Development and validation of a fallow deer (Dama dama)-specific TaqMan real-time PCR assay for the detection of food adulteration

Accepted Manuscript Development and validation of a fallow deer (Dama dama)-specific TaqMan real-time PCR assay for the detection of food adulteration Maria Kaltenbrunner, Rupert Hochegger, Margit Cichna-Markl PII: DOI: Reference:

S0308-8146(17)31564-9 http://dx.doi.org/10.1016/j.foodchem.2017.09.087 FOCH 21756

To appear in:

Food Chemistry

Received Date: Revised Date: Accepted Date:

25 February 2017 25 July 2017 17 September 2017

Please cite this article as: Kaltenbrunner, M., Hochegger, R., Cichna-Markl, M., Development and validation of a fallow deer (Dama dama)-specific TaqMan real-time PCR assay for the detection of food adulteration, Food Chemistry (2017), doi: http://dx.doi.org/10.1016/j.foodchem.2017.09.087

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Development and validation of a fallow deer (Dama dama)-specific TaqMan real-time PCR assay for the detection of food adulteration

Maria Kaltenbrunnera,b, Rupert Hocheggera and Margit Cichna-Marklb,*

a

Austrian Agency for Health and Food Safety, Institute for Food Safety, Department

of Molecular Biology and Microbiology, Spargelfeldstraße 191, 1220 Vienna, Austria b

Department of Analytical Chemistry, University of Vienna, Währinger Straße 38,

1090 Vienna, Austria

*

Corresponding author

Tel.: +43-1-4277-52374 Fax: +43-1-4277-9523 E-mail: [email protected]

The co-authors e-mail addresses: Maria Kaltenbrunner: [email protected] Rupert Hochegger: [email protected]

1

Abstract The aim of the present study was to develop a real-time PCR assay for the identification and quantification of fallow deer (Dama dama) in food to detect food adulteration. Despite high sequence homology among different deer species, a fallow deer-specific primer/probe system targeting a fragment of the nuclear MC1-R gene was designed. This primer/probe system did not amplify DNA from 19 other animals and 50 edible plant species. Moderate cross-reactivity was observed for sika deer, red deer, roe deer, reindeer and wild boar. The LOD and LOQ of the real-time PCR assay were 0.1% and 0.4%, respectively. To validate the assay, DNA mixtures, meat extract mixtures, meat mixtures and model game sausages were analyzed. Satisfactory quantitative results were obtained when the calibration mixture was similar to the analyzed sample in both the composition and concentration of the animal species of interest.

Keywords Fallow deer, Dama dama, real-time PCR, game meat, food adulteration, quantification

2

1

Introduction

Some consumers favor game meat over meat from domesticated animals because of its characteristic flavor and tenderness (Hoffman & Wiklund, 2006). Health-conscious individuals might also choose game meat because of its higher content of omega-3 fatty acids compared with, for example, beef (Hoffman & Wiklund, 2006; Poławska, Cooper, Jóźwik, & Pomianowski, 2013). Game meat is popular because it does not contain residues of antibiotics or growth hormones (Fajardo, González, López-Calleja, Martín, Hernández, García, et al., 2006; Hoffman & Wiklund, 2006). Red deer (Cervus elaphus) and roe deer (Capreolus capreolus) are the most frequently consumed deer species in Europe. In addition, meat from fallow deer (Dama dama) and sika deer (Cervus nippon) is commercially available (Fajardo, et al., 2006; Hoffman & Wiklund, 2006; Obidziński, Kiełtyk, Borkowski, Bolibok, & Remuszko, 2013). To meet the worldwide increasing demand for game meat, in addition to wild animals living in the forest, deer herds are frequently raised in deer farms (Hoffman & Cawthorn, 2013; Hoffman & Wiklund, 2006). Since game meat is expensive, it is prone to substitutions with cheaper meat from domesticated animals, such as pigs (Fajardo, González Isabel, Rojas, García, & Martín, 2010). According to the Codex Alimentarius Austriacus, 38% (w/w) of the meat in “game” sausage must originate from game species (Codex Alimentarius Austriacus, 2005). If food producers or restaurant owners specify the deer species, e.g., if the product is declared as “fallow deer goulash”, then the meat from fallow deer must not be substituted with meat from other deer species, e.g., meat from red deer.

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Various methods have previously been developed to detect deer meat adulteration in food, e.g., a PCR-RFLP (restriction-fragment length polymorphism) method (Fajardo, et al., 2006), an end-point PCR assay (Fajardo, González, LópezCalleja, Martín, Rojas, Hernández, et al., 2007) and a real-time PCR assay (Fajardo, González, Martín, Rojas, Hernández, García, et al., 2008). These assays target mitochondrial DNA. Since the copy number of mitochondrial DNA varies between different species and even between different tissue types of the same species, methods targeting mitochondrial DNA are not suitable for quantification (Ballin, Vogensen, & Karlsson, 2009). To accurately determine the content of deer meat in food, we developed realtime PCR assays targeting single copy genes. Previously, we have presented assays for the quantification of roe deer (Druml, Mayer, Cichna-Markl, & Hochegger, 2015) and the sum of fallow deer, red deer and sika deer (Druml, Grandits, Mayer, Hochegger, & Cichna-Markl, 2015). Since the latter method does not allow distinction between red deer, fallow deer and sika deer, it is not applicable for the verification of the declaration of a certain deer species. In the present study, we developed a real-time PCR assay for the specific detection and quantification of fallow deer in food. Despite the limited number of gene sequences for fallow deer in the National Center for Biotechnology Information (NCBI) database and the high sequence homology between different deer species, we successfully designed a fallow deer-specific primer/probe system and developed a real-time PCR assay for the quantification of the fallow deer content in food.

2

Materials and methods

4

2.1

Chemicals and food samples Ethylenediaminetetraacetic acid (EDTA), tris(hydroxymethyl)aminomethane

(Tris), sodium chloride (NaCl), hydrochloric acid (HCl), isoamyl alcohol, proteinase K (600 mAnsonU/mL) and 2-propanol were purchased from Merck (Darmstadt, Germany). Hexadecyltrimethylammonium bromide (CTAB) was obtained from Sigma Aldrich (St. Louis, Missouri, USA), chloroform was obtained from Ampresco (Solon, Ohio, USA) and ethanol was obtained from VWR Chemicals (Leuven, Belgium). RNase A (85.5 U/mg, 10 mg/mL) and phenol/chloroform/isoamyl alcohol (25:24:1) were purchased from AppliChem (Darmstadt, Germany). Ultrapure water was produced in-house (purity 18.2 MΩ cm at 25 °C). Game meat samples were provided from Wildpark Ernstbrunn (Ernstbrunn, Austria), the Research Institute of Wildlife Ecology (Vienna, Austria) and the University of Veterinary Medicine Vienna (Vienna, Austria). Meat from domesticated animals, such as pigs, was purchased at local supermarkets and meat markets. The samples were collected from lean muscle meat. Meat was collected between 2015 and 2016 and stored at − 20 °C. All animal species were verified through sequencing. “DNA mixtures” were produced by mixing DNA isolated from the muscle meat of the respective animal species. “Meat extract mixtures” were prepared by mixing the extracts from the meat flesh of the respective animal species prior to DNA isolation. In the case of “meat mixtures”, the meat of the respective animal species was weighed and mixed prior to extraction and isolation of the DNA. Model sausages were produced according to the Codex Alimentarius Austriacus. “Fallow deer sausages” containing different proportions of fallow deer (2%, 10%, 25%, 38.5% and 50% (w/w)) and pig meat were produced at the Austrian Agency for Health and Food Safety (AGES, Vienna, Austria). A “game sausage” was 5

prepared at the Higher Technical College for Food Technology Hollabrunn (Hollabrunn, Austria). This game sausage contained 21% fallow deer, 21% red deer, 21% roe deer, 7% wild boar, 30% (w/w) bacon (domesticated pig) and common sausage ingredients, such as nitrite curing salt (28 g/kg sausage), sucrose (2 g/kg sausage) and dextrose (3 g/kg sausage). In addition, common food allergens were added (soy; celery; white, brown and black mustard; wheat; sesame; milk powder and egg powder; 27 g/15 kg sausage).

2.2

DNA isolation DNA was isolated from raw meat, meat mixtures and model sausages using a

CTAB method as previously described (Druml, Mayer, Cichna-Markl, & Hochegger, 2015). The concentration and purity of the isolated DNA were determined by measuring

the

absorbance

at

260 nm

and

280 nm

using

a

QIAxpert

spectrophotometer (Qiagen, Hilden, Germany). The DNA isolates were stored at − 20°C.

2.3

Primer and probes Genomic DNA sequences were downloaded from the NCBI database (NCBI,

2016). Sequence similarity was detected via online BLAST (Basic Local Alignment Algorithm Search Tool) (BLAST, 2016). CLC Genomics Workbench 8.0 (Qiagen, Hilden, Germany) was used for sequence alignment. The primers and probes were designed using Primer Express 3.0 (Applied Biosystems, Foster City, CA, USA). All probes were labeled with VIC at the 5’ end and a non-fluorescent minor groove binding quencher (MGBNFQ) at the 3’ end. The primers were synthesized at Sigma Aldrich or Eurofins (Ebersberg, Germany), and

6

probes were generated at Applied Biosystems. The sequences of the primers and probes and the primer/probe systems tested are shown in Table 1.

2.4

Real-time PCR Real-time PCR was performed in strip tubes with caps (0.1 mL, Qiagen) on a

72-well rotor (Qiagen) in a Rotor Gene Q cycler (Qiagen) or in an optical 96-well reaction plate (0.2 mL, Applied Biosystems) sealed with optical adhesive film (Applied Biosystems) on the ABI 7500 Real-time PCR System (Applied Biosystems). The PCR was conducted in a total volume of 25 µL. Unless otherwise indicated, the reaction mix contained 12.5 µL of QuantiTect Multiplex PCR NoROX Master Mix (Qiagen), 2.5 µL of ultrapure water, 5 µL of 5x primer/probe mix and 5 µL of isolated DNA (DNA concentration between 5 and 20 ng/µL). The standard temperature program was initiated with a denaturation step at 95 °C for 15 min, followed by 40 cycles at 94 °C for 1 min and 60 °C for 1 min. This temperature program was used unless otherwise indicated.

2.5

Specificity of the primer/probe systems All primer/probe systems were tested for cross-reactivity with DNA isolated from

the following 24 animal species: alpine ibex (Capra ibex), cattle (Bos taurus), chamois (Rupicapra rupicapra), chicken (Gallus gallus), crocodile (Crocodylus niloticus), donkey (11%) (Equus asinus), duck (Anatidae), goat (Capra hircus), goose (Anserinae),

hare

(Lepus

europaeus),

horse

(Equus

caballus),

kangaroo

(Macropodidae), moose (Alces alces), mouflon (Ovis orientalis), ostrich (Struthio camelus), pig (Sus scrofa domestica), rabbit (Oryctolagus cuniculus), red deer (Cervus elaphus), reindeer (Rangifer tarandus), roe deer (Capreolus capreolus),

7

sheep (Ovis aries), sika deer (Cervus nippon), turkey (Meleagris gallopavo) and wild boar (Sus scrofa scrofa). Primer/probe system 5b (Table 1) was also tested for cross-reactivity with DNA isolates from the following 50 plant species: allspice (Pimenta dioica), anise (Pimpinella anisum), bay leaf (Laurus nobilis), bean (Phaseolus vulgaris), black mustard (Brassica nigra), black pepper (Piper nigrum), broccoli (Brassica oleracea), buckwheat (Fagopyrum esculentum), caraway (Carum carvi), cardamom (Elettaria cardamomum), carrot (Daucus carota), celery (Apium graveolens), chili pepper (Capsicum sp.), chives (Allium schoenoprasum), coriander (Coriandrum sativum), cumin (Cuminum cyminum), curcuma (Curcuma longa/domestica), dill (Anethum graveolens), fennel (Foeniculum vulgare), garlic (Allium sativum), ginger (Zingiber officinale), green pea (Pisum sativum), horseradish (Armoracia rusticana), leek (Allium ampeloprasum), lentil (Lens culinaris), lovage (Levisticum officinale), maize (Zea mays), marjoram (Origanum majorana), onion (Allium cepa), oregano (Origanum vulgare), parsley (Petroselinum crispum), parsnip (Pastinaca sativa), peanut (Arachis hypogaea), pearl millet (Pennisetum glaucum), potato (Solanum tuberosum), radish (Raphanus sativus), rapeseed (Brassica napus), rice (Oryza sativa), rosemary (Rosmarinus officinalis), rye (Secale cereale), sage (Salvia officinalis), savory (Satureja hortensis), sesame (Sesamum indicum), sweet pepper (Capsicum annuum), tarragon (Artemisia dracunculus), thyme (Thymus vulgaris), tomato (Solanum lycopersicum), walnut (Juglans regia), wheat (Triticum aestivum) and white mustard (Sinapis alba). DNA isolates from animal and plant species were used at concentrations of 10 and 20 ng/µL, respectively. The concentrations of forward primer, reverse primer and probe were 200 nM, 200 nM and 100 nM, respectively. Cross-reactivity tests were performed using QuantiTect® Multiplex PCR NoROX Master Mix (Qiagen) and the 8

following temperature program: initial denaturation at 95 °C for 15 min, followed by 40x (94 °C for 1 min and 60 °C for 1°min). To extend the cross-reactivity tests for primer/probe system 5b DNA isolates from the 24 animal species were analyzed using other commercial master mixes. The following master mixes were tested (the respective temperature protocols are shown in brackets): TaqMan® Universal PCR Master Mix (Applied Biosystems; 95 °C for 10 min; 45x (95 °C for 15 s, 60 °C for 1 min)); GoTaq® Probe qPCR Master Mix (Promega, Madison, Wisconsin, USA; 95 °C for 2 min; 40x (95 °C for 3 s, 60 °C for 31 s)); PerfeCTa® qPCR ToughMix TM UNG (Quanta Biosciences, Gaithersburg, Maryland, USA; 95 °C for 10 min; 45x (95 °C for 5 s, 60 °C for 30 s)) and TakyonTM No Rox Probe MasterMix dTTP Blue (Eurogentec, Seraing, Belgium; 95°C for 3 min; 40x (95 °C for 3 s, 60 °C for 32 s)).

2.6

Optimization of the concentration of primer/probe system 5b To determine the optimal primer and probe concentration, the following

combinations (forward primer/ reverse primer/ probe concentration in nM) were tested:

100/100/50;

100/100/100;

100/100/250;

100/200/100;

100/400/100;

100/800/100; 200/200/50; 200/200/100; 200/200/250; 200/400/100; 200/800/100; 200/1000/100; 400/400/50; 400/400/100; 400/400/250; 800/800/50; 800/800/100 and 800/800/250. Optimization experiments were performed with DNA isolates (10 ng/µL) from fallow deer and red deer.

2.7

Robustness of the real-time PCR assay To determine the robustness of the real-time PCR assay, the annealing

temperature was varied ± 1 °C and the volume of the reaction mix (containing QuantiTect® Multiplex PCR NoROX Master Mix, ultrapure water, primers and probe) 9

was varied ± 5%. In addition, the PCR was performed on another thermal cycler (ABI 7500 Real-time PCR System, Applied Biosystems).

2.8

Working range, linear range and amplification efficiency The working range of the real-time PCR assay was determined by serially

diluting the fallow deer DNA isolate (337 ng/µL) with water (1:2 to 1:524,288). For determination of the linear range, the fallow deer DNA isolate (20 ng/µL) was diluted with pig DNA (20 ng/µL; 1:2 to 1:16,384). After plotting the Ct values against the logarithm of the fallow deer DNA concentrations, the equation of the standard curve was obtained via linear regression. From the slope of the standard curve, the amplification efficiency was calculated as follows:  % = 10

2.9

 



− 1 ∙ 100.

Limit of detection (LOD), limit of quantification (LOQ) and repeatability The LOD and LOQ of the real-time PCR assay were determined according to

the guidelines of the European Network of GMO Laboratories (ENGL, 2015). A DNA isolate (5 ng/µL) containing 1% (w/w) fallow deer and 99% (w/w) non-target pig DNA, was serially diluted with pig DNA to obtain mixtures containing 0.5%, 0.4%, 0.25%, 0.2%, 0.1% or 0.05% (w/w) fallow deer DNA. The concentration generating a Ct value < 37 in at least 19 out of 20 measurements was defined as LOD. To determine the LOQ, the real-time PCR assay was calibrated by analyzing a DNA mixture containing 25% (w/w) fallow deer in pork DNA (20 ng/µL) and dilutions (1:4, 1:16, 1:64, 1:256 and 1:1,024) thereof in triplicates. The LOQ was defined as the lowest concentration for which a relative standard deviation (RSD) of ≤ 25% was achieved. 10

The repeatability of the real-time PCR assay was investigated by analyzing the DNA mixture containing 25% (w/w) fallow deer in pig DNA (20 ng/µL) and five dilutions thereof (1:4 to 1:1,024) in triplicates on two consecutive days.

2.10 Quantification The relative content of fallow deer in DNA mixtures, meat extract mixtures, meat mixtures and model sausages was determined. The DNA isolates were analyzed using two real-time PCR systems targeting single copy genes: the fallow deerspecific real-time PCR assay and a reference real-time PCR assay, to determine the total amount of mammalian and poultry species. The reference assay (MY70) has previously been published (Druml, Kaltenbrunner, Hochegger, & Cichna-Markl, 2016). Both real-time PCR assays were calibrated. To analyze the DNA mixtures, DNA mixtures containing 50%, 25%, 10% or 5% (w/w) fallow deer DNA in pig DNA were used for calibration. DNA isolates from meat extract mixtures containing 50%, 25%, 10% or 5% (w/w) fallow deer in pig were used to determine the fallow deer content in meat extract mixtures. To analyze the meat mixtures, real-time PCR assays were calibrated with DNA isolates from meat mixtures containing 50%, 25%, 10% or 5% (w/w) fallow deer and pig meat. To quantify the fallow deer content in model sausages, the DNA isolated from a meat extract mixture containing 25% (w/w) fallow deer, 25% (w/w) red deer and 50% (w/w) pig was used for calibration. For fallow deer sausage containing 2% (w/w) fallow deer, DNA isolated from the meat extract mixture containing 10% (w/w) fallow deer in pig was used. Calibration mixtures were adjusted to a DNA concentration of 20 ng/µL, serially diluted (1:4, 1:16, 1:64, 1:256, 1:1,024) in water and used for the calibration of both real-time PCR assays. 11

Calibration curves were obtained by plotting the logarithm of the DNA concentration against the respective Ct value. After establishing the calibration curves for both PCR assays using linear regression, the percentage of fallow deer was calculated according to the following formulas: &'(.*(. +, (.

  

"#  = 10 ! μ%

 '' -

"#  = 10 '  μ%

0"12"1 

! %

=

&'./. *./. +, ./.

    '' -

"#   μ% "# ∙ 100 '  μ%  !

where cDNA fallow deer and c DNA total meat are the concentrations of fallow deer DNA and total meat DNA, respectively; Ctspec. and Ctref. are the Ct values obtained with the fallow deer-specific and the reference PCR assay, respectively; dspec. and dref. are the intercepts of the standard curves of the fallow deer-specific and the reference PCR assay, respectively and slopespec. and sloperef. are the slopes of the calibration curves of the fallow deer-specific and the reference PCR assay, respectively.

2.11 Influence of the presence of red deer on the accuracy of the real-time PCR assay To investigate whether the presence of red deer influences the accuracy of the quantitative results, DNA mixtures, meat extract mixtures and meat mixtures containing different contents (ranging from 0% to 38%; Table 3) of red deer (DNA, meat extract, meat) were produced. The DNA isolates from these mixtures were diluted to a DNA concentration of 5 ng/µL and analyzed using both real-time PCR assays.

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3

Results and Discussion

3.1 Design of a fallow deer-specific primer/probe system The design of a fallow deer-specific primer/probe system was difficult since the number of fallow deer gene sequences available in the NCBI database was low. In addition, the high sequence homology with genes of other species, particularly red deer and sika deer, made the design challenging. The present strategy involved the identification of genes not only sequenced for fallow deer but also for red deer and sika deer. Among these genes, we selected those for which the BLAST algorithm indicated a sequence identity ≤ 99% at high query coverage (percent of the query sequence overlapping the subject sequence). In total, we designed primers and probes targeting five regions in four genes. The genes encoded the fallow deer proteins prion protein PrP (Prnp), lactoferrin, alpha lactalbumin and melanocyte stimulating hormone receptor (MC1-R), respectively. We used short amplicons (to enable the detection and quantification of fallow deer in processed foods) containing as many fallow deer-specific bases as possible. However, with one exception, the regions selected contained only one fallow deer-specific base. Thus, the fallow deerspecific bases were located in either the forward primer, the reverse primer or the probe. The primers were designed to contain the fallow deer-specific base in the next to last position. We attempted to increase the binding specificity by implementing a mismatch in the last but two position of the primer. Enhancing the binding specificity through a base mismatch next to the specific base, called mismatch amplification mutation assay (MAMA), has previously been described for allelic discrimination (Cha, Zarbl, Keohavong, & Thilly, 1992). In 2004, Li et al. reported the combination of MAMA with a 5’-exonuclease (TaqMan) assay, called TaqMAMA (Li, Kadura, Fu, & Watson, 2004) with potential for discriminating between sequences differing in only 13

one base. Consistent with previous studies (Druml, Grandits, Mayer, Hochegger, & Cichna-Markl, 2015; Druml, Kaltenbrunner, Hochegger, & Cichna-Markl, 2016; Druml, Mayer, Cichna-Markl, & Hochegger, 2015), we used MGB-labeled probes because of the higher stability of duplexes formed with single-stranded DNA compared with alternative probes. The advantage is that reflecting the increase in the melting temperature, shorter probes can be used (Kutyavin, Afonina, Mills, Gorn, Lukhtanov, Belousov, et al., 2000). The primers and probes were combined, generating 18 primer and probe systems (Table 1), and tested for cross-reactivity. The first specificity test with primer/probe system 1a was performed using the TaqMan® Universal PCR Master Mix (Applied Biosystems). The fallow deer DNA isolate (10 ng/µL) resulted in a Ct value of 24.39 (n = 2), whereas DNA isolates from the other 24 animal species tested did not lead to an increase in the fluorescence signal within 45 cycles (Figure 1A). However, when we tested the same primer/probe system using the QuantiTect® Multiplex PCR NoROX Kit Master Mix (Qiagen), which is particularly applicable for multiplexing, in addition to fallow deer DNA (Ct value 24.89, n = 2), DNA from alpine ibex (Ct value 31.76, n = 2) and goat (Ct value 32.56, n = 2) was amplified (Figure 1B). Efforts to increase the specificity by slightly modifying the forward and/or reverse primer (primer/probe systems 1b – 1k, Table 1) were not successful. Primer/probe systems 2, 3a, 3b, 4a, 4b and 5a (Table 1) were not suitable because in addition to fallow deer DNA, DNA from red deer, sika deer and roe deer was amplified. The ∆Ct values between fallow deer and the cross-reacting species ranged from 0.72 to 4.80. However, when we modified the reverse primer of primer/probe system 5a (targeting the MC1-R gene) by altering the position of the fallow deer-specific base and including a base mismatch (primer/probe system 5b, Figure 2), the specificity was markedly increased. Primer/probe system 5b showed 14

some cross-reactivity with sika deer, red deer, roe deer, reindeer and wild boar (Figure 1 D). However, compared to the Ct value of 24.18 (n = 10) for fallow deer, the Ct values (37.07, 36.30, 37.50, 37.60 and 37.45, respectively (n ≥ 2)) were high, resulting in ∆Ct values > 12. For DNA from the other 19 animals and the 50 plant species tested, an increase of the fluorescence signal was not observed within 40 cycles. Thus, the primer/probe system 5b was considered applicable for use in practice. To investigate the influence of the master mix on the specificity of primer/probe system 5b, DNA isolates from the 24 animal species were additionally analyzed using other commercial master mixes. ∆Ct values > 12 (between fallow deer and the crossreacting species) were achieved using the GoTaq® Probe qPCR Master Mix (Promega), the PerfeCTa® qPCR ToughMix TM UNG (Quanta Biosciences) and the TakyonTM No Rox Probe MasterMix dTTP Blue (Eurogentec). However, ∆Ct values < 12 (Ct values: fallow deer 31.39, red deer 43.18, sika deer 40.65, roe deer 39.87 and moose 42.15 (n = 4)) were achieved using TaqMan® Universal PCR Master Mix (Applied Biosystems) (Figure 1 C).

3.2 Optimization of the primer/probe concentrations All cross-reactivity tests described above were performed using the same primer/probe concentrations (forward primer: 200 nM, reverse primer: 200 nM, probe: 100 nM). Next, we investigated how the primer/probe concentrations of primer/probe system 5b influence the ∆Ct value between fallow deer and the cross-reacting species. Among all primer/probe concentrations tested, the previously used concentrations resulted in the lowest Ct value for fallow deer and the highest ∆Ct value between fallow deer and the cross-reacting species. Thus, these primer/probe concentrations were used in all subsequent experiments. 15

3.3 Robustness To examine the robustness of the fallow deer real-time PCR assay, crossreactivity tests with DNA isolates from fallow deer, red deer and sika deer were performed by varying the annealing temperature ± 1 °C or the volume of the reaction mix (consisting of QuantiTect Multiplex PCR NoROX Master Mix (Qiagen), primer/probe mix and water) per PCR ± 5%. To determine whether the real-time PCR assay can be used on another thermal cycler, additional experiments were performed on the ABI 7500 thermal cycler (Applied Biosystems). The real-time PCR assay was robust. Neither slight modifications of the PCR conditions nor the use of another thermal cycler affected the ∆Ct values between fallow deer and the cross-reacting animal species.

3.4 Working range, linear range and amplification efficiency The working range of the real-time PCR assay was determined by serially diluting the fallow deer DNA isolate (337 ng/µL) with water (1:2 to 1:524,288). A linear relationship (R2 = 0.997, slope = − 3.41) between the logarithm of the fallow deer DNA concentration and the Ct value was observed between 42.1 µg/mL and 10.3 ng/mL. From the slope of the standard curve, the amplification efficiency was calculated as 96%. The range of linearity was further determined by serially diluting the fallow deer DNA extract (20 ng/µL) with pig DNA (20 ng/µL, 1:2 to 1:16,384). A linear relationship (R2 = 0.999) was observed for fallow deer concentrations between 20 µg/mL and 9.8 ng/mL. The slope of the standard curve was − 3.31, corresponding to an amplification efficiency of 100%.

16

3.5 LOD, LOQ and repeatability For determining the LOD and LOQ of the real-time PCR assay, we prepared a mixture containing fallow deer DNA in pig DNA (1:99, w/w). We diluted the DNA mixture with pig DNA down to a fallow deer DNA content of 0.05% and analyzed the diluted mixtures in 20 replicates. The LOD was defined as the lowest fallow deer DNA concentration with a Ct value < 37 in at least 19 out of 20 replicates. The LOQ was defined as the lowest concentration for which a RSD ≤ 25% was obtained. The Ct values and fallow deer DNA concentrations (including means, standard deviations and relative standard deviations) are shown in Table 2. LOD and LOQ were determined as 0.1% and 0.4%, respectively. DNA isolates (5 ng/µL) from red deer, sika deer, moose, reindeer, roe deer and wild boar, which were analyzed in duplicates in the same PCR, led to Ct values > 37, confirming the previous finding that the presence of these species will not limit the applicability of the real-time PCR assay. The repeatability of the real-time PCR assay was assessed by analyzing a serially diluted DNA mixture containing 25% (w/w) fallow deer and 75% (w/w) pig DNA in triplicates on two consecutive days. The RSD of the Ct values was < 2%, indicating that the real-time PCR is highly repeatable.

3.6 Quantification results Sixteen DNA mixtures, 16 meat extract mixtures, 16 meat mixtures and six model sausages containing various fallow deer contents were analyzed to investigate the applicability of the real-time PCR assay for quantification. For calibration, four calibrators differing in fallow deer content were tested. Using a calibrator containing fallow deer, red deer and pig DNA at ratios of 10:10:80 (w/w/w) or 5:5:90 (w/w/w), the fallow deer recovery in the 16 DNA mixtures 17

ranged from 78% to 114% (Table 3). When the calibrator contained 25% (w/w) fallow deer DNA in pig DNA, the analysis of DNA mixtures containing 5% to 50% fallow deer resulted in recoveries ranging from 79% to 124%. For DNA mixtures with fallow deer contents of 1% or 2%, recoveries of 101% or 144% were obtained, respectively. Calibration with a mixture containing 50% fallow deer/50% pig DNA resulted in recoveries ranging from 99% to 137% for DNA mixtures containing 20% to 50% fallow deer DNA. Recoveries of 120% and 188% were obtained for mixtures containing 1% or 2% fallow deer DNA, respectively. These results clearly indicate that the composition of the calibration mixture should be adjusted to the composition of the sample to obtain accurate quantitative results. Similar results were obtained for meat extract mixtures (Supplementary Table 1). In general, calibrators with higher fallow deer contents resulted in better recoveries for samples containing higher concentrations of fallow deer, whereas lower concentrated calibration mixtures were applicable to samples with low fallow deer contents. The results obtained for meat mixtures are summarized in Table 3. For meat mixtures with fallow deer contents of 1% or 2%, recoveries ranging from 180% to 446% were obtained when the calibration mixture contained 50% fallow deer/50% pig (w/w), 125% to 404% when the calibration mixture contained 25% fallow deer/75% pig (w/w), 93% to 166% (with one exception of 326%) when the calibration mixture contained 10% fallow deer/90% pig (w/w) and 87% to 178% (with one exception of 231%) when the calibration mixture contained 5% fallow deer/33% red deer/62% pig (w/w). In general, the fallow deer contents determined for meat mixtures were less accurate than those determined for DNA mixtures and meat extract mixtures. However, deviations from the theoretical fallow deer concentrations reflected, at least in part, the fact that the meat mixtures were prepared by weighing out small (mg) 18

amounts of meat. Therefore, differences in the water content of the meat samples will result in some inaccuracy, particularly in the case of meat mixtures with low fallow deer contents. Previous studies have shown that in case of the analysis of complex matrices, it is important to adjust the matrix of the calibrator to the sample matrix (Eugster, Ruf, Rentsch, & Köppel, 2009). In addition, the results of the present study demonstrated that the proportion of the target animal species in the calibrator should be as close as possible to that in the sample. Thus, we suggest the following strategy for determining the fallow deer content in routine analysis. In a first step, the food product should be qualitatively analyzed. In addition to the DNA isolate from the sample, a standard containing 0.1% fallow deer should be analyzed. This cut-off standard will not only serve as a positive control near the LOD of the real-time PCR assay, but also enable the rough estimation of the fallow deer content in the sample. Based on this information, an appropriate calibrator can be selected for subsequent quantitative analysis. The real-time PCR assay for fallow deer was further validated after analyzing model sausages with known compositions. For calibration, either a meat extract mixture or a meat mixture was used. For all model sausages, recoveries ranging from 91% to 110% were obtained (Table 4). To determine whether the presence of cross-reacting species affects the accuracy of the quantitative results, red deer (either DNA, meat extract or meat) was added to the samples at proportions up to 38%. Table 3 indicates that the presence of red deer did not decrease the accuracy.

4

Conclusion

19

The development of a real-time PCR assay for detecting fallow deer adulteration was challenging. In total, we designed eleven forward primers, ten reverse primers and five probes and combined these sequences into 18 primer/probe systems. Among these systems, a primer/probe system targeting a 64-bp fragment of the Dama dama MC1-R gene was selective. In addition to DNA from fallow deer, this system amplified DNA from sika deer, red deer, roe deer, reindeer and wild boar. However, since the Ct values between fallow deer and the cross-reacting species were > 12, the presence of these species in food products will not lead to systematic errors. The real-time PCR was sensitive (LOD: 0.1%, LOQ: 0.4%). To determine the applicability of the assay for quantifying the fallow deer content, DNA mixtures, meat extract mixtures, meat mixtures and model game sausages containing known contents of fallow deer were analyzed. Quantification was performed using a previously published reference PCR system. The results indicate that the highest accuracy was obtained when the composition and the fallow deer concentration in the calibration mixture were similar to the composition and the fallow deer content in the sample. The robustness of the real-time PCR assay was examined using five different commercially available master mixes and slightly varying the annealing temperature and the volume of the master mix and changing the thermocycler used. The real-time PCR assay was robust. Neither slight modifications of the PCR conditions nor the use of another thermal cycler affected the ∆Ct values between fallow deer and the cross-reacting animal species. With the exception of one master mix, the ∆Ct values between fallow deer and the cross-reacting species were > 12.

20

Conflict of interest statement The authors declare no conflicts of interest.

Supplementary Table 1

Acknowledgments The authors would like to thank the Research Institute of Wildlife Ecology (Vienna, Vienna, Austria), the Wildpark Ernstbrunn (Ernstbrunn, Lower Austria, Austria) and the University of Veterinary Medicine Vienna (Vienna, Vienna, Austria) for providing meat samples from different animal species. The authors would also like to thank Walter Mayer (Austrian Agency for Health and Food Safety, Institute for Food Safety, Department of Molecular Biology and Microbiology, Vienna, Vienna, Austria) for sharing his expertise.

References

Ballin, N. Z., Vogensen, F. K., & Karlsson, A. H. (2009). Species determination - Can we detect and quantify meat adulteration? Meat Science, 83(2), 165-174. BLAST.

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Algorithm

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Druml, B., Grandits, S., Mayer, W., Hochegger, R., & Cichna-Markl, M. (2015). Authenticity control of game meat products – A single method to detect and quantify adulteration of fallow deer (Dama dama), red deer (Cervus elaphus) and sika deer (Cervus nippon) by real-time PCR. Food Chemistry, 170, 508517. Druml, B., Kaltenbrunner, M., Hochegger, R., & Cichna-Markl, M. (2016). A novel reference real-time PCR assay for the relative quantification of (game) meat species in raw and heat-processed food. Food Control, 70, 392-400. Druml, B., Mayer, W., Cichna-Markl, M., & Hochegger, R. (2015). Development and validation of a TaqMan real-time PCR assay for the identification and quantification of roe deer (Capreolus capreolus) in food to detect food adulteration. Food Chemistry, 178, 319-326. ENGL. (2015). European Network of GMO Laboratories. Definition of Minimum Performance Requirements for Analytical Methods of GMO Testing. . Accessed October 31, 2016. Eugster, A., Ruf, J., Rentsch, J., & Köppel, R. (2009). Quantification of beef, pork, chicken and turkey proportions in sausages: Use of matrix-adapted standards and comparison of single versus multiplex PCR in an interlaboratory trial. European Food Research and Technology, 230(1), 55-61. Fajardo, V., González, I., López-Calleja, I., Martín, I., Hernández, P. E., García, T., & Martín, R. (2006). PCR-RFLP authentication of meats from red deer (Cervus elaphus), fallow deer (Dama dama), roe deer (Capreolus capreolus), cattle (Bos taurus), sheep (Ovis aries), and goat (Capra hircus). Journal of Agricultural and Food Chemistry, 54(4), 1144-1150.

22

Fajardo, V., González, I., López-Calleja, I., Martín, I., Rojas, M., Hernández, P. E., García, T., & Martín, R. (2007). Identification of meats from red deer (Cervus elaphus), fallow deer (Dama dama), and roe deer (Capreolus capreolus) using polymerase chain reaction targeting specific sequences from the mitochondrial 12S rRNA gene. Meat Science, 76(2), 234-240. Fajardo, V., González, I., Martín, I., Rojas, M., Hernández, P. E., García, T., & Martín, R. (2008). Real-time PCR for detection and quantification of red deer (Cervus elaphus), fallow deer (Dama dama), and roe deer (Capreolus capreolus) in meat mixtures. Meat Science, 79(2), 289-298. Fajardo, V., González Isabel, I., Rojas, M., García, T., & Martín, R. (2010). A review of current PCR-based methodologies for the authentication of meats from game animal species. Trends in Food Science and Technology, 21(8), 408421. Hoffman, L. C., & Cawthorn, D. (2013). Exotic protein sources to meet all needs. Meat Science, 95(4), 764-771. Hoffman, L. C., & Wiklund, E. (2006). Game and venison - meat for the modern consumer. Meat Science, 74(1), 197-208. Kutyavin, I. V., Afonina, I. A., Mills, A., Gorn, V. V., Lukhtanov, E. A., Belousov, E. S., Singer, M. J., Walburger, D. K., Lokhov, S. G., Gall, A. A., Dempcy, R., Reed, M. W., Meyer, R. B., & Hedgpeth, J. (2000). 3'-minor groove binder-DNA probes increase sequence specificity at PCR extension temperatures. Nucleic Acids Research, 28(2), 655-661. Li, B., Kadura, I., Fu, D. J., & Watson, D. E. (2004). Genotyping with TaqMAMA. Genomics, 83(2), 311-320. NCBI. (2016). National Center for Biotechnology Information Search database. . Accessed October 25, 2016. 23

Obidziński, A., Kiełtyk, P., Borkowski, J., Bolibok, L., & Remuszko, K. (2013). Autumn-winter diet overlap of fallow, red, and roe deer in forest ecosystems, Southern Poland. Central European journal of biology, 8(1), 8-17. Poławska, E., Cooper, R. G., Jóźwik, A., & Pomianowski, J. (2013). Meat from alternative species - Nutritive and dietetic value, and its benefit for human health - A review. CYTA - Journal of Food, 11(1), 37-42.

24

Gene (NCBI acc. no.) Prion protein PrP (Prnp) gene, partial intron 2 sequence and complete cds (AY286007.1)

System 1a

Primer/probe sequence (5' - 3') fw 1 GGTGGCTACATGCTGGGAAGT rev 1 GTCCTCATAGTCATTGCCAAAATG probe CCATGAATAGGCCTCTTA 1b fw 1 GGTGGCTACATGCTGGGAAGT rev 2 GTCCTCATAGTCATTGCCAAAATGT probe CCATGAATAGGCCTCTTA 1c fw 2 CGGTGGCTACATGCTGGG rev 2 GTCCTCATAGTCATTGCCAAAATGT probe CCATGAATAGGCCTCTTA 1d fw 2 CGGTGGCTACATGCTGGG rev 1 GTCCTCATAGTCATTGCCAAAATG probe CCATGAATAGGCCTCTTA 1e fw 3 CTCGGTGGCTACATGCTG rev 3 CCTCATAGTCATTGCCAAAATGT probe CCATGAATAGGCCTCTTA 1f fw 5 CTCCGTGGCTACATGCTG rev 4 CCTCATAGTCATTGCCAAAATCT probe CCATGAATAGGCCTCTTA 1g fw 4 CTCTGTGGCTACATGCTG rev 5 CCTCATAGTCATTGCCAAAATTT probe CCATGAATAGGCCTCTTA 1h fw 3 CTCGGTGGCTACATGCTG rev 1 GTCCTCATAGTCATTGCCAAAATG probe CCATGAATAGGCCTCTTA 1i fw 5 CTCCGTGGCTACATGCTG rev 1 GTCCTCATAGTCATTGCCAAAATG probe CCATGAATAGGCCTCTTA 1j fw 1 GGTGGCTACATGCTGGGAAGT rev 3 CCTCATAGTCATTGCCAAAATGT probe CCATGAATAGGCCTCTTA 1k fw 1 GGTGGCTACATGCTGGGAAGT rev 4 CCTCATAGTCATTGCCAAAATCT probe CCATGAATAGGCCTCTTA Lactoferrin 2 fw GACTGGGAGACAGCCTTTGG (Sanger sequencing rev GCAGTACAGCTCCCAGAAAACA of a ~200bp fragment) probe CACTGAGGCCACCG Alpha lactalbumin gene, 3a fw 1 TCAGTAACACCAAATTCCCAGAAA intron rev ACATCAAGATTCCCAAACAGCTC (DQ379356.1) probe TTTCTTAAAGTTCATGGGTAG 3b fw 2 TCAGTAACACCAAATTCCCAGTAA rev ACATCAAGATTCCCAAACAGCTC probe TTTCTTAAAGTTCATGGGTAG Alpha lactalbumin gene, 4a fw 1 GGCTTATGTGAGAAATCTAATACAGTAATGC GCGCTGCGCCAGAGAAGT intron rev (DQ379356.1) probe CCCTAGGTAAGAACTCCCT 4b fw 2 TGAGAAATCTAATACAGTAATGCCTA GCGCTGCGCCAGAGAAGT rev probe CCCTAGGTAAGAACTCCCT MC1-R gene 5a fw GACACCATGGAGCCACAGATAA (Y13963.1) rev 1 CAGGCAGCTGTGGTGCAA probe CGTCGATGACATTGTCCAG 5b fw GACACCATGGAGCCACAGATAA rev 2 AGGCAGCTGTGGTGCTAC probe CGTCGATGACATTGTCCAG Bold letters in the primer and probe sequences indicate fallow deer specific bases Underlined letters in the primer and probe sequences indicate mismatch bases

Length [nt] 21 24 18 21 25 18 18 25 18 18 24 18 18 23 18 18 23 18 18 23 18 18 24 18 18 24 18 21 23 18 21 23 18 20 22 14 24 23 21 24 23 21 31 18 19 26 18 19 22 18 19 22 18 19

Amplicon [nt] 66

66

67

67

67

67

67

69

69

66

66

61

76

76

74

66

65

64

Table 2: Determination of LOD and LOQ by analyzing a serially diluted DNA mixture containing 1% (w/w) fallow deer DNA in 99% (w/w) pig DNA (non-target DNA). Measurements were carried out in 20 replicates. Fallow deer DNA content (%)

Mean SD RSD (%)

1.00 Ct value

c (ng/mL)

0.50 Ct value

c (ng/mL)

0.40 Ct value

c (ng/mL)

0.25 Ct value

c (ng/mL)

0.20 Ct value

c (ng/mL)

0.10 Ct value

0.05 Ct value

c (ng/mL)

34.15

45.2

35.60

17.4

35.05

24.9

35.36

19.7

35.38

12.0

36.31

6.5

36.49

10.1

34.01

49.5

35.66

16.7

35.11

24.0

36.03

13.3

35.82

9.0

35.97

8.1

36.76

8.6

33.88

54.2

35.02

25.5

35.27

21.6

35.39

19.4

34.76

33.50

69.8

35.00

25.8

35.05

24.9

35.69

16.2

36.42

17.9

37.10

3.9

36.76

8.5

6.1

35.90

8.5

37.37

6.0

34.12

46.2

34.87

28.0

35.30

21.1

34.52

32.6

35.94

8.3

36.22

6.9

37.38

5.9

34.13

45.8

35.44

19.2

35.82

15.0

36.02

13.4

35.12

14.2

35.68

9.8

37.71

4.9

34.16

44.9

35.48

18.7

34.91

27.3

33.79

57.4

34.58

33.9

36.15

12.0

35.02

24.3

36.45

6.0

36.56

5.5

37.92

4.3

35.33

20.1

36.03

7.9

36.28

6.7

38.19

3.7

33.76

58.6

34.74

30.5

35.04

25.1

34.47

33.7

35.17

13.7

35.35

12.2

38.19

3.7

33.60

65.0

35.13

23.6

34.96

26.4

34.43

34.5

35.31

12.5

36.42

6.1

37.20

6.6

33.68

61.6

34.62

34.11

46.6

34.63

33.2

35.21

22.4

35.62

17.0

35.04

14.9

36.46

5.9

37.22

6.5

32.9

35.06

24.7

34.85

26.8

35.54

10.8

36.87

4.5

38.13

3.8

33.77

58.1

35.06

24.8

35.04

25.1

34.59

31.4

35.76

9.3

37.11

3.9

36.46

10.3

34.02

49.5

34.73

30.9

34.83

28.8

35.20

21.8

35.00

15.3

36.76

4.9

37.27

6.3

34.00

49.8

35.13

23.6

35.39

19.9

35.25

21.1

35.27

12.9

35.71

9.7

37.36

6.0

33.71

60.8

35.62

17.1

35.90

14.2

35.05

23.8

35.85

8.8

36.08

7.6

35.91

14.2

33.79

57.6

35.03

25.3

34.55

34.6

34.96

25.2

35.23

13.2

37.15

3.8

35.69

16.3

33.87

54.4

34.58

34.0

34.77

30.0

35.22

21.5

35.09

14.5

35.58

10.5

38.10

3.9

33.47

71.2

35.02

25.5

34.97

26.3

34.95

25.3

36.01

8.0

36.11

7.4

37.61

5.2

33.85 33.87 0.22 0.64

55.1 55.1 8.0 14.5

34.99 35.05 0.35 1.01

25.9 25.6 5.7 22.4

34.67 35.15 0.41 1.15

32.1 24.0 5.7 23.8

35.39 35.17 0.47 1.33

19.4 23.0 6.3 27.4

35.13 35.52 0.49 1.38

14.1 11.5 3.4 29.4

36.31 36.30 0.52 1.44

6.5 7.0 2.3 33.5

37.90 37.28 0.74 1.99

4.3 6.9 3.5 50.3

c Concentration of fallow deer determined SD Standard deviation RSD Relative standard deviation

c (ng/mL)

Table 3: Quantification results obtained for DNA mixtures and DNA isolates from meat mixtures with different fallow deer content using different calibration mixtures. (n=2)

Content (%) Fallow Red deer deer (%) (%) DNA mixture

Pig (%)

Calibrator containing 50% fallow deera Mean fallow deer Mean recovery content determined (%) (%)

Calibrator containing 25% fallow deera Mean fallow deer Mean recovery content determined (%) (%)

Calibrator containing 10% fallow deerb, c Mean fallow deer Mean recovery content determined (%) (%)

Calibrator containing 5% fallow deerb Mean fallow deer Mean recovery content determined (%) (%)

1

0

99

1.8

178

1.4

136

0.9

87

0.9

94

1

1

98

1.9

188

1.4

144

1.0

101

1.0

96

1

38

61

1.2

120

1.0

101

1.0

103

0.8

78

2

0

98

3.4

169

2.7

137

1.8

89

1.9

93

2

2

96

3.2

158

2.7

135

2.0

100

1.8

91

5

33

62

5.3

106

5.6

112

4.8

95

4.6

92

10

0

90

14.0

140

12.4

124

10.0

100

9.6

96

10

10

80

13.4

134

10.9

109

10.3

103

9.0

90

20

0

80

27.3

137

20.3

102

19.2

96

20.2

101

20

20

60

21.2

106

19.0

95

21.4

107

19.3

96

25

0

75

31.3

125

23.9

95

28.6

114

24.3

97

25

25

50

29.1

117

19.7

79

23.1

92

24.3

97

33

5

62

32.7

99

28.5

86

36.0

109

33.6

102

38

1

61

50.6

133

34.1

90

39.2

103

39.5

104 97

39

0

61

46.8

120

34.2

88

38.9

100

37.8

50

0

50

52.5

105

42.1

84

48.2

96

52.2

104

Meat mixture 1.1

0.0

98.9

5.0

446

4.5

404

3.6

326

2.6

231

1.2

1.1

97.8

2.7

234

1.8

152

1.1

98

1.4

118

1.0

37.6

61.4

1.9

180

1.3

127

1.0

93

0.9

87

1.9

0.0

98.1

4.1

215

2.4

125

2.6

136

1.7

89

2.1

1.9

96.0

8.4

399

5.6

266

3.5

166

3.7

178

5.3

32.5

62.2

16.8

315

12.1

227

9.1

171

7.5

140

10.1

0.0

89.9

15.1

150

11.3

112

12.1

119

8.4

83

10.8

10.4

78.7

13.8

128

10.0

92

7.6

70

7.4

69

19.6

0.0

80.4

53.8

274

33.2

169

34.5

176

24.1

123 94

19.6

19.8

60.7

33.8

173

21.3

109

16.5

85

18.4

24.8

0.0

75.2

37.3

150

24.4

98

23.9

96

18.0

73

24.6

24.6

50.7

28.5

116

23.0

93

19.1

77

13.8

56

29.9

5.1

65.0

60.2

201

43.1

144

31.8

106

35.7

119

36.6

0.9

62.5

63.9

175

44.7

122

39.1

107

29.5

81

38.4

0.0

61.6

69.9

182

51.5

134

46.7

121

32.6

85

49.7

0.0

50.3

55.2

111

39.2

79

35.0

70

24.9

50

a

Calibrators containing fallow deer (concentration given in the table) and pig. b Calibration mixtures contained fallow deer (concentration is given in the table), red deer and pig. c Calibrator for the meat mixtures contained 10% fallow deer and 90% pig.

Table 4: Quantification results obtained for DNA isolates from game sausages with different fallow deer contents. (n ≥ 4)

Content

Fallow deer content determined mean ± SD (%) 2.0 ± 0.2

Fallow deer (%) 2.0

Red deer (%) -

Roe deer (%) -

Wild boar (%) -

Pig (%) 98.0

10.0

-

-

-

90.0

10.8 ±

1.3

108 ±

13

25.0

-

-

-

75.0

27.6 ±

2.3

110 ±

9

38.5

-

-

-

61.5

40.4 ±

8.1

105 ±

21

50.0

-

-

-

50.0

45.7 ±

7.8

91 ±

16

Game sausageCAAb 21.0 21.0 21.0 7.0 30.0 20.0 ± 6.1 a DNA isolate from meat extract mixture containing 10% fallow deer and 90% pig was used for calibration b DNA isolate from meat extract mixture containing 25% fallow deer, 25% red deer and 50% pig was used for calibration c DNA isolate from meat mixture containing 25% fallow deer, 25% red deer and 50% pig was used for calibration

95 ±

29

Fallow deer sausage meat 2%a Fallow deer sausage meat 10%b b

Fallow deer sausage meat 25%

b

Fallow deer sausage meat 38.5% Fallow deer sausage meat 50%

b

Recovery mean ± SD (%) 101 ± 8

Figure captures: Figure 1 Specificity tests carried out with two different commercial master mixes (TaqMan Universal PCR Master Mix (A and C) and QuantiTect Multiplex PCR NoROX Master Mix (B and D)) using primer/probe system 1 (A and B) and primer/probe system 5b (C and D).

Figure 2 Sequence alignment of the MC1-R gene. Arrows indicate the position of the forward primer (“primer/probe system 5 fw”), two reverse primers (“primer/probe system 5 rev 1” and “primer/probe system 5 rev 2”) and the probe (“primer/probe system 5 probe) of primer/probe set 5. Both reverse primers contain the fallow deer specific base (indicated by the vertical red arrow). In addition, primer/probe system 5 rev 2 contains a mismatch base (indicated by the vertical blue arrow). Sequences were aligned using the CLC Genomics Workbench 8.0 (Qiagen).

Figure 1 A

B Fallow deer Goat

Fallow deer Goat

Alpine ibex ΔCt = 6.9

C

D Fallow deer Ct = 31.39

Red deer Moose Sika deer Roe deer ΔCt = 9.3

Fallow deer

Sika deer Reindeer Roe deer Red deer ΔCt = 12.1

Figure 2

Research highlights: •

A new quantitative fallow deer specific real-time PCR method

•

All performance criteria recommended by the ENGL network were achieved

•

DNA from cross-reacting species amplified with ∆Ct value > 12

•

Successful quantification of DNA-, meat extract-, meat mixtures and model sausages

25