by-product
meal, dried egg product
50 Castor and Pollux Natural Ultramix Grain-Free Dry Beef, pork meal, lamb meal, natural flavors 1.3 ± 0.031
Red Meat Recipe with Raw Bites
51 Natural Balance Wild Pursuit Beef and Lamb Dry Beef, chicken meal, lamb meal, natural flavors 0.062 ± 0.052
Meal Formula Dry Dog Food
54 Paws Premium Cat Food Dry Chicken by-product, animal fat, meat and bone meal, 2.0 ± 0.027
animal digest (chicken, turkey, liver)
55 Iams Dog Food Patty with Chicken Canned Chicken, meat by-products, chicken by-products, dried 2.2 ± 0.16
egg product
816 THIENES ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 101, NO. 3, 2018
and bone meal, or meat by-products which includes extracts or
protein hydrolysates of meat (potentially including pork), pork
was also detected in three (dry Royal Canin; sample ID No. 1,
Paws Premium Cat Food ID No. 54, and Iams Dog Food Patty
with Chicken ID No. 55) but was 0.062% (w/w) in one (sample
ID No. 51; Natural Balance dry dog foods). No pork was detected
in Max Cat canned cat food with chicken, beef, and lamb (sample
ID No. 28) or in the canned Eukanuba Adult Chicken and Rice
Entrée (sample ID No. 32), neither of which have pork or
unspecified meat by-products listed in the ingredients.
Conclusions
The Microbiologique Cooked Pork ELISA kit has an
analytical sensitivity of 0.00014 and 0.00040% (w/v) for
cooked and autoclaved pork, respectively, and an ROQ of
0.05–3.2% (w/v) in the absence of a background meat matrix.
It can detect and quantify pork contamination in horse, beef,
chicken, goat, and lamb meat background down to 0.1% (w/w).
The Microbiologique Cooked Pork ELISA kit is a significant
improvement over the current USDA-tested protocol (9) for the
detection of pork, which is qualitative and reported to have a
sensitivity of at least 4% (w/w) by the USDA. The assay can be
completed in as little as 70 min, whereas the USDA-tested
protocol takes 2.5–3 h to complete.
The Microbiologique Cooked Pork ELISA kit is reproducible
within a single run, between separate runs, and between
operators, with CVs well below 10%. It is robust, having CVs
below 10%, with varying incubation periods and temperatures.
The kit performed as expected when tested against commercially
prepared meat products, showing no cross-reactivity with other
meats. It could detect pork in pet foods containing pork or animal
products that potentially contained pork.
Acknowledgments
We thank Lourdes Nadala for helping with the experimental
design and preparation of the manuscript, Youngsil Ha for meat
species identification by PCR, Harish Janagama and Kristina
Pratt for veterinary help, and Phil Goodwin for advice with assay
development.
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