Journal of Food Composition and Analysis 25 (2012) 83–87

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Journal of Food Composition and Analysis

journal homepage: www.elsevier.com/locate/jfca

Short Communication

Real-time PCR assays for detection and quantitation of porcine and bovine DNA in
gelatin mixtures and gelatin capsules
Hui Cai *, Xuelin Gu, Mary S. Scanlan, Dintletse H. Ramatlapeng, Chris R. Lively
cGMP Laboratories, PPD Inc., 8551 Research Way, Suite 90, Middleton, WI 53562, USA

A R T I C L E I N F O A B S T R A C T

Article history: Detection and quantitation of material from porcine and bovine species in gelatin and gelatin capsules is
Received 21 February 2011 required for health safety concerns and for some religious practices. In this study, two species-specific
Received in revised form 23 June 2011 qPCR assays were developed based upon repetitive elements. They allowed sensitive detection of
Accepted 24 June 2011
porcine and bovine DNA at as low as 1 pg/mL. The lack of cross-reactivity when the sets were used to
Available online 23 July 2011
amplify DNA from the other species indicates high specificity of the assay. When binary gelatin blends
containing various amounts of porcine and bovine gelatin were prepared and analyzed by the qPCR
Keywords:
assays, the determined ratios of porcine material to bovine material were very close to their theoretical
Gelatin
Gelatin capsule
values, and a contamination level as low as 1% of the other species in the gelatin blends could be
Quantitative PCR determined. When evaluated in gelatin capsules, although significantly less DNA was detected,
Real time PCR determination of porcine and bovine species identities and estimation of the relative abundance of each
Species detection species was possible. Therefore, the porcine and bovine species-specific qPCR assays described here
Species quantitation represent simple, reliable and sensitive DNA-based tests for determination and quantitation of the
Food analysis species of origin from highly processed products.
Food composition ß 2011 Elsevier Inc. All rights reserved.

1. Introduction gelatin capsules free of bovine by-products because cows are

Gelatin is a protein derived from the selective hydrolysis of
collagen, the principal constituent of connective tissues and bones
in vertebrates. Because of its gelling, thickening and stabilizing
properties, gelatin is commonly used as a binding and/or glazing
agent in a wide variety of food products such as marshmallows,
gummy candies, meat and aspics (Jones, 1977). In the pharmaceu-
tical industry, gelatin is used to make hard and soft capsules,
tablets, granulation, dietary supplements and protective coatings
for medicines, etc. (B.E. Jones, 2004).
The raw materials for gelatin manufacture come from
slaughterhouse by-products of various animals, primarily porcine
and bovine species (Hinterwaldner, 1977; R.T. Jones, 2004). The
largest source of gelatin is pigskins. However, a significant portion
of gelatin comes from bovine hides and splits. The remainder is
from bone materials extracted from porcine, bovine, poultry, or
fish species (Hinterwaldner, 1977; R.T. Jones, 2004).
Detection and quantitation of porcine and bovine species
material in gelatin and gelatin capsules is required for religious
reasons and health safety concerns. For example, Muslim halal and
Jewish kosher dietary laws require gelatin or gelatin capsules free
of porcine material. Likewise, Hindu customs require gelatin or
* Corresponding author. Tel.: +1 608 203 2831; fax: +1 608 827 8807.
E-mail address: hui.cai@ppdi.com (H. Cai).
considered sacred. Concerns about bovine by-products used in
gelatin have risen since the emergence of Bovine Spongiform
Encephalopathy (BSE). BSE, commonly known as mad cow disease,
is a fatal and neurodegenerative disease in cattle that causes a
spongy degeneration of the brain and spinal cord (Harman and
Silva, 2009; Smith and Bradley, 2003). First recognized in the
United Kingdom in 1986, to date, over 180,000 cases of BSE have
been reported in cattle (OIE, 2011). In the United States, the Food
and Drug Administration (FDA) released in 1997 guidelines for the
sourcing and processing of gelatin to reduce the potential risk
posed by BSE (FDA, 1997). These guidelines include provisions for
documented confirmation that the raw materials used in gelatin
production are from animals that are safe for human consumption
and that no bovine gelatin from countries reporting BSE is used in
injectable, ophthalmic, or implanted FDA-regulated products (FDA,
1997).
Species determination in animal by-products has been achieved
mainly through analyses of proteins and DNA. Protein-based
species determination often relies on methods such as enzyme-
linked immunosorbent assay (ELISA) (Giovannacci et al., 2004),
HPLC (Chou et al., 2007), mass spectrometry (Buckley et al., 2009)
and isoelectric focusing (Tepedino et al., 2001). However, for highly
processed products such as gelatin and gelatin capsules, protein-
based analyses may be hindered by progressive denaturation of the
protein markers caused by extreme temperature and pH treat-
ments during manufacture, resulting in the loss of heterogeneity

0889-1575/$ – see front matter ß 2011 Elsevier Inc. All rights reserved.
doi:10.1016/j.jfca.2011.06.008
84 H. Cai et al. / Journal of Food Composition and Analysis 25 (2012) 83–87
and antigenicity. Because of the greater stability of DNA, DNA-
based analyses such as PCR may offer an alternative. To date, PCR
analysis with species-specific primers has been used for sensitive
and specific detection of various animal species from highly
processed products (Dalmasso et al., 2004; Fumiere et al., 2006). In
addition, quantitative PCR (also called qPCR or real-time PCR) has
been increasingly used for fast, sensitive and specific detection of
species-specific DNA in a variety of processed products (Jonker
et al., 2008; Kesmen et al., 2009). Two types of chemistries
commonly used for detection of qPCR products accumulated
during PCR cycles are fluorescent dyes (e.g. SYBR green) and
sequence-specific DNA probes (e.g. Taqman probe). Fluorescent
dyes (e.g. SYBR green) intercalate with any double-stranded DNA.
The Taqman probe consists of oligonucleotide that is linked to a
fluorescent dye and quencher. Its degradation frees the fluorescent
dye from the quencher resulting in a fluorescence emission
proportional to the amount of template. The fluorescence signal
after each PCR cycle is measured and used for DNA quantitation
(Lovatt, 2002).
Despite the importance of the topic, as far as we know,
literature on determination and quantitation of porcine and bovine
DNA from gelatin mixtures and gelatin capsules is limited. For
example, Doi et al. (2009) reported an ELISA method that employed
antisera generated by immunizing rabbits and goats with bovine
gelatin. The method allowed for detection of bovine and porcine
gelatin from processed foods. Zhang et al. (2009) developed a mass
spectrometric method based on the differential sequences present
in bovine and porcine Type I collagen. Analysis of tryptic digests of
gelatin by high performance liquid chromatography/tandem mass
spectrometry (HPLC–MS/MS) allowed for differentiation of bovine
and porcine gelatin (Zhang et al., 2009). Tasara et al. (2005)
reported a qPCR-based approach for detection and quantitation of
bovine species material in pork or fish gelatin, where sensible
determination of bovine DNA at as low as 0.1% was achieved.
The objective of this study was to develop porcine- and bovine-
specific qPCR assays to determine and quantitate the species of
origin in bulk gelatin and gelatin capsules. To achieve this
objective: (i) two species-specific qPCR assays were developed
based upon repetitive elements of the porcine and bovine
genomes; (ii) species determination and quantitation were
evaluated using binary gelatin blends that contain various
amounts of porcine and bovine gelatin; and (iii) species
determination and quantitation were further evaluated in gelatin
capsules.
2. Materials and methods
2.1. Gelatin and gelatin capsules
Porcine and bovine species-specific gelatin was purchased from
Sigma–Aldrich (St. Louis, MO). Binary gelatin admixtures were
prepared by spiking gelatin of one species into gelatin of the other
species at 0%, 1%, 5%, 10%, 20% and 40% (w/w) to a total weight of
2000 mg. Porcine and bovine species-specific gelatin capsules
were purchased from Torpac (Fairfield, NJ). Binary gelatin capsule
admixtures (50%, w/w) were prepared by mixing 1000 mg of each
capsule. If necessary, gelatin capsules were minced using scissors
to obtain an accurate weight.
2.2. DNA isolation from gelatin and gelatin capsules
To isolate DNA, 2000 mg gelatin or gelatin capsules were added
to 40 mL of nuclease-free water (Ambion, Austin, TX) and
incubated at 65 8C for 30 min or until completely dissolved.
DNA was isolated from a 300 mL aliquot of the solution using
MasterPureTM DNA purification kit (EpiCentre, Madison, WI). The
manufacturer’s protocols were followed except for the addition of
glycogen (Sigma–Aldrich, St. Louis, MO), addition of carrier tRNA
(Invitrogen, Carlsbad, CA), and modification to DNA resuspension
procedures. A total of 2 mL of glycogen solution was added to
isopropanol. Glycogen was used as DNA carrier to assist DNA
precipitation. In addition, 0.5 mL of tRNA (50 ng/mL) was added to
lysis solution that contained proteinase K. The carrier tRNA was
added to prevent non-specific absorbance and loss of DNA
(Gallagher et al., 1987). Finally, the DNA pellet was resuspended
in equal volume (300 mL) of nuclease-free water rather than the
buffer and volume suggested by the manufacturer.
2.3. Porcine and bovine genomic DNA and qPCR standard curve
Porcine and bovine genomic DNA were purchased from
Zyagen (San Diego, CA). The initial concentrations for both
genomic DNA were 1 mg/mL. The qPCR standard curve was
generated from a series of 10-fold dilutions of porcine or bovine
genomic DNA in nuclease-free water ranging in concentration
from 1 to 107 pg/mL.
2.4. Primer and probe design
The primer and probe sets were designed using online software
PrimerQuestTM (http://www.idtdna.com/) (Integrated DNA Tech-
nologies, IDT, Coralville, IA). The primers used for species-specific
amplification of porcine genomic DNA were 50 -ATT TCC ATC CCA
CAG CCC-30 (forward) and 50 -AAC AGA TGC TGA CTC ACA GAC-30
(reverse). The probe used was 50 -CCC AAC CCC CAA ACT GTC TCC T-
30 . The primers used for species-specific amplification of bovine
genomic DNA were 50 -CTA AGA TCA TGG CAT CAG GTC C-30
(forward) and 50 -CCC CAA AAT AAA GTC AGC CAC-30 (reverse). The
probe used was 50 -TCC ACT GTT TCC CCA TCT ATT TGC CA-30 . Both
probes were labeled with a fluorescent reporter dye FAM at the 50
end and a double quencher dye Zen/Iowa black FQ in the middle
(Zen) and at the 30 end (Iowa black FQ). Custom synthetic
oligonucleotide primers and probes were obtained from IDT. All
primers and probes were HPLC-purified. The sizes of the expected
porcine and bovine amplicons were small (95 bp and 83 bp,
respectively), which were essential given the extent of DNA
degradation possible in highly processed products like gelatin and
gelatin capsules.
2.5. qPCR amplification and data analysis
After DNA isolation, each sample was analyzed in duplicate
wells of a 96-well optical reaction plate (Applied Biosystems,
Forster City, CA) by qPCR. On the same plate, the DNA standard
solutions for the standard curve and a no-template control were
each analyzed in 4 replicate wells. qPCR was performed with an
Applied Biosystems 7500 Real-Time PCR system (Life Technolo-
gies, Forster City, CA) using the following thermal cycling
conditions: initial heat denaturation at 50 8C for 2 min, 95 8C for
10 min, followed by 40 cycles each of 95 8C for 15 s and 60 8C for
1 min. Four microliters of genomic DNA were amplified in a total
volume of 20 mL mixture containing 2 Taqman Universal PCR
Master Mix (Life Technologies), and final concentrations of 250 nM
probe and 500 nM of each primer. Following amplification, the
data were analyzed using the 7500 Real-Time System Sequence
Detection Software version 1.3 (Applied Biosystems), which
automatically generates the standard curve and calculates the
DNA concentration. The standard curve was generated by plotting
CT (threshold cycle) against log DNA concentration (pg/mL) of the
DNA standard solutions. CT (threshold cycle) is the cycle at which
the fluorescence crosses the threshold value. Runs were deemed
acceptable if all 4 wells of no-template control had undetermined
 H. Cai et al. / Journal of Food Composition and Analysis 25 (2012) 83–87 85

CT values or CT values  38, and the standard curve had a linear Porcine genomic DNA standard curve
coefficient of determination (r2) equal to or greater than 0.99. a 40

35
3. Results and discussion

Ct value
y = -3.3987x + 37.874
30 2
R = 0.9981
3.1. Development of porcine and bovine species-specific qPCR assays 25

Many published species-specific primer and probe sets 20

amplifying housekeeping genes or rRNA sequences cannot discern 15
the animal species source of gelatin, possibly due to the extensive -0.5 0.5 1.5 2.5 3.5 4.5 5.5 6.5
degradation of these low copy number amplification templates Log DNA concentration
(Tasara et al., 2005). Therefore, in this study, species-specific qPCR
primer and probe sets were designed to amplify repetitive Bovine genomic DNA standard curve
elements in the porcine and bovine genomes. b 40
The repetitive elements were selected because (i) many
35
repetitive elements arose independently during evolution, provid-

ing unique genome tags for the species of interest; (ii) the multiple
amplification sites provided by the repetitive elements may
significantly enhance amplification sensitivity; and (iii) may assist
amplification when using degraded DNA as a template. In this
study, the bovine species-specific primer and probe set was
designed to target a Long Interspersed Repetitive DNA element
(LINE) from the bovine genome. The porcine species-specific
primer and probe set targets MPRE42, one of the Meat Animal
Research Center Porcine Repetitive Elements (Wiedmann et al.,
2006). The sequences of both repetitive elements were retrieved
from RepeatMasker server (http://www.repeatmasker.org/) and
were searched (BLAST) against the bovine and porcine genomes at
the National Center for Biotechnology Information (http://
www.ncbi.nlm.nih.gov/) to determine the relative abundance,
homology and species-specificity of the regions of interests.
Information obtained was implemented into primer and probe
design.
Since repetitive elements can be less stable and may undergo
changes in copy number during evolution (Lovatt, 2002), concerns
about DNA quantitation targeting repetitive elements arise due to
possible variations between different copies of repetitive elements
as well as variations between different animal subjects of the same
species. To minimize the impact of variations from repetitive
elements on DNA quantitation, based on the results from
bioinformatics analyses, the genomic regions chosen were (i) well
conserved between different copies of repetitive elements,
assisting PCR amplification using DNA from highly processed
sample; and (ii) had a low level of intra-species polymorphisms,
minimizing variations between porcine or bovine subjects of the
same species.
Specificity of the qPCR assay was provided by selected design of
primers and probe to DNA sequences that are unique to the target
genome but have a low level of intra-species polymorphism. For
both primer and probe sets, the target genomic regions chosen
were porcine or bovine species-specific, minimizing cross reactiv-
ity between different species; to evaluate the specificities of each
qPCR assay, porcine or bovine primer and probe set was amplified
against the genomic DNA of the other species at three different
concentrations (104, 105 and 106 pg/mL). No cross-reactivity was
observed even at the 106 pg/mL level (data not shown), indicating
high specificity of these primer and probe sets for their own species
and minimal cross-reactivity between species evaluated.
The porcine and bovine species-specific qPCR assays were also
evaluated for their sensitivity and efficiency. When serial 10-fold
dilutions of porcine and bovine genomic DNA from 1 to 107 pg/mL
were prepared, linear relationships were obtained between the CT
values and the log DNA concentrations (coefficient of determina-
tion r2 = 0.9981 for both standard curves, Fig. 1). The optimized
porcine and bovine primer and probe sets allowed detection of
porcine or bovine genomic DNA at 1 pg/mL or lower (detection
Ct value
30 y = -3.5248x + 37.473
2
R = 0.9981
25
20
15
-0.5 0.5 1.5 2.5 3.5 4.5 5.5 6.5
Log DNA concentration
Fig. 1. Porcine (a) and bovine (b) genomic DNA standard curves where CT (threshold
cycle) was plotted against Log DNA concentration (pg/mL) of DNA standard
solutions. CT represented the PCR cycle at which fluorescence reaches threshold
value.
limit not tested). The y-intercept, which corresponds to the CT
value for a single copy of the target DNA, was 37.874 and 37.482 for
porcine and bovine standard curves, respectively, indicating
superior amplification sensitivities. The slopes of the porcine
and bovine standard curves were 3.3987 and 3.5248, respec-
tively. Good amplification efficiency is typically 90–110% (Adams,
2006). Calculated from the slopes of the standard curves
(efficiency = 10[1/slope]  1) (Adams, 2006), the porcine and
bovine qPCR efficiencies were 97% and 92%, respectively.
3.2. qPCR detection and quantitation of porcine and bovine material
in gelatin
To evaluate the qPCR assays for detection and quantitation of
porcine and bovine material in bulk gelatin, binary gelatin blends
containing known amount of species-specific porcine gelatin and
bovine gelatin were prepared by blending gelatin of one species
into the other species at 0%, 1%, 5%, 10%, 20% and 40% (w/w). The
DNA isolated from the gelatin blends was quantitated by qPCR.
The qPCR assays allowed sensitive detection and accurate
quantitation of porcine and bovine material in gelatin (Tables 1 and
2). Overall, the concentration of bovine DNA in bovine gelatin was
16 times that of porcine DNA in porcine gelatin. To our surprise,
the species-specific gelatins tested were not 100% free of DNA of
the other species. While only trace amount of bovine DNA (0.01 pg/
mg, Table 1) was detected in porcine gelatin, a significant amount
of porcine DNA (17.83 pg/mg, Table 2) was determined in bovine
gelatin. To allow accurate quantitation of porcine gelatin in bovine
gelatin, porcine DNA that came from bovine gelatin was subtracted
from the total porcine DNA quantified by qPCR (Table 2). The
corrected porcine DNA values were then used for porcine gelatin
quantitation. Overall, as shown in Tables 1 and 2, the determined
gelatin percentages from gelatin blends of various concentrations
were close to their theoretical values. In addition, the qPCR assays
were able to detect a contamination level as low as 1% of the other
species in the gelatin blends.
86 H. Cai et al. / Journal of Food Composition and Analysis 25 (2012) 83–87

Table 1
Quantitation of porcine and bovine gelatin by qPCR in gelatin blends containing a known amount of bovine gelatin spiked into porcine gelatin.

Gelatin blend Bovine DNA % Bovine gelatin Porcine DNA % Porcine gelatin
determined (pg/mg)a determinedc determined (pg/mg)b determinedc
% Bovine gelatin % Porcine gelatin

0 100 0.01 0.00 83.30 100.00

1 99 13.32 0.98 84.18 99.02
5 95 66.10 4.91 80.00 95.09
10 90 133.78 9.56 79.03 90.44
20 80 266.22 19.92 66.86 80.08
40 60 546.19 40.20 50.75 59.80
a
Concentration of bovine DNA in bovine species-specific gelatin was 1333.71 pg/mg. This concentration was determined from unspiked bovine gelatin by qPCR.
b
Concentration of porcine DNA in porcine species-specific gelatin was 83.30 pg/mg. This concentration was determined from unspiked porcine gelatin by qPCR.
c
% Bovine (or porcine) gelatin determined was calculated as:

Bovine ðor porcineÞ gelatin determined ðmgÞ

% Bovine ðor porcineÞ gelatin determined ¼  100
Bovine gelatin determined ðmgÞ þ porcine gelatin determined ðmgÞ

where

Bovine DNA determined ðpg=mgÞ  total gelatin weight ð2000 mgÞ

Bovine gelatin determined ðmgÞ ¼
Concentration of bovine DNA in bovine gelatin ð1333:71 pg=mgÞ
Porcine DNA determined ðpg=mgÞ  total gelatin weight ð2000 mgÞ
Porcine gelatin determined ðmgÞ ¼ :
Concentration of porcine DNA in porcine gelatin ð83:30 pg=mgÞ

Table 2
Quantitation of porcine and bovine gelatin by qPCR in gelatin blends containing a known amount of porcine gelatin spiked into bovine gelatin.

Gelatin Blend Porcine DNA Porcine DNA from % Porcine gelatin Bovine DNA % Bovine gelatin
determined (pg/mg)a porcine gelatin (pg/mg)b determinedd determined (pg/mg)c determinedd
% Porcine gelatin % Bovine gelatin

0 100 17.83 0.00 0.00 1606.24 100.00

1 99 18.91 1.26 1.28 1557.26 98.72
5 95 23.75 6.81 6.66 1533.26 93.34
10 90 26.48 10.43 9.64 1570.14 90.36
20 80 35.72 21.46 19.50 1422.00 80.50
40 60 50.44 39.74 38.32 1027.30 61.68
a
Concentration of porcine DNA in porcine species-specific gelatin was 100.02 pg/mg. This concentration was determined from unspiked porcine gelatin by qPCR.
b
Concentration of porcine DNA in bovine species-specific gelatin was 17.83 pg/mg. This concentration was determined from unspiked bovine gelatin by qPCR.
c
Concentration of bovine DNA in bovine species-specific gelatin was 1606.25 pg/mg. This concentration was determined from unspiked bovine gelatin by qPCR.
d
% Bovine (or porcine) gelatin determined was calculated as:

Bovine ðor porcineÞ gelatin determined ðmgÞ

% Bovine ðor porcineÞ gelatin determined  100
Bovine gelatin determined ðmgÞ þ porcine gelatin determined ðmgÞ
where

Bovine DNA determined ðpg=mgÞ  total gelatin weight ð2000 mgÞ

Bovine gelatin determined ðmgÞ ¼
Concentration of bovine DNA in bovine gelatin ð1606:25 pg=mgÞ
Porcine DNA from porcine gelatin ðpg=mgÞ  total gelatin weight ð2000 mgÞ
Porcine gelatin determined ðmgÞ ¼ :
Concentration of porcine DNA in porcine gelatin ð100:02 pg=mgÞ

3.3. qPCR detection and quantitation of porcine and bovine gelatin
capsules
The qPCR assays were further evaluated for species determina-
tion and quantitation from gelatin capsules. Significantly less DNA
was detected in gelatin capsules (1.10 pg/mg of porcine DNA in
porcine gelatin capsules; 0.30 pg/mg of bovine DNA in bovine
gelatin capsules) than gelatin, possibly due to the further
degradation of DNA during gelatin capsule manufacture. As was
observed in bulk gelatin, a negligible amount of bovine DNA was
detected in porcine gelatin capsules and a small amount of porcine
DNA was detected in bovine gelatin capsules (data not shown).
This is likely due to the presence of porcine DNA in bulk bovine
gelatin, the raw material for bovine gelatin capsule manufacture.
When equal amounts of porcine gelatin capsules and bovine
gelatin capsules were mixed and analyzed by qPCR, the mixture
was determined to contain 55% of porcine gelatin capsules and
45% of bovine gelatin capsules. Therefore, the qPCR assays
permitted sensitive species determination in gelatin capsules. The
relative abundance of porcine and bovine gelatin may be estimated
as well.
4. Conclusions
Two species-specific qPCR assays were developed based upon
repetitive elements of the porcine and bovine genomes, and they
allowed sensitive detection of porcine and bovine DNA at
concentrations as low as 1 pg/mL. In addition, the lack of cross-
reactivity when the sets were used to amplify DNA from the other
species indicates high specificity of the qPCR assay for its own
species. When binary gelatin blends containing various amounts of
porcine and bovine gelatin were prepared and analyzed by the
qPCR assays, the determined ratios of porcine material to bovine
material were very close to their theoretical values, and a
contamination level of as low as 1% of the other species in the
gelatin blends could be determined. When evaluated in gelatin
capsules, although significantly less DNA was detected, determi-
nation of porcine and bovine species identities and estimation of
the relative abundance of each species was possible. The data
reported in this study are from DNA samples that were potentially
degraded during manufacturing of gelatin and gelatin capsules,
which are highly processed products. Therefore, the porcine and
bovine species-specific qPCR assays described here represent a
 H. Cai et al. / Journal of Food Composition and Analysis 25 (2012) 83–87
simple, reliable and sensitive DNA-based test for determining the
species of origin of highly processed products. They may be used as
routine molecular tools for confirmation of presence or absence of a
particular species in bulk gelatin and gelatin capsules. We expect
broad applicability of the assay with minor modifications to other
food matrices as well. However, since the extent of DNA degradation
that occurs during gelatin and gelatin capsule manufacture may vary
and the copy number of repetitive elements between different
animal subjects within the same species may vary (although the data
generated from this study suggested, with careful qPCR assay design,
there were minimal impact from amplifying repetitive elements on
DNA quantitation), the DNA quantity determined may not always
represent the amount of porcine and bovine species-specific
materials in gelatin and gelatin capsules. Thus the quantitative
data obtained would only be an approximation.
Acknowledgement
We thank Joel Galang for assistance with proofreading.
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