Food Control 96 (2019) 53–58

Contents lists available at ScienceDirect

Food Control
journal homepage: www.elsevier.com/locate/foodcont

Applicability of the poultry qPCR method to detect DNA of poultry T

processed animal protein materials
Ingrid M.J. Scholtensa,∗, Theo W. Prinsa, Rob J.C.F. Margryb, Harald Dahlmansb,
Leo W.D. van Raamsdonka
a
RIKILT Wageningen University & Research, Akkermaalsbos 2, 6708 WB, P.O. Box 230, 6700, AE, Wageningen, Netherlands
b
NutriControl, P.O. Box 107, 5460, AC, Veghel, Netherlands

A R T I C LE I N FO A B S T R A C T

Keywords: After the Bovine Spongiform Encephalitis (BSE) crisis most processed animal proteins (PAPs) were banned from
Sensitivity use in animal feed. For the foreseen reintroduction of pork PAPs in poultry feed, and poultry PAPs in pork feed
Poultry and to comply with the species-to-species ban that prohibits cannibalism, a sensitive and specific TaqMan PCR
Chicken detection method for poultry DNA has been designed and published. This poultry method is able to detect DNA
Turkey
of chicken, turkey, duck and geese in one PCR reaction. PAPs however, are a difficult and variable matrix.
Duck
Therefore, the usability of the poultry method was investigated on a range of different poultry PAPs. It was
Geese
Species-to-species ban shown that the poultry detection method is capable of detecting poultry DNA in eight out of nine different
Feed poultry PAPs mixed at a 0.1% level in chicken feed. The method can also detect at least 0.1% poultry PAPs mixed
Processed animal proteins (PAPs) in pork PAPs. These results show that the poultry method fulfils the 0.1% detection limit requirement in the EU
Transmissible spongiform encephalopathy legislation.
(TSE)
Bovine spongiform encephalopathy (BSE)

1. Introduction to a prohibition of all animal material in ruminant feed in 2006

After the outbreak of the Bovine Spongiform Encephalopathy (BSE)
crisis, with its highest occurrence in the European Union in 1992, vir-
tually all processed animal proteins (PAPs) were banned from animal
feed in 2001 (European Commission, 2001). The most likely cause of
the outbreak was the feeding of bovine proteins to cattle (Prince et al.,
2003). After installing severe measures in the areas of monitoring the
presence of BSE in ruminants at slaughter, of separate processing of risk
materials and of the mentioned restrictions in feeding, the risk of BSE is
now considered to be very low. In this framework exemptions and re-
laxations to the feed bans in EU legislation have been considered and
agreed in some cases. In 2013 pig and poultry PAPs were allowed in fish
feed (European Commission, 2013b). The use of poultry PAP and pork
PAP in feed for non-ruminant terrestrial animals was considered, but is
still under discussion. Both the desire for relaxation and the require-
ment of specific detection methods are parts of this discussion.
It is necessary to emphasize the presence of several types of bans for
PAPs in animal feed. The major ban is targeting on feed and food safety.
The ban on the use of mammalian PAPs in ruminant feed was amended
(European Commission, 2006). In the view of a lack of species specific
monitoring methods, the extended feed ban, prohibiting most applica-
tions of animal by-products, was installed in 2001 with the intention to
finally lift this extended feed ban in the future, see Regulation (EC)
999/2001 article 7 (3) and (4) (European Commission, 2001). In order
to facilitate this temporary nature, the species-to-species ban was in-
stalled in 2002 (European Commission, 2002), Regulation (EC) 1774/
2002, replaced by Regulation (EC) 1069/2009 article 11 (European
Commission, 2009). This ban prevents that a species will be fed with
material of the same species, so pigs are not allowed to eat pork and
poultry is not allowed to eat poultry. Also a series of additional lim-
itations and relaxations are installed, e.g. for fish, for blood and blood
products, gelatine, hides and skins, etc. This quite specific though still
concise overview is presented for the following reason. The intention of
the ruminant feed ban and, partly, the extended feed ban is to ensure
feed and food safety. In contrast, the species-to-species ban is installed
for reasons of ethics and authenticity.
In order to enforce the absence of pork material in pig feed, and of
poultry material in poultry feed, sensitive and specific detection

∗
Corresponding author.
E-mail addresses: ingrid.scholtens@wur.nl (I.M.J. Scholtens), theo.prins@wur.nl (T.W. Prins), rob.margry@gmail.com (R.J.C.F. Margry),
harald.dahlmans@nutricontrol.nl (H. Dahlmans), leo.vanraamsdonk@wur.nl (L.W.D. van Raamsdonk).

https://doi.org/10.1016/j.foodcont.2018.08.035
Received 14 May 2018; Received in revised form 28 August 2018; Accepted 29 August 2018
Available online 01 September 2018
0956-7135/ © 2018 The Author(s). Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license
(http://creativecommons.org/licenses/BY-NC-ND/4.0/).
I.M.J. Scholtens et al. Food Control 96 (2019) 53–58

methods for pork material and for poultry material are needed.
Following the basic choice to apply DNA based methods for the iden-
tification of animal proteins in feed (O. Fumière, Dubois, Baeten, von
Holst, & Berben, 2006), a PCR method has been developed for pigs (O.
Fumière, Marien, A., Maljean, O., Berben, G., 2016) and a specific
method to detect the four poultry species chicken, turkey, ducks and
geese in one PCR reaction was published (Scholtens, Prins, & van
Raamsdonk, 2017). This species range was the closest match achievable
for monitoring the complicated definition of “poultry” in order to meet
the legal requirement for specificity (for further discussion see L.W.D.
van Raamsdonk et al. (2018). In general, as a relic from the past
(European Commission, 1998) the technical limit of 0.1% PAPs in feed
and in feed materials generally applies to all detection methods
(European Commission, 2010). The PCR ruminant method is designed
according to the ALARA principles, an acronym for ‘As Low As Rea-
sonably Achievable’ used in safety critical applications, as monitoring
method for a ban targeting feed and food safety issues. In this paper,
however, the sensitivity of the designed poultry method will be ad-
justed close to the technical limit of 0.1%, which is sufficient for
monitoring authenticity and cannibalism.
In order to demonstrate the applicability of the poultry method
targeting four different species (-groups) to support the two legal re-
quirements, specificity and sensitivity are being extensively evaluated
and presented independently (Scholtens et al., 2017 and current paper,
respectively).
The quality of the DNA that can be extracted from different PAPs is
known to be variable, due to severe rendering methods (Regulation
(EC) 142/2011 Annex IV) (European Commission, 2011), which influ-
ences the sensitivity of detection methods. The method sensitivity in
real-life samples is not necessarily the same as the sensitivity in DNA
extracted from model matrices used to evaluate the specificity of a
method (Zagon, Uzuner, Lampen, & Braeuning, 2017). Therefore, the
sensitivity of the poultry method has been investigated in several dif-
ferent commercial poultry PAP materials and poultry PAPs mixed in
feed or in pork PAPs. The influence of different mastermixes on the
sensitivity as part of robustness testing was investigated as well.
2. Materials and methods
2.1. PAPs and feed materials for sensitivity testing
PAPs and feed materials were provided by NutriControl (Veghel, the
Netherlands). One duck PAP, three chicken PAPs, four poultry PAPs and
one hydrolysed protein material were tested (Table 1). These PAP
materials were all different in composition (e.g. species, ratios of meat,
organ tissues and carcass). The codes between brackets refer to the
origin of the material. CCL13, CCL25 and sterilizer NC were made
Table 1
PAPs and hydrolysed protein materials.
Name or indication Background Declared composition
Duck meal (ISI) commercial Duck MBM
Chicken soft 133 °C, pre- experimental Chicken MM
cooked (CCL13)
Chicken bones 133 °C, pre- experimental Chicken MBM
cooked (CCL25)
Pure chicken, 128-133 °C, experimental Chicken MBM
(sterilizer NC)
Poultry meal (DL) commercial Chicken/turkey MBM
Poultry meal (BG) commercial Chicken/turkey MBM
Poultry meat meal (ER25) commercial Chicken/turkey MM
Poultry meal standard commercial Chicken/turkey MBM
(ER30)
Poultry hydrolysed protein commercial Chicken/turkey hydrolysed
(ER32) proteins (no feather meal)
MBM: meat and bone meal; MM: meat meal.
under controlled circumstances as part of the EU FP6 SAFEED-PAP
project (https://cordis.europa.eu/project/rcn/84692_en.html). For
these samples the rendering temperatures were known and therefore
they are added to the sample names.
The remainder of the samples were obtained from different ren-
dering factories and exact details were not known.
For sensitivity testing, PAPs or hydrolysed protein material were
diluted in chicken feed (tested negative for poultry DNA). Three pork
blood meals (A, B and C) and one pork greaves meal were tested for
absence of poultry material to be used as matrix. The chicken feed
samples spiked with the eight poultry PAP materials and one hydro-
lysed protein as well as the poultry PAP spikes in pork blood meal A
were prepared according to Table 2.
Poultry material of samples 1-6A, 9A, 11A and 13A (Table 1) were
used to make spiked samples of 1% w/w in a chicken feed (samples 1-
6B, 9B, 11B and 13B; Table 2). A part of this material was used to dilute
1:9 in feed material for producing a set of spiked samples at 0.1% w/w
(samples 1-6C, 9C, 11C and 13C). Subsequently, a part of this 0.1%
material was diluted 1:1 with chicken feed to obtain a set of spiked
samples at 0.05% w/w (samples 1-6D, 9D, 11D and 13D). Commercial
material of duck (1A) and of chicken/turkey (5A) was used to produce
mixtures of these two materials in chicken feed at 0.1% and 0.05%
(samples 14C and 14D,; Table 2). Material of meat meal (9A) and of
hydrolysed protein (13A) was used for spiking in a pork blood meal at
2% and 0.1% w/w (samples 9E, 13E and 9F, 13F respectively; Table 2).
The level of 2% was based on an EFSA indication (EFSA Panel on
Biological Hazards (BIOHAZ), 2011).
To ascertain the correctness of the samples, the mixtures were
carefully mixed with a spatula in between the dilution steps. Also, five
batches per sample per dilution were used for DNA isolation to study
the homogeneity of these samples.
2.2. DNA extraction
The poultry method was developed and specificity was tested
(Scholtens et al., 2017) using DNA extracted with a CTAB DNA ex-
traction protocol derived from Murray and Thompson (1980). To link
up with the mandatory ruminant DNA extraction method (EURL-AP,
2014) in this study the Promega Wizard Kit for Food was used for DNA
extraction according to the protocol as described by the EURL-AP
(EURL-AP, 2014) The DNA was dissolved in 300 μl water and 5 μl was
subsequently used in the PCR reaction in two different dilutions (un-
diluted (1x) and diluted in water (10x).
2.3. Primers and probes, mastermix and PCR program
Primers and probes, mastermix, PCR machine, software and PCR
program were as described before (Scholtens et al., 2017). Although an
arbitrary cut-off of 36 was taken, the total number of cycles was kept at
45. In addition to the poultry method also the two parts of this method,
chicken/turkey and duck/geese method were tested separately. This
Code was accomplished by using either the turkey/chicken primers (100 mM
each) and probes (50 mM each) or the duck/geese primers and probe in
1A
the same concentrations. Water was used to correct for the final volume
2A
of 25 μl. Diagenode mastermix DMMM2xA300 (GMO-MM2x-A300) was
3A used. Eurogentec qPCR mastermix Plus (RT-QP2X-03-075+), Diag-
enode DMMLD2D600 (GMO-UN-A600), Qiagen QuantiTect multiplex
4A PCR mastermix (1036670) and Applied Biosystems TaqMan Multiplex
5A
mastermix (4461881) were also tested for robustness testing.
6A
9A 3. Results and discussion
11A
3.1. Characterisation of poultry PAP materials
13A
Eight different poultry PAP materials and one poultry hydrolysed
protein material were tested with the poultry method. These materials

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Table 2
Preparation of the chicken feed and pork blood materials spiked with poultry PAP or poultry hydrolysed protein (HP).
Matrix Spike Result Code

49.50 g feed 0.50 g 100% poultry PAP/HP 1% poultry PAP/HP in feed 1-6B, 9B, 11B, 13B
9.00 g feed 1.00 g 1% poultry PAP/HP in feed 0.1% poultry PAP/HP in feed 1-6C, 9C, 11C, 13C
2.00 g feed 2.00 g 0.1% poultry PAP/HP in feed 0.05% poultry PAP/HP in feed 1-6D, 9D, 11D, 13D
8.00 g feed 1.00 g 1% PAP 1A in feed +1.00 g 1% PAP 5A in feed 0.1% duck +0.1% chicken in feed 14C
5.00 g feed 5.00 g 0.1% duck +0.1% chicken in feed 0.05% duck +0.05% chicken in feed 14D
49.50 g pork blood meal 1.00 g 100% poultry PAP 2% poultry PAP in pork blood meal 9E, 13E
9.50 g pork blood meal 0.500 g of 2% poultry PAP in pork blood meal 0.1% poultry PAP in pork blood meal 9F, 13F

Feed is a chicken feed (laying hen feed) in all cases.

consisted of one duck meal, three chicken meals, four commercial
poultry meals and one commercial poultry hydrolysed protein (see also
Materials and Methods section). In order to understand and properly
evaluate the sensitivity of the poultry method, the poultry species
present in the PAP materials were characterized using the chicken/
turkey and duck/geese methods separately. It was first investigated
whether the separate chicken/turkey and duck/geese methods could be
applied for this. The specificity of the two separate methods towards the
targeted poultry species and the non-target species ostrich (Struthio
camelus) was verified using good quality DNA extracted from meat
(50 ng per reaction). Other non-targets were not included because it
was already demonstrated that the poultry method does not detect
other species, and therefore neither the duck/geese nor the chicken/
turkey parts are expected to give any signal (Scholtens et al., 2017).
DNA extracted from meat material was chosen for the specificity testing
because the quality is much better than DNA isolated from PAPs. A-
specific signals will thus be more visible in DNA extracted from meat
than extracted from PAPs.
The results are shown in Table 3. The results and Cq values of the
poultry method were similar to those obtained by Scholtens et al.
(2017). The poultry method detects chicken, turkey, duck and geese
and gives only late signals for five other Galliformes species that were
not included in the design of the primers and probes (also the sequences
were not actively excluded). Also late signals are seen with the ostrich
meat DNA. The ostrich sequence was excluded as a target when de-
signing the poultry method.
The duck/geese method did not detect chicken or turkey. The other
Galliformes species gave only late signals similar to the Cq values of the
poultry method. This means that the duck/geese method is specific for
ducks and geese species and it can be used to detect duck/geese in the
100% poultry PAP materials.
The chicken/turkey method gave slight signals with some duck
species, e.g. Mallard duck (Anas platyrhynchos) and Northern pintail (A.
acuta) at Cq values around 29-30. Although it cannot be excluded that
the chicken/turkey method detects late signals originating from duck
species, the DNA applied here is isolated from pure meat. Autoclaved
(rendered) non-target species are not likely to be detected for the lower
quality of the DNA, in a presence of an adulteration level of less than
100% in feed.
Nine 100% poultry PAP materials (Table 1) were tested with the
poultry method and with the separate chicken/turkey and duck/geese
methods. The results are shown in Table 3. The poultry method is able
to detect poultry DNA in all 100% poultry PAP materials investigated
here. It can be seen that there is a large difference in Cq values ranging
from around 22 to 32. This is probably due to different rendering
conditions and also to differences in composition of the poultry protein
materials (e.g. different ratios of meat, organ tissues and carcass). In
some samples inhibition is observed with the undiluted DNA, since in
the 10x diluted DNA the Cq value is earlier (e.g. sample 2A).
The late signals (1x 36.6, 10x 37.06) obtained with the chicken/
turkey method in the commercial duck meal sample 1A can either be
explained by a non-specific reaction, or by the presence of low amounts
of either chicken or turkey. This was investigated further by applying
also the chicken specific method as described by Pegels et al. (2012).
Here, the chicken signal in the commercial duck meal sample 1A could
not be confirmed. In all other samples, chicken was confirmed. The
conclusion is that the duck meal most likely does not contain chicken/
turkey DNA.
The duck/geese method was found positive in the duck meal and
also in three poultry meals and the poultry hydrolysed protein. In the
four poultry samples also the chicken/turkey method was positive in-
dicating that these four PAPs are a mixture of chicken/turkey and duck/

Table 3
Specificity testing of the poultry method, chicken/turkey method and duck/geese method on DNA isolated from raw meat material to verify if the chicken/turkey
method and the duck/geese method can be used to distinguish between chicken/turkey and duck/geese DNA in 100% poultry PAP materials (see Table 4).
Poultry Method Chicken/Turkey method Duck/Geese method

Order Species (Cq) Conclusion (Cq) Conclusion (Cq) Conclusion

Galliformes Chicken (Gallus gallus) 18.77 Detected 18.67 Detected N/A Not detected
Turkey (Meleagris gallopavo) 16.54 16.39 N/A
Anseriformes Muscovy duck (Cairina moschata) 16.18 Detected 35.27 Late or no signals 16.14 Detected
Mallard duck (Anas platyrhynchos) 15.39 29.66 15.43
Northern pintail (Anas acuta) 15.14 29.27 15.03
Gadwall (Anas strepera) 15.07 38.52 15.01
White-fronted goose (Anser albifrons) 14.71 N/A 14.55
Greylag goose (Anser anser) 14.93 N/A 14.58
Galliformes (sequences excluded in design) Common pheasant (Phasianus colchicus) 29.40 Late signals N/A Late or no signals 29.42 Late signals
Helmeted guineafowl (Numida meleagris) 27.30 38.39 27.22
Japanese quail (Coturnix japonica) 37.02 N/A 38.59
Chukar partridge (Alectoris chukar) 30.80 35.40 30.78
Red-legged partridge (Alectoris rufa) 30.51 N/A 30.49
Struthionidae (sequence excluded in design, non-target) Ostrich (Struthio camelus) 34.58 Late signal N/A No signal 34.75 Late signal
PCR control water N/A N/A N/A

Cq values are averages of two PCR reactions with the same DNA; N/A: no signal or signal > 40.

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I.M.J. Scholtens et al. Food Control 96 (2019) 53–58

Table 5
0.1% poultry PAP and poultry hydrolysed protein in chicken feed, mean Cq values in undiluted (1x) and 10x diluted DNA extracts with the poultry method, chicken/
turkey method and duck/geese method. More detailed results can be seen in Supplemental Material 2A.
Sample name No. Poultry method Chicken/Turkey method Duck/Geese method

1x (Cq) 10x (Cq) Sample result 1x (Cq) 10x (Cq) Sample result 1x (Cq) 10x (Cq) Sample result

Duck meal (ISI) 1C 33.31 36.80 D N/A N/A ND 33.36 36.83 D

Chicken soft 133 °C, pre-cooked (CCL 13) 2C 34.42 37.72 D 33.44 36.34 D N/A N/A ND
Chicken bones 133 °C, pre-cooked (CCL 25) 3C 33.18 36.12 D 31.91 35.03 D N/A N/A ND
Pure chicken, 128-133 °C, (sterilizer NC) 4C 34.65 38.98 D 34.65 ND D N/A N/A ND
Poultry meal (DL) 5C 33.36 36.20 D 32.30 35.44 D N/A N/A ND
Poultry meal (BG) 6C 38.75 N/A ND N/A N/A ND N/A N/A ND
Poultry meat meal (ER25) 9C 33.48 36.44 D 33.31 36.35 D N/A N/A ND
Poultry meal standard (ER30) 11C 32.74 35.50 D 32.48 35.12 D 36.47 N/A ND
Poultry hydrolysed protein (ER32) 13C 30.39 33.07 D 30.36 32.84 D 33.12 37.03 D
0.1% duck meal (ISI) (1A) + 0.1% poultry meal (DL) (5A) 14C 31.95 35.39 D 34.09 36.48 D 32.18 35.70 D
0.05% duck meal (ISI) (1A) + 0.05% poultry meal (DL) (5A) 15C 28.62 31.20 D 29.03 31.18 D 30.11 33.58 D

Values are averages of 5 PCR reactions; N/A: no signal or Cq > 40; D: detected, Cq ≤ 36; sample result ND: not detected cut off Cq > 36.

geese material. This is not in disagreement with the label ‘poultry’. The
signals with the duck/geese method in the samples 1A, 9A, 11A and
13A were confirmed by the duck method of Pegels et al. (2012) (Sup-
plemental Material 1). As positive control, only the Mallard duck (A.
platyrhynchos) appeared to be positive with the duck method of Pegels
et al. while the Muscovy duck (C. moschata) gave very late signals with
the duck method of Pegels et al. (Supplemental Material 1). The ap-
plication of the geese method of Pegels et al. resulted in exclusively late
signals.
3.2. Sensitivity in mixtures of poultry PAPs and chicken feed
According to the EU legislation the sensitivity of a poultry detection
method needs to be at least 0.1% of poultry PAPs in feed material
(European Commission, 2013a). Therefore, the detection of eight dif-
ferent poultry PAPs and one poultry hydrolysed protein spiked at 0.1%
and 0.05% in chicken feed (Table 2) was investigated. Also, to in-
vestigate detection of two different species, one sample with 0.1% duck
meal and 0.1% poultry meal and another sample wit 0.05% duck meal
and 0.05% poultry meal were tested. A summary of the results can be
seen in Tables 5 and 6. More detailed data including individual Cq
values per repetition and standard deviations are given in Supplemental
Material 2A and 2B.
Table 5 shows that the 0.1% poultry PAP and 0.1% poultry hydro-
lysed protein diluted in chicken feed is detected in eight of the nine feed
materials. Only sample 6C, poultry meal (BG), did not test positive. A
reason that this sample tested negative could be that the material has
been rendered more severely. It was already seen in Table 4 that 100%
of this material gave late Cq values compared to the other PAPs tested,
also in 10x diluted DNA. The separate chicken/turkey and duck/geese
methods gave Cq values comparable to the poultry method. From the
results on the 100% PAP materials as presented in Table 4 it was al-
ready known that five of the 100% poultry PAP materials contain duck
and/or geese material: 1A, 5A, 9A, 11A, 13A. This was confirmed for
the 0.1% mixtures of sample 1C, 11C and 13C, 14C (1A+5A: each at
0.1%) and 14D (1A+5A: each at 0.05%, together at 0.1%) (Table 5).
In Table 6 poultry PAP percentages below the technical limit, i.e. of
0.05% mixed in chicken feed were tested. At the 0.05% level poultry
DNA was detected in six of the nine materials tested instead of eight of
the nine materials at the 0.1% level (Table 4). The chicken/turkey
method was a little bit more sensitive than the poultry method as it
detected one sample more (2D). At the 0.05% level duck/geese DNA
was only detected in samples 1D and 13D at late Cq values. This is
sufficient with respect to legislation.
3.3. Sensitivity of poultry PAPs mixed with pork PAPs
The sensitivity of the poultry method was also investigated for
poultry PAPs mixed in three different pork PAPs. At first it was in-
vestigated if the pork PAPs did not contain any poultry material. The
results of the poultry PCR on four pure pork PAP materials are shown in
Table 7. It is demonstrated that pork greaves meal contains some
poultry PAP. Also pork blood meal C contains a low amount of poultry
material. Only pork blood meal A and B were found to be negative in
the poultry PCR. Pork blood meal A was used as matrix to test adul-
terations with poultry materials. From Table 7 it can be concluded that
adulteration at levels of 2% and 0.1% poultry PAP can be detected in
pork blood meal. Detailed data can be found in Supplemental Material
3.

Table 6
0.05% PAP and poultry hydrolysed protein in chicken feed, mean Cq values in undiluted (1x) and 10x diluted DNA extracts with the poultry method, chicken/turkey
method and duck/geese method. More detailed results can be seen in Supplemental Material 2B.
Sample name Nr Poultry method Chicken/Turkey method Duck/Geese method

1x (Cq) 10x (Cq) Sample result 1x (Cq) 10x (Cq) Sample result 1x (Cq) 10x (Cq) Sample result

Duck meal (ISI) 1D 34.53 37.23 D N/A N/A ND 33.74 37.75 D

Chicken soft 133 °C, pre-cooked (CCL 13) 2D 38.35 N/A ND 36.61 38.25 ND N/A N/A ND
Chicken bones 133 °C, pre-cooked (CCL 25) 3D 35.30 37.57 D 33.48 36.46 D N/A N/A ND
Pure chicken, 128-133 °C, (sterilizer NC) 4D N/A N/A ND 37.76 N/A ND N/A N/A ND
Poultry meal (DL) 5D 35.52 38.98 D 33.99 37.19 D N/A N/A ND
Poultry meal (BG) 6D N/A N/A ND 38.82 39.07 ND N/A N/A ND
Poultry meat meal (ER25) 9D 35.24 37.57 D 34.03 36.67 D N/A N/A ND
Poultry meal standard (ER30) 11D 35.04 37.61 D 34.02 36.11 D N/A N/A ND
Poultry hydrolysed protein (ER32) 13D 33.12 35.60 D 32.10 34.57 D 35.08 N/A D

Values are averages of 5 PCR reactions; N/A: no signal or Cq > 40; D: detected, Cq ≤ 36; ND: not detected Cq > 36; ND: not detected sample results that are
different from those in Table 5.

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Table 4
Characterisation of the 100% poultry PAP materials and one poultry hydrolysed protein tested with the poultry method, chicken/turkey method and duck/geese
method.
Sample name No. Poultry method Chicken/Turkey method Duck/Geese method

1x (Cq) 10x (Cq) Result 1x (Cq) 10x (Cq) Result 1x (Cq) 10x (Cq) Result

Duck meal (ISI) 1A 19.75 23.14 D 36.60 37.06 ND 18.93 22.24 D

Chicken soft 133 °C, pre-cooked (CCL 13) 2A 30.40 24.23 D 35.18 24.59 D N/A N/A ND
Chicken bones 133 °C, pre-cooked (CCL 25) 3A 22.84 25.59 D 22.86 25.53 D N/A N/A ND
Pure chicken, 128-133 °C, (sterilizer NC) 4A 25.71 28.78 D 25.70 28.70 D N/A N/A ND
Poultry meal (DL) 5A 32.73 23.01 D 35.44 22.79 D 33.96 32.60 D
Poultry meal (BG) 6A 30.49 33.03 D 30.41 28.82 D N/A N/A ND
Poultry meat meal (ER25) 9A 22.53 25.15 D 22.50 32.94 D 31.00 34.22 D
Poultry meal standard (ER30) 11A 21.63 23.59 D 21.60 24.98 D 24.68 28.06 D
Poultry hydrolysed protein (ER32) 13A 22.69 20.47 D 26.91 20.40 D 22.27 23.57 D

Cq values are averages of two PCR reactions on undiluted (1x) and 10x diluted DNA. N/A: no signal or Cq > 40; D: detected, Cq ≤ 36; ND: not detected.

Table 7
PCR results of the poultry method on four pork PAPs and on mixtures of 2% and
0.1% poultry PAP in pork blood meal.
Sample No. 1x (Cq) 10x (Cq) Sample result

Pork blood meal A N/A N/A ND

Pork blood meal B N/A N/A ND
Pork blood meal C 35.05 39.21 D
Pork greaves meal 29.34 31.36 D
2% Poultry meat meal (ER25)* 9E 27.69 30.32 D
0.1% Poultry meat meal (ER25)* 9F 30.38 33.71 D
2% Poultry hydrolysed protein 13E 24.18 26.99 D
(ER32)*
0.1% Poultry hydrolysed protein 13F 31.62 35.11 D
(ER32)*

Values are averages of two PCR reactions (matrices) or five PCR reactions
(spiked material); N/A no signal or Cq > 40; D: detected, Cq ≤ 36; ND: not
detected; * in Pork blood meal A.

3.4. Different mastermixes

Since it is the intention to transfer this method to a range of other
laboratories, it is important to establish the performance of the method
with other brands of mastermixes than applied in the previous experi-
ments. Four extra mastermixes were tested with the same primer and
probe concentrations and PCR programs as were used when applying
the Diagenode DMMM2xA300 mastermix. The results are shown in
Table 8 and Fig. 1. In Table 8, Cq data of all samples are shown. It can
be seen that the Eurogentec and Diagenode DMMLD2D600 mastermix
do not work satisfactory with some samples (2 and 5) for the absence of
Fig. 1. Comparison of the performance of the poultry method on 100% poultry
PAP materials using five different mastermixes.
signals, and therefore these two samples are omitted from Fig. 1 where
average Cq values are shown to visualize the differences in perfor-
mance. Qiagen QuantiTect Multiplex PCR and Diagenode
DMMM2xA300 are performing in a nearly identical way. The average
Cq values for Applied Biosystems TaqMan Multiplex are later than those

Table 8
PCR results with different brands of mastermix.
Sample name Sample no. Diagenode Eurogentec qPCR Qiagen QuantiTect Applied Biosystems Diagenode
DMMM2xA300 mastermix Plus Multiplex PCR TaqMan Multiplex DMMLD2D600

1x (Cq) 10x (Cq) 1x (Cq) 10x (Cq) 1x (Cq) 10x (Cq) 1x (Cq) 10x (Cq) 1x (Cq) 10x (Cq)

duck meal ISI 1A 19.75 23.14 23.60 25.65 19.93 25.20 20.48 29.72 23.42 25.00
chicken soft 133 °C, pre- 2A 30.40 24.23 N/A 30.36 24.86 23.41 24.98 26.65 N/A 30.82
cooked (CCL 13)
chicken bones 133 °C, pre- 3A 22.84 25.59 31.39 31.15 23.36 25.57 26.30 30.61 32.42 29.47
cooked (CCL 25)
pure chicken, 128-133 °C, 4A 25.71 28.78 34.09 34.43 26.31 29.02 30.71 34.71 30.76 31.68
(sterilizer NC)
poultry meal (DL) 5A 32.73 23.01 N/A 30.19 25.14 22.59 24.00 25.73 N/A N/A
poultry meal (BG) 6A 30.49 33.03 40.58 39.84 30.81 33.21 37.22 40.36 39.31 37.83
Poultry meat meal (ER25) 9A 22.53 25.15 30.04 30.78 22.74 23.11 25.40 23.83 29.23 28.60
Poultry meal standard 11A 21.63 23.59 28.12 28.30 21.38 24.16 22.86 28.30 31.62 26.93
(ER30)
Poultry hydrolysed protein 13A 22.69 20.47 28.79 24.85 20.04 20.50 18.81 22.45 33.91 25.25
(ER32)

Values are averages of two PCR reactions; N/A no signal.

57
I.M.J. Scholtens et al.
of the Qiagen QuantiTect Multiplex PCR and Diagenode
DMMM2xA300, but in Table 8 it can be seen that for one samples the
Cq values are earlier (sample 13).
4. Conclusions
The applicability of a previously published detection method for
poultry DNA has been investigated in feed and real-life pork PAP ma-
terials. The detection limit of the poultry method was shown to be at
least 0.1% in a background of chicken feed and in 100% poultry or pork
PAPs. This means that the method fulfils the legal requirement of a
0.1% limit of detection. The poultry method performs well with
Diagenode DMMM2xA300 and Qiagen QuantiTect Multiplex PCR
mastermix. Applied Biosystems TaqMan Multiplex mastermix can also
be used, although most Cq values are later.
The commercial poultry PAP materials and one hydrolysed protein
investigated in this study did not always consist of pure chicken/turkey
material. Four of the nine investigated poultry PAPs appeared to con-
tain a mixture of chicken/turkey DNA as well as duck/geese DNA. It
also proved to be hard to obtain pure pork PAP materials. Two out of
three pork PAP materials tested positive with the poultry method and
are therefore suspected to contain poultry material.
An arbitrary cut-off at Cq 36 for the poultry method was used in this
study. This Cq value is dependent on the baseline setting of the PCR
machine, mastermix, brand of PCR machine and other factors. As a
consequence, the transferability to other laboratories needs to be tested.
Universal material for calibration is needed to use the poultry method
in a harmonised way in other laboratories.
The current study demonstrates an optimal sensitivity for the de-
tection method for poultry DNA in poultry feeds and pork PAPs in the
framework of the EU species-to-species ban. The complementary ele-
ments of this method, detection of chicken/turkey and of duck/geese,
show a sufficient applicability as well.
Funding
This research was funded by the Topsector Agri & Food, Privaat
Publieke Samenwerking ‘Veilige valorisatie van slachtbijproducten’ (in
English: Private Public Collaboration ‘Safe utilization of slaughter by-
products’).
Acknowledgements
The authors like to thank Monique Bremer for very useful discus-
sions during the research presented in this article.
Appendix A. Supplementary data
Supplementary data related to this article can be found at https://
doi.org/10.1016/j.foodcont.2018.08.035.
58
Food Control 96 (2019) 53–58
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