- Email: [email protected]
Identification of poultry species using polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) and capillary electrophoresis-single strand conformation polymorphism (CE-SSCP) methods
Accepted Manuscript Identification of poultry species using polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) and capillary electrophoresis-single strand conformation polymorphism (CE-SSCP) methods Ákos Tisza, Ádám Csikós, Ádám Simon, Gabriella Gulyás, András Jávor, Levente Czeglédi PII:
S0956-7135(15)30039-6
DOI:
10.1016/j.foodcont.2015.06.006
Reference:
JFCO 4486
To appear in:
Food Control
Received Date: 23 April 2015 Revised Date:
1 June 2015
Accepted Date: 2 June 2015
Please cite this article as: Tisza Á., Csikós Á., Simon Á., Gulyás G., Jávor A. & Czeglédi L., Identification of poultry species using polymerase chain reaction-single strand conformation polymorphism (PCRSSCP) and capillary electrophoresis-single strand conformation polymorphism (CE-SSCP) methods, Food Control (2015), doi: 10.1016/j.foodcont.2015.06.006. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT 1
1 2 3
Identification of poultry species using polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) and capillary electrophoresis-single strand conformation polymorphism (CE-SSCP) methods
4 5 6 7 8 9
Ákos Tisza – Ádám Csikós – Ádám Simon – Gabriella Gulyás - András Jávor – Levente Czeglédi * Institute of Animal Science, Biotechnology and Nature Conservation, University of Debrecen, 138. Boszormenyi Street, 4032 Debrecen. Hungary
RI PT
*Corresponding author. Tel. +36 52508444/88199; e-mail address: [email protected] (L. Czeglédi)
Abstract
In the last decades, animal species identification became more important to prevent food adulteration.
11
Here, we demonstrate the identification of seven poultry species, chicken, guinea fowl, pheasant,
12
turkey, goose, duck and muscovy duck, through the use of the polymerase chain reaction-single
13
strand conformation polymorphism (PCR-SSCP) and capillary electrophoresis-single strand
14
conformation polymorphism (CE-SSCP) methods. DNA were isolated from poultry meat and meat
15
products and were amplified with universal primers, designed for the mitochondrial 12S rRNA.
16
Species-specific patterns and the reliable detection limit were identified as 0.5% for PCR and CE
17
applications. Analyses of commercially available poultry products revealed fraud, as 6 of 36 contained
18
undeclared species. The above-mentioned techniques are sensitive, reproducible and reliable for
19
poultry species identification from foodstuffs.
21
Highlights
EP
20
TE D
M AN U
SC
10
• PCR-SSCP and CE-SSCP methods were developed for poultry species identification
22
in raw meat and processed meat products as well.
AC C
23 24
• Detection limit of the assays was 0.5%.
25
• Fraud was revealed, 6 of 36 meat products contained undeclared species.
26
• Assays are sensitive, reproducible and reliable.
27
1
Abbreviations: PCR–SSCP - polymerase chain reaction-single strand conformation polymorphism; CE–SSCP - capillary electrophoresis-
single strand conformation polymorphism; IEF - isoelectric focusing; PAGE - polyacrylamide gel electrophoresis; ELISA - enzyme-linked immunosorbent assay; RAPD–PCR - random amplified polymorphic DNA; RFLP - restriction fragment length polymorphism; TGGE temperature gradient gel electrophoresis; DGGE - denaturing gradient gel electrophoresis; 6-FAM - 6-carboxyfluorescein
1
ACCEPTED MANUSCRIPT 28
Keywords: species identification; PCR; SSCP; CE-SSCP; poultry species
29 30
1. Introduction
31 Identification of animal species in foodstuffs has become a substantial issue in the last
33
decades, in order to prevent substitutions and admixtures in animal products for religious
34
reasons, as well as to meet health and government regulations (Meyer et al., 1995; Arslan et
35
al., 2006; Mane et al., 2006). Procedures for food labeling have been legislated by the
36
European Union, which ensure that consumers receive truthful and important information
37
about the quality of animal products. There are several methods for identifying species in
38
foodstuffs, including analysis of fatty acid, protein or DNA. Fatty acid detection methods are
39
usually used for identification in dairy products. Protein based methods, such as isoelectric
40
focusing (IEF) (King & Kurth, 1982), polyacrylamide gel electrophoresis (PAGE) (Craig et
41
al., 1995), enzyme-linked immunosorbent assays (ELISA) (Chen & Hsieh, 2000) and Western
42
blot are time consuming, expensive and inaccurate methods, due to the processing of meat
43
products and tissue dependency. DNA-based methods are more reliable, because of high
44
specificity, sensitivity, rapidity and cost effectivity (Bottero et al., 2003b; Dalmasso et al.,
45
2004; Calvo et al., 2001b). Recently, mitochondrial DNA (mtDNA) investigations have
46
become more frequent, due to its structure (no introns in the sequence), compared to nuclear
47
DNA. The inheritance of mtDNA is maternal, and because of the lack of recombination
48
events and its conservative sequences, it is advantageous for DNA-based experiments (Rokas
49
et al., 2003). The average number of mitochondria is about 1000 mitochondria/cell; therefore,
50
the amplification procedures are easily feasible (Kocher et al., 1989; Greenwood &
51
1999). Several different mitochondrial regions are usually amplified for meat species
52
identification, such as the mitochondrial D-loop region (Montiel-Sosa et al., 2000), 12S rRNA
53
(Stamoulis et al., 2010; Rojas et al., 2012), 16S rRNA (Borgo et al., 1996; Bottero et al.,
AC C
EP
TE D
M AN U
SC
RI PT
32
2
Pääbo,
ACCEPTED MANUSCRIPT 2003b; Dalmasso et al., 2004) and cytochrome b (Matsunaga et al., 1999; Girish et al., 2004).
55
In the last three decades, several polymerase chain reaction (PCR)-based methods were
56
developed (Mullis & Faloona, 1987). The most commonly used techniques for species
57
identification are species-specific PCR (Man et al., 2007; Arslan et al., 2006), multiplex PCR
58
(Dalmasso et al., 2004; Matsunaga et al., 1999), random amplified polymorphic DNA
59
(RAPD–PCR) (Calvo et al., 2001a), DNA hybridization (Ebbehøj & Thomsen, 1991a;
60
Ebbehøj & Thomsen,1991b; Hunt et al., 1997), PCR - restriction fragment length
61
polymorphism (PCR-RFLP) analysis (Fajardo et al., 2006) and real-time PCR (Sawyer et al.,
62
2003; Zhang et al., 2007), mutation scanning methods, such as temperature gradient gel
63
electrophoresis (TGGE), denaturing gradient gel electrophoresis (DGGE) and single strand
64
conformation polymorphism (SSCP) perform unique methods (Gasser, R. B., 1997; Cotton,
65
R. G., 1997). SSCP is an appropriate tool for fraud detection, due to its low cost and
66
sensitivity, which is based on the electrophoretic mobility of the single-stranded DNA
67
molecule under denaturing conditions in a polyacrylamide gel, where the mobility depends on
68
the DNA conformation (Orita et al., 1989; Hayashi K., 1991). Recently, the PCR-based
69
capillary electrophoresis (CE) applications have begun to gain more interest, owing to their
70
advantages, such as automation, higher resolution, faster speed and reproducibility. Presently,
71
CE based analytical methods are more relevant for animal species identification, especially
72
meat and meat products, fish and seafood products (Rodríguez-Ramírez et al., 2011).
SC
M AN U
TE D
EP
AC C
73
RI PT
54
74
The aims of this study were to develop a simple, sensitive PCR-SSCP protocol and a fast and
75
sensitive CE-SSCP method for the identification of poultry species in meat and meat
76
products.
77 78 3
ACCEPTED MANUSCRIPT 79
2. Materials and methods
80 81
2.1 Sample preparation
82 Muscle tissues were collected from chicken, guinea fowl, pheasant, turkey, goose, duck and
84
muscovy duck. 3 non-relative individuals per species were included in the study. Bird species
85
were confirmed by visual inspection. Poultry products were purchased in department stores.
86
Each sample was stored at -20 °C until further analysis. DNA was prepared from 70 mg of
87
muscle or meat products by phenol-chloroform extraction method as described by De et al.
88
(2011). Concentration and quality of DNA samples was determined using NanoDrop 1000
89
spectrophotometer (Thermo Fischer Scientific, USA).
M AN U
SC
RI PT
83
90 91
93
2.2 Primer design
TE D
92
Nucleotide sequences of 12S rRNA mitochondrial gene of chicken (GenBank: FJ610339.1),
95
guinea fowl (GenBank: FN675566.1), pheasant (GenBank: U83742.1), turkey (GenBank:
96
AJ490508.1), goose (GenBank: JN695752.1), duck (GenBank: JN695762.1) and muscovy
97
duck (GenBank: AM902523.1) were downloaded from the NCBI GenBank database.
98
Nucleotide sequences were aligned using the CLUSTAL OMEGA algorithm of European
99
Bioinformatics Institute (EBI) (Figure 1). The phylogenetic trees of eight poultry species
100
were generated using the amplified region of 12S rRNA using the neighbor-joining method of
101
CLUSTALW2 PHYLOGENY algorithm of EBI (Figure 2). The universal forward and
102
reverse primers produce 277 bp amplicons for pheasant and turkey, 278 bp amplicons for
103
chicken, guinea fowl and goose, 280 bp and 281 bp amplicons for duck and muscovy duck,
AC C
EP
94
4
ACCEPTED MANUSCRIPT 104
respectively. Primers were tested by Oligoanalyzer software (Integrated DNA Technologies,
105
Inc.) for hairpin, self-dimer and hetero-dimer structures.
153 154
155
ACTCTAAGGACTTGGCGGTG.............................A.......... .................................................A...C...... .................................................A.......... .......................T.........................G.......... .................................................AC......... .......................T.........................G........G. .......................T.........................G..........
Chicken Guinea fowl Pheasant Turkey Goose Duck Muscovy duck
FJ610339.1| FN675566.1| U83742.1| AJ490508.1| JN695752.1| JN695762.1| AM902523.1|
........T...................-.G............................. .....................T......-............................... ............................-............................... .....................T......-............................... ...C....TA..................-............................... .........A........G.........A.G............................. C........A..................-..A............................
Chicken Guinea fowl Pheasant Turkey Goose Duck Muscovy duck
FJ610339.1| FN675566.1| U83742.1| AJ490508.1| JN695752.1| JN695762.1| AM902523.1|
.....T.....A..A............T..----ATAGC...T......T.......... ....AC.TGA.AGCGCA.CAG-....-CTC----AACAGT....A....C.......... ....AA.TGA.AG..CA.CAGTGAGC-T..----ACAGT..A..A...GC.......... ....AA.....A..AT...T......-CTC----AATAGT....A....C.......... .....G.....G............GA.A...T..C---..-........T.......... .....G.....G...G........G..G......C---..-........T.......... .....G.....G...............G......C---...........T..........
Chicken Guinea fowl Pheasant Turkey Goose Duck Muscovy duck
FJ610339.1| FN675566.1| U83742.1| AJ490508.1| JN695752.1| JN695762.1| AM902523.1|
...........T.........G......................CA.....C.A.--CGA ...........C...A...........................GCA.....C.CTCACGA ...........C................................TA.....T.A.--CGA ...........C...A............................CA.....C.G.--CGA ...........T....A.....................CC...TTCATAG--GGCA.ACG ...........T.....AC...................CC...TGCA-T.GGGC.A.ACG ...........C......C...................CC...CACACT.GGGC.G.ACG
Chicken Guinea fowl Pheasant Turkey Goose Duck Muscovy duck
FJ610339.1| FN675566.1| U83742.1| AJ490508.1| JN695752.1| JN695762.1| AM902523.1|
.....G.CG.GA..C......T.A.AAGGAGGATTTAGCAGTAAA .....AGCA.GA..C.T....T.A..................... .....G.CG.GA..C...T..T.G..................... .....GGCG.GA..CT.....T.G..................... G....A.GCGTG..A..A.TTC.G.....C............... G....A.GTATG..A.T..TTC.A..................... .....A.GCATG..A.T..TTC.G.....................
TE D
M AN U
SC
RI PT
FJ610339.1| FN675566.1| U83742.1| AJ490508.1| JN695752.1| JN695762.1| AM902523.1|
Figure 1. Multiple sequence alignment for 12S rRNA of seven poultry species
EP
152
Chicken Guinea fowl Pheasant Turkey Goose Duck Muscovy duck
AC C
106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130 131 132 133 134 135 136 137 138 139 140 141 142 143 144 145 146 147 148 149 150 151
156
Figure 2. Phylogenetic tree of seven poultry species based on 12S rRNA target sequence Phylogenetic tree was generated using the
157
neighbor-joining method by CLUSTALW2 PHYLOGENY algorithm of EBI
158 159
5
ACCEPTED MANUSCRIPT 160
2.3 Polymerase chain reaction with universal primers
161 The PCR amplification of DNA from meat samples was performed in 30 µl volume
163
containing 1x Dream Taq Buffer (Fermentas), 200 µM dNTP mix (Fermentas), 4 mM MgCl2
164
(Promega), 0.1 µM forward primer (5’-ACTCTAAGGACTTGGCGGTG-3’) (Sigma-
165
Aldrich), 0.1 µM reverse primer (5’-TTTACTGCTAAATCCTCCTT-3’) (Sigma-Aldrich),
166
1.5 U Dream Taq polymerase (Fermentas) and 150 ng DNA template. PCR was carried out in
167
a thermal cycler PTC-200 (Bio-Rad, USA). Amplification protocol was as follows: first step
168
is denaturation at 95 °C for 1.5 min, followed by 35 cycles consisting of denaturation at 95 °C
169
for 30 sec, primer annealing at 60 °C for 30 sec and extension at 72 °C for 30 sec. The final
170
extension step was 5 min at 72 °C. Amplified PCR products were stained by Ethidium
171
Bromide (Bio-Rad, USA) and were analyzed by electrophoresis in 2% agarose gel (Lonza) for
172
1 h with 10 V/cm in TAE (Tris-Acetate-EDTA, pH:8) (Lonza) buffer.
173
According to the CE application, primers were labeled with fluorescent dyes (Life
174
Technologies). Forward primer was 5’-labeled with 6-carboxyfluorescein (6-FAM) (5’-
175
ACTCTAAGGACTTGGCGGTG-3’). Reverse primer was 5’-labeled with VIC (5’-
176
TTTACTGCTAAATCCTCCTT-3’). The PCR mixtures consisted of 1x DreamTaq Buffer
177
(Fermentas), 4 mM MgCl2 (Promega), 200 µM of each dNTP (Fermentas), 0.1 µM of each
178
labeled primers (Sigma-Aldrich), 0.05 U/µl of DreamTaq DNA Polymerase (Fermentas) and
179
150 ng of template DNA. The steps of the PCR reaction were the same, as described above.
181
SC
M AN U
TE D
EP
AC C
180
RI PT
162
2.4 PCR-single strand conformation polymorphism (PCR-SSCP)
182 183
Amplified PCR products were diluted in denaturing solution containing 90 v/v% formamide-
184
dye (8 v/v% bromophenol blue stain (Sigma-Aldrich); 92 v/v% formamide). The solution was
6
ACCEPTED MANUSCRIPT heat-denatured at 95 °C for 5 min and chilled immediately on ice. Denatured DNA fragments
186
were loaded onto a 10% acrylamide:bis-acrylamide (37.5:1) non-denaturing polyacrylamide
187
gel (20 cm × 16 cm × 0.75 mm). Polyacrylamide gel electrophoresis was performed at 4 °C
188
for 8 h with 25 V/cm on a Protean II xi Cell vertical format electrophoresis system (Bio-Rad,
189
USA) with a Power Pac Universal Power Supply (Bio-Rad, USA).
190
After electrophoresis, the polyacrylamide gel was stained using the silver staining method
191
according to Merril et al. (1984) and documented with the Uvipro Platinum (Uvitec) gel
192
documentation system.
SC
RI PT
185
193
2.5 Sample preparation and conditions of capillary electrophoresis-single strand
195
conformation polymorphism (CE-SSCP)
M AN U
194
196
CE was performed on an ABI Prism 310 Genetic Analyzer (Life Technologies), equipped
198
with an argon-ion laser, light emitted at 488 – 514 nm. Samples were electro-kinetically-
199
injected at 15 kV for 10 sec to a 47 cm (effective length: 30 cm) long diameter 50 µm (Life
200
Technologies) capillary filled with 15 wt% solution of Pluronic F108 polymer (Sigma-
201
Aldrich), containing 0.7x Genetic Analyzer buffer (Life Technologies). Electrophoresis was
202
performed at 35 °C with 15 kV. Signal detection was used in all separations between 525-650
203
nm wavelengths. Results were collected using the Data Collection Software Version 3.1.0
204
program, and were analyzed with GeneMapper® 3.7 (Life Technologies) software. To correct
205
run-to-run variations, electropherograms were normalized by fixing the positions of peaks
206
produced by GeneScanTM 500 LIZTM dye Size Standard (Life Technologies). Samples were
207
prepared for capillary electrophoresis analysis as follows: 0.5 µl PCR product + 0.5 µl 500
208
LIZTM dye + 9 µl of deionized Hi-Di formamide (Life Technologies) were mixed, then
209
denatured at 95°C for 3 minutes followed by quick cooling on ice. Electrophoresis was
AC C
EP
TE D
197
7
ACCEPTED MANUSCRIPT 210
performed at 35 °C, using 15 kV electrophoresis voltage with 5 sec injection time and 40 min
211
electrophoresis running time.
212 213
2.6 DNA sequencing
RI PT
214 The amplified fragments of 12S rRNA gene were purified using the PCR Advanced™ PCR
216
Clean Up System (Viogene, Taiwan) according to the manufacturer’s instructions. The
217
purified PCR amplicons were sequenced by Macrogen Europe Inc. in Amsterdam, The
218
Netherlands. Obtained sequence data were aligned with sequences from the NCBI GenBank
219
database.
220 221
3. Results
222
224
3.1 Polymerase chain reaction
TE D
223
M AN U
SC
215
Primers were designed to amplify a 277-285 bp region of mitochondrial 12S rRNA of poultry
226
species. Following PCR reaction, amplicons were separated on agarose gel using 50 bp
227
GeneRulerTM DNA ladder (Life Technologies). PCR products of each species were of sharp,
228
good quality and no artifacts were detected at PCR with either unlabeled nor labeled primers.
230 231
AC C
229
EP
225
3.2 PCR-single strand conformation polymorphism (PCR-SSCP)
232 233
In this study, PCR-SSCP and CE-SSCP were tested using the DNA of seven poultry species,
234
chicken, turkey, duck, muscovy duck, goose, guinea fowl and pheasant and a mixture of
8
ACCEPTED MANUSCRIPT chicken and duck DNA samples. The applicability of these methods was tested using meat
236
products.
237
Double-stranded PCR amplicons were transformed into single-stranded conformers with
238
unique secondary structure after denaturation. In the case of the PCR-SSCP method, single-
239
stranded DNA molecules were separated on polyacrylamide gel, in contrast to CE-SSCP,
240
where running of denaturated amplicons took place in a polymer matrix. Species-specific
241
conformations were visualized by silver staining (Merril et al., 1984). With SSCP analysis,
242
each poultry species had distinct patterns and could be differentiated from each other (Figure
243
3). Polyacrylamide gel electrophoresis resulted in no false positives.
244
Chicken-duck DNA mixtures were tested, to determine the sensitivity of this method. Duck
245
DNA samples contained chicken DNA in 20; 10; 5; 1; 0.5%, respectively (Figure 4). Positive
246
control samples contained chicken and duck DNA in 100%. The detection threshold of PCR-
247
SSCP method was 0.5% presence of chicken DNA in the chicken-duck DNA mixture (Table
248
1).
249
The applicability of the PCR-SSCP method was tested on 36 commercial meat products, such
250
as sausages, salamis and frankfurters, as shown in Table 2. Chicken, turkey, duck and goose
251
DNA were used as positive controls on polyacrylamide gel. Species-specific patterns were
252
obtained in these products and the presence of undeclared species was detected in six cases,
253
that is 16.67% of all the analyzed samples. 35.7% of indicated turkey meat products (5 of 14
254
meats) contained turkey and chicken DNA. 12.5% of meat products labeled as chicken meat
255
(1 of 8 meats) contained turkey and chicken DNA. 83.4% of all meat products, 30 of 36,
256
exclusively contained indicated species; consequently, fraud was not detected by PCR-SSCP
257
(Table 2).
AC C
EP
TE D
M AN U
SC
RI PT
235
9
RI PT
ACCEPTED MANUSCRIPT
258
SC
Figure 3. PCR-SSCP pattern of 12S rRNA of seven poultry species. 1.: duck; 2.: muscovy duck; 3.: chicken; 4.: goose; 5.: guinea fowl; 6.: pheasant; 7.: turkey.
Figure 4. Electrophoretic analysis of PCR-SSCP from chicken and duck meat DNA. 1.: chicken; 2.: duck; 3, 4, 5, 6 and 7 DNA mixture of chicken and duck DNA containing 20, 10, 5, 1, 0.5% of chicken DNA, respectively. Table 1. Determination of detection limit of PCR-SSCP and CE-SSCP methods analyzing 10 DNA mixtures containing 0.5% and 99.5%, and 10 DNA mixtures containing 0.1% and 99.9% chicken and duck DNA, respectively.
AC C
263 264 265 266 267 268
EP
262
TE D
M AN U
259 260 261
Number of detectable chicken specific DNA band by PCR-SSCP/ all samples Number of detectable chicken specific DNA band by CE-SSCP/ all samples
0.5% chicken and 99.5% duck
0.1% chicken and 99.9% duck
10/10
0/10
10/10
0/10
269 270
3.3 Capillary electrophoresis-single strand conformation polymorphism (CE-SSCP)
271
10
ACCEPTED MANUSCRIPT PCR products were heat denatured and prepared for CE-SSCP analysis. Fluorescently labeled
273
primers were used to detect the unique single strand conformers. Seven poultry species were
274
distinguished from each other, which demonstrates that our fluorescently labeled universal
275
primers are capable of use for pattern recognition. Two single strand conformers appeared for
276
each poultry species. Species-specific patterns are shown on Figure 5.
277
The sensitivity of the CE-SSCP method for analyzing the chicken-duck DNA mixture,
278
containing decreasing concentrations of chicken DNA, was determined in the same way as
279
was set for PCR-SSCP (Figure 6). The detection threshold of chicken was 0.5% DNA in the
280
chicken-duck DNA mixture, similar to the PCR-SSCP method (Table 1).
281
The applicability of the CE-SSCP was tested on the same commercial meat products used in
282
the PCR-SSCP analyses, and the same chicken, turkey, duck and goose positive control
283
samples were used in the CE-SSCP analysis, as well. After CE, species-specific patterns were
284
obtained and the presence of undeclared species were detected in six products, similarly to the
285
findings when using PCR-SSCP, which is 16.67% of commercial products included in this
286
study. All of the 6 fraudulent findings revealed the presence of chicken, as this was the non-
287
labeled species in the “poultry-duck” meat product. The presence of a PCR conformer
288
reflecting turkey was identified, in contrast to PCR-SSCP, where only duck and chicken
289
patterns were observed. We found an additional VIC labeled conformer in the same poultry
290
meat product, but it was not identical to any of the 7 bird species. No fraud was detected in
291
the rest of meat products using the CE-SSCP method (Table 2).
SC
M AN U
TE D
EP
AC C
292
RI PT
272
293 294 295
3.4 DNA sequencing
296
11
ACCEPTED MANUSCRIPT We performed sequence alignment (EBI, CLUSTAL OMEGA algorithm) with sequences
298
from the NCBI GenBank database and sequenced DNA regions from the meat products. We
299
established that one product, labeled as turkey, contained turkey and also chicken DNA.
300
Thymine (turkey) and cytosine (chicken) bases were found at the 56th position of the
301
sequenced 12S rRNA amplicons. The presence of turkey and chicken DNA were verified with
302
PCR-SSCP and CE-SSCP methods as well (Figure 7).
303
← VIC
data points
TE D
RFU
B Numida meleagris (Guinea fowl)
6-FAM →
304
SC
← 6-FAM
M AN U
RFU
A Gallus gallus domestica (Chicken)
← VIC
EP
data points
305
AC C
RFU
C Phasianus colchicus (Pheasant)
6-FAM →
RI PT
297
← VIC
data points
12
ACCEPTED MANUSCRIPT
RFU
D Meleagris galoppavo (Turkey)
306
← VIC
RI PT
6-FAM →
data points
← VIC
307
M AN U
6-FAM →
SC
RFU
E Anser anser domestica (Goose)
F Anas platyrhynchos domestica (Duck)
TE D
RFU
data points
6-FAM →
308
← VIC
EP
data points
AC C
RFU
G Cairina moschata (Muscovy duck)
6-FAM →
309
← VIC
data points
13
ACCEPTED MANUSCRIPT
310
data points
Figure 5. Species-specific patterns of 12S rRNA of seven poultry species on capillary electrophoresis electropherograms: Chicken (A), Guinea fowl (B), Pheasant (C), Turkey (D), Goose (E), Duck (F), Muscovy duck (G), No Template Control (H). GeneScanTM 500 LIZTM dye Size Standard is shown as reference peaks (pale gray), 6-FAM and VIC labeled strands are shown as gray and dark gray peaks, respectively. Y- and X-axis shows relative fluorescence unit (RFU) and data points (1 data point is equal with 220 msec migration time), respectively.
SC
311 312 313 314 315 316
RI PT
RFU
H No Template Control
M AN U
RFU
A Chicken control C→ 6-FAM
CVIC
317 318
→
data points
RFU
TE D
B Duck control D→ 6-FAM
321 322
AC C
RFU
C 80% Duck, 20% Chicken
data points
EP
319 320
C→ 6-FAM
D→ 6-FAM
DVIC
CVIC
DVIC
→
→
data points
RFU
D 90% Duck, 10% Chicken
323 324
C→ 6-FAM
D→ 6-FAM
CVIC
DVIC
→
data points
14
→
→
ACCEPTED MANUSCRIPT
RFU
E 95% Duck, 5% Chicken C→ 6-FAM
D→ 6-FAM
325 326
CVIC
DVIC
→
→
CD- → 6-FAM 6-FAM ↓
327 328
→
M AN U
RFU
CD- → 6-FAM 6-FAM ↓
329 330
CVIC ↓
DVIC
→
data points
TE D
RFU
H No Template Control
EP
data points
Figure 6. Mixture of duck and chicken 12S rRNA samples. 100% Chicken; C (A), 100% Duck; D (B). Percentage of the DNA mixtures: 80% Duck, 20% Chicken (C), 90% Duck, 10% Chicken (D), 95% Duck, 5% Chicken (E), 99% Duck, 1% Chicken (F), 99.5% Duck, 0.5% Chicken (G), No Template Control (H). Y- and X-axis shows relative fluorescence unit (RFU) and data points (1 data point is equal with 220 msec migration time), respectively.
AC C
339
DVIC
data points G 99.5% Duck, 0.5% Chicken
331 332 333 334 335 336 337 338
CVIC ↓
SC
RFU
F 99% Duck, 1% Chicken
RI PT
data points
15
ACCEPTED MANUSCRIPT A
B
C
T
RI PT
Ch
340
Figure 7. Meat product was labeled as made of turkey meat, sequencing, PCR-SSCP and CE-SSCP proved the fraud, as it contained chicken DNA as well. The presence of turkey (Thymine) and chicken (Cytosine) DNA were confirmed by DNA sequencing (A); PCR-SSCP, from left turkey control (T), chicken control (Ch), meat product (M) (B); and CE-SSCP, turkey (T), chicken (Ch) (C).
SC
341 342 343 344 345
347
Table 2. Identification of species in commercial meat products with PCR-SSCP and CE-SSCP methods.
Label (species)
Number of meat products
9
Chicken
8
Duck, poultry
349
EP
AC C
Poultry
Detected species by
PCR-SSCP method
CE-SSCP method
7 chicken
1 chicken+turkey
14
Turkey, chicken, poultry Turkey, poultry
Detected species by
9 turkey+chicken
TE D
Turkey, chicken
Turkey
348
M AN U
346
9 turkey 5 turkey+chicken
1
1 turkey+chicken
2
2 turkey+chicken
1
1 chicken
1
1 chicken+duck
1 turkey+chicken+duck
4. Discussion
350 351
In our study, we were able to identify seven poultry species by polymerase chain reaction-
352
single strand conformation polymorphism (PCR-SSCP) and capillary electrophoresis-single
353
strand conformation polymorphism (CE-SSCP) methods, applied to analyze commercially 16
ACCEPTED MANUSCRIPT available poultry products. In the last decades, the importance of identification of food species
355
and the composition of foods has increased. Species identification DNA methods are based on
356
nuclear and mtDNA markers. In meat species identification, mtDNA regions are frequently
357
used. Design of universal primers for the simultaneous amplification of poultry species played
358
a critical role. Commonly used mitochondrial regions have strong conservative sequences,
359
despite the diversity of species, which are capable for use in the design of universal primers,
360
and also have variable regions, which can be used to differentiate species (Horreo et al.,
361
2013). In the literature, one of the most commonly used mitochondrial regions is the 12S
362
rRNA (Stamoulis et al., 2010; Rojas et al., 2012). Among the PCR-based species detection
363
methods, SSCP is a cost effective and rapid technique (Orita et al., 1989; Hayashi K., 1991)
364
and has the ability to detect single nucleotide polymorphisms (Oohara I., 1997). Due to this
365
advantage, the SSCP method can be effective and appropriate for poultry species
366
identification. Studies using CE-SSCP focused on medical fields, such as pathogen
367
identification, use 16S rRNA for the separation of microorganisms (Chung et al., 2007; Shin
368
et al., 2010). CE-based techniques were developed for food authentication in the last decades,
369
such as in fish (Dooley et al., 2005), and in meat and seafood (Rodríguez-Ramírez et al.,
370
2011).
371
In our work, we compared PCR-based SSCP and CE-SSCP methods with the identification of
372
seven poultry species (Gallus gallus domestica, Numida meleagris, Phasianus colchicus,
373
Meleagris galoppavo, Anser anser domestica, Anas platyrhynchos domestica, Cairina
374
moschata). Designed 12S rRNA primers successfully amplified the poultry DNA in each
375
species. DNA patterns were distinguished successfully using both applications. Sensitivity of
376
methods was tested with DNA mixtures using duck and chicken. The detection limit of
377
chicken DNA in the duck-chicken mixture was 0.5% for PCR-SSCP and CE-SSCP as well,
378
which is equal to 0.75 ng chicken DNA, as the total DNA were 150 ng in the PCR reaction. In
AC C
EP
TE D
M AN U
SC
RI PT
354
17
ACCEPTED MANUSCRIPT this case, the sensitivity of both methods was the same, unlike in some other studies. Ru et al.
380
(2000) have described that the accuracy of CE-SSCP in the detection of colon tumor is better,
381
than conventional SSCP. Andersen et al., (2003) have reported, that capillary array
382
electrophoresis-SSCP (CAE-SSCP) has increased sensitivity, compared to traditional SSCP.
383
López-Calleja et al. (2007) detected 1% cows’ milk in sheep’s and goats’ milk cheeses,
384
Colgan et al. (2001) proved the presence of 0.3% bovine and ovine species and the presence
385
of 1% of porcine species in meat and bone meal with species specific PCR. Rodríguez et al.
386
(2005) have detected pork in pork-beef DNA mixtures in the range of 0.5-5% with real-time
387
PCR assay. Another study referred that 2% cow milk is detectable in buffalo milk (Dalmasso
388
et al., 2011) and with species-specific real-time PCR, 1% of target species is able to be
389
detected in the mixture (Cammà et al., 2012). In multiplex PCR reaction, the detection limits
390
of goats’, sheep’s and cows’ milk were 0.5% in dairy products (Bottero et al., 2003a). With
391
the PCR-RFLP method, sensitivity was 1% for pig presence among the studied species (Partis
392
et al., 2000). Species-specific PCR techniques have the most effective sensitivity, where the
393
detection limit is even 0.25 ng DNA for all meat samples (Matsunaga et al., 1999). The
394
sensitivity threshold is 0.1 % for the DNA detection of cow in water buffalo milk and
395
mozzarella cheese (López-Calleja et al., 2005) and, in the case of ruminant species, in raw and
396
heat treated foodstuffs (Martín et al., 2007). Compared with these studies, sensitivity of PCR-
397
SSCP due to the silver staining method is very high, around 1 pg/mm2 (Bassam et al., 1991).
398
CE-SSCP has the same sensitivity, because of laser-induced detection (Albin et al., 1991).
399
These detection limit values are suitable for fraud detection.
400
In our study, 36 commercially available poultry products were tested and 6 of them contained
401
undeclared species.
AC C
EP
TE D
M AN U
SC
RI PT
379
402 403
18
ACCEPTED MANUSCRIPT 404
5. Conclusions
405 In summary, a polymerase chain reaction-single strand conformation polymorphism (PCR-
407
SSCP) and capillary electrophoresis-single strand conformation polymorphism (CE-SSCP)
408
methods were developed to identify seven poultry species. A reliable detection limit was 0.5%
409
DNA for both applications, which reflect 0.75 ng DNA. 36 commercially available poultry
410
products were analyzed using the PCR-SSCP and CE-SSCP techniques, on the bases of which
411
we identified 6 products with undeclared species. In conclusion, the sensitivity,
412
reproducibility and reliability of PCR-SSCP and CE-SSCP are close to each other, cost
413
efficiency is better in the case of PCR-SSCP. On the other hand, the latter is more labor-
414
intensive and the environmental impact is higher, than for CE-SSCP. Both methods proved to
415
be appropriate tools for poultry species identification in raw meat and processed meat
416
products.
M AN U
SC
RI PT
406
TE D
417 Acknowledgements
419
This work was supported by the TÁMOP-4.1.1.C-12/1/KONV-2012-0014 project, Hungary.
420 421
423
References
AC C
422
EP
418
424 425
Albin, M., Weinberger, R., Sapp, E., & Moring, S. (1991). Fluorescence detection in capillary electrophoresis: evaluation of derivatizing reagents and techniques. Analytical Chemistry, 63(5), 417-422.
426 427 428
Andersen, P. S., Jespersgaard, C., Vuust, J., Christiansen, M., & Larsen, L. A. (2003). High‐throughput single strand conformation polymorphism mutation detection by automated capillary array electrophoresis: validation of the method. Human mutation, 21(2), 116-122.
429 430
Arslan, A., Ilhak, O. I., & Calicioglu, M. (2006). Effect of method of cooking on identification of heat processed beef using polymerase chain reaction (PCR) technique. Meat Science, 72(2), 326-330.
431 432
Bassam, B. J., Caetano-Anollés, G., & Gresshoff, P. M. (1991). Fast and sensitive silver staining of DNA in polyacrylamide gels. Analytical Biochemistry, 196(1), 80-83.
19
ACCEPTED MANUSCRIPT Borgo, R., Souty-Grosset C., Bouchon, D., & Gomot, L. (1996). PCR‐RFLP Analysis of Mitochondrial DNA for Identification of Snail Meat Species. Journal of Food Science, 61(1), 1-4.
435 436 437
Bottero, M., Civera, T., Nucera, D., Rosati, S., Sacchi, P., & Turi, R. (2003a). A multiplex polymerase chain reaction for the identification of cows’, goats’ and sheep's milk in dairy products. International Dairy Journal, 13(4), 277-282.
438 439 440
Bottero, M., Dalmasso, A., Nucera, D., Turi, R., Rosati, S., Squadrone, S., Goria, M., & Civera, T. (2003b). Development of a PCR assay for the detection of animal tissues in ruminant feeds. Journal of Food Protection®, 66(12), 2307-2312.
441 442
Calvo, J. H., Zaragoza, P., & Osta, R. (2001a). Random amplified polymorphic DNA fingerprints for identification of species in poultry pate. Poultry science, 80(4), 522-524.
443 444 445
Calvo, J., Zaragoza, P., & Osta, R. (2001b). Technical note: A quick and more sensitive method to identify pork in processed and unprocessed food by PCR amplification of a new specific DNA fragment. JOURNAL OF ANIMAL SCIENCE-MENASHA THEN ALBANY THEN CHAMPAIGN ILLINOIS-, 79(8), 2108-2112.
446 447
Cammà, C., Di Domenico, M., & Monaco, F. (2012). Development and validation of fast real-time PCR assays for species identification in raw and cooked meat mixtures. Food Control, 23(2), 400-404.
448 449
Chen, F., & Hsieh, Y. (2000). Detection of pork in heat-processed meat products by monoclonal antibody-based ELISA. Journal of AOAC International, 83(1), 79-85.
450 451 452
Chung, J. H., Park, Y. S., Kim, J., Shin, G. W., Nam, M. H., Oh, M., Kim, C. W., Jung, G. Y., & Hyun Park, J. (2007). Parallel analysis of antimicrobial activities in microbial community by SSCP based on CE. Electrophoresis, 28(14), 2416-2423.
453 454
Colgan, S., O’brien, L., Maher, M., Shilton, N., McDonnell, K., & Ward, S. (2001). Development of a DNAbased assay for species identification in meat and bone meal. Food Research International, 34(5), 409-414.
455
Cotton, R. G. (1997). Slowly but surely towards better scanning for mutations. Trends in genetics, 13(2), 43-46.
456 457
Craig, A., Ritchie, A., & Mackie, I. (1995). Determining the authenticity of raw reformed breaded scampi (Nephrops norvegicus) by electrophoretic techniques. Food Chemistry, 52(4), 451-454.
458 459
Dalmasso, A., Civera, T., La Neve, F., & Bottero, M. T. (2011). Simultaneous detection of cow and buffalo milk in mozzarella cheese by real-time PCR assay. Food Chemistry, 124(1), 362-366.
460 461
Dalmasso, A., Fontanella, E., Piatti, P., Civera, T., Rosati, S., & Bottero, M. (2004). A multiplex PCR assay for the identification of animal species in feedstuffs. Molecular and cellular probes, 18(2), 81-87.
462 463 464
De, S., Brahma, B., Polley, S., Mukherjee, A., Banerjee, D., Gohaina, M., Singh, K. P., Singh, R., Datta, T. K., & Goswami, S. L. (2011). Simplex and duplex PCR assays for species specific identification of cattle and buffalo milk and cheese. Food Control, 22(5), 690-696.
465 466
Dooley, J. J., Sage, H. D., Brown, H. M., & Garrett, S. D. (2005). Improved fish species identification by use of lab-on-a-chip technology. Food Control, 16(7), 601-607.
467 468
Ebbehøj, K. F., & Thomsen, P. D. (1991a). Species differentiation of heated meat products by DNA hybridization. Meat Science, 30(3), 221-234.
469 470
Ebbehøj, K. F., & Thomsen, P. D. (1991b). Differentiation of closely related species by DNA hybridization. Meat Science, 30(4), 359-366.
471 472
Fajardo, V., González, I., López-Calleja, I., Martín, I., Hernández, P. E., García, T., & Martín R. (2006). PCRRFLP authentication of meats from red deer (Cervus elaphus), fallow deer (Dama dama), roe deer (Capreolus
AC C
EP
TE D
M AN U
SC
RI PT
433 434
20
ACCEPTED MANUSCRIPT capreolus), cattle (Bos taurus), sheep (Ovis aries), and goat (Capra hircus). Journal of Agricultural and Food Chemistry, 54(4), 1144-1150.
475 476
Gasser, R. B. (1997). Mutation scanning methods for the analysis of parasite genes. International journal for parasitology, 27(12), 1449-1463.
477 478
Girish, P., Anjaneyulu, A., Viswas, K., Anand, M., Rajkumar, N., Shivakumar, B., & Bhaskar, S. (2004). Sequence analysis of mitochondrial 12S rRNA gene can identify meat species. Meat Science, 66(3), 551-556.
479 480
Greenwood, A. D., & Pääbo, S. (1999). Nuclear insertion sequences of mitochondrial DNA predominate in hair but not in blood of elephants. Molecular ecology, 8(1), 133-137.
481 482
Hayashi, K. (1991). PCR-SSCP: a simple and sensitive method for detection of mutations in the genomic DNA. PCR methods and applications, 1(1), 34-38.
483 484 485
Horreo, J. L., Ardura, A., Pola, I. G., Martinez, J. L., & Garcia‐Vazquez, E. (2013). Universal primers for species authentication of animal foodstuff in a single polymerase chain reaction. Journal of the science of food and agriculture, 93(2), 354-361.
486 487
Hunt, D. J., Parkes, H. C. & Lumley, I. D. (1997). Identification of the species of origin of raw and cooked meat products using oligonucleotide probes. Food Chemistry, 60(3), 437-442.
488 489
King, N., & Kurth, L. (1982). Analysis of raw beef samples for adulterant meat species by enzyme‐staining of isoelectric focusing gels. Journal of Food Science, 47(5), 1608-1612.
490 491 492
Kocher, T. D., Thomas, W. K., Meyer, A., Edwards, S. V., Paabo, S., Villablanca, F. X., & Wilson, A. C. (1989). Dynamics of mitochondrial DNA evolution in animals: amplification and sequencing with conserved primers. Proceedings of the National Academy of Sciences of the United States of America, 86(16), 6196-6200.
493 494 495
López-Calleja, I. M., González, I., Fajardo, V., Hernandez, P. E., García, T., & Martín, R. (2007). Application of an indirect ELISA and a PCR technique for detection of cows’ milk in sheep's and goats’ milk cheeses. International Dairy Journal, 17(1), 87-93.
496 497 498
López-Calleja, I., Alonso, I. G., Fajardo, V., Rodríguez, M., Hernández, P., García, T., & Martín, R. (2005). PCR detection of cows’ milk in water buffalo milk and mozzarella cheese. International Dairy Journal, 15(11), 1122-1129.
499 500
Man, Y. C., Aida, A., Raha, A., & Son, R. (2007). Identification of pork derivatives in food products by speciesspecific polymerase chain reaction (PCR) for halal verification. Food Control, 18(7), 885-889.
501 502
Mane, B., Tanwar, V., Girish, P., & Dixit, V. (2006). Identification of species origin of meat by RAPD–PCR technique. Journal of Veterinary Public Health, 4(2), 87-90.
503 504
Martín, I., García, T., Fajardo, V., López-Calleja, I., Hernández, P. E., González, I., & Martín, R. (2007). Species-specific PCR for the identification of ruminant species in feedstuffs. Meat Science, 75(1), 120-127.
505 506 507
Matsunaga, T., Chikuni, K., Tanabe, R., Muroya, S., Shibata, K., Yamada, J., & Shinmura, Y. (1999). A quick and simple method for the identification of meat species and meat products by PCR assay. Meat Science, 51(2), 143-148.
508 509
Merril, C. R., Goldman, D., & Van Keuren, M. L. (1984). [30] Gel protein stains: Silver stain. Methods in enzymology, 104, 441-447.
510 511 512
Meyer, R., Hofelein, C., Luthy, J., & Candrian, U. (1995). Polymerase chain reaction-restriction fragment length polymorphism analysis: a simple method for species identification in food. Journal of AOAC International, 78(6), 1542-1551.
AC C
EP
TE D
M AN U
SC
RI PT
473 474
21
ACCEPTED MANUSCRIPT Montiel-Sosa, J., Ruiz-Pesini, E., Montoya, J., Roncales, P., López-Pérez, M., & Pérez-Martos, A. (2000). Direct and highly species-specific detection of pork meat and fat in meat products by PCR amplification of mitochondrial DNA. Journal of Agricultural and Food Chemistry, 48(7), 2829-2832.
516 517
Mullis, K. B., Faloona, F. A. (1987) Specific synthesis of DNA in vitro via a polymerase-catalyzed chain reaction. Methods Enzymol. 155, 335-50
518 519 520
Oohara, I. (1997). Detection of single strand conformation polymorphisms (SSCPs) on mitochondrial DNA fragments between two domesticated strains of rainbow trout Oncorhynchus mykiss. Fisheries science, 63(1), 151-152.
521 522
Orita, M., Suzuki, Y., Sekiya, T., & Hayashi, K. (1989). Rapid and sensitive detection of point mutations and DNA polymorphisms using the polymerase chain reaction. Genomics, 5(4), 874-879.
523 524
Partis, L., Croan, D, Guo, Z., Clark, R., Coldham, T., & Murby, J. (2000). Evaluation of a DNA fingerprinting method for determining the species origin of meats. Meat Science, 54(4), 369-376.
525 526
Rodríguez, M. A., García, T., González, I., Hernández, P. E., & Martín, R. (2005). TaqMan real-time PCR for the detection and quantitation of pork in meat mixtures. Meat Science, 70(1), 113-120.
527 528 529
Rodríguez-Ramírez, R., González-Córdova, A. F., & Vallejo-Cordoba, B. (2011). Review: Authentication and traceability of foods from animal origin by polymerase chain reaction-based capillary electrophoresis. Analytica Chimica Acta, 685(2), 120-126.
530 531 532
Rojas, M., González, I., García, T., Hernández, P. E., & Martín, R. (2012). Authentication of meat and commercial meat products from common pigeon (Columba livia) woodpigeon (Columba palumbus) and stock pigeon (Columba oenas) using a TaqMan® real-time PCR assay. Food Control, 23(2), 369-376.
533 534
Rokas, A., Ladoukakis, E., & Zouros, E. (2003). Animal mitochondrial DNA recombination revisited. Trends in Ecology & Evolution, 18(8), 411-417.
535 536 537
Ru, Q., Jing, H., Luo, G., & Huang, Q. (2000). Single-strand conformation polymorphism analysis to detect the p53 mutation in colon tumor samples by capillary electrophoresis. Journal of Chromatography A, 894(1), 171177.
538 539
Sawyer, J., Wood, C., Shanahan, D., Gout, S., & McDowell, D. (2003). Real-time PCR for quantitative meat species testing. Food Control, 14(8), 579-583.
540 541
Shin, G. W., Hwang, H. S., Oh, M., Doh, J., & Jung, G. Y. (2010). Simultaneous quantitative detection of 12 pathogens using high‐resolution CE‐SSCP. Electrophoresis, 31(14), 2405-2410.
542 543
Stamoulis, P., Stamatis, C., Sarafidou, T., & Mamuris, Z. (2010). Development and application of molecular markers for poultry meat identification in food chain. Food Control, 21(7), 1061-1065.
544 545 546
Zhang, C., Fowler, M. R., Scott, N. W., Lawson, G., & Slater, A. (2007). A TaqMan real-time PCR system for the identification and quantification of bovine DNA in meats, milks and cheeses. Food Control, 18(9), 11491158.
AC C
EP
TE D
M AN U
SC
RI PT
513 514 515
22
S0956-7135(15)30039-6
DOI:
10.1016/j.foodcont.2015.06.006
Reference:
JFCO 4486
To appear in:
Food Control
Received Date: 23 April 2015 Revised Date:
1 June 2015
Accepted Date: 2 June 2015
Please cite this article as: Tisza Á., Csikós Á., Simon Á., Gulyás G., Jávor A. & Czeglédi L., Identification of poultry species using polymerase chain reaction-single strand conformation polymorphism (PCRSSCP) and capillary electrophoresis-single strand conformation polymorphism (CE-SSCP) methods, Food Control (2015), doi: 10.1016/j.foodcont.2015.06.006. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT 1
1 2 3
Identification of poultry species using polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) and capillary electrophoresis-single strand conformation polymorphism (CE-SSCP) methods
4 5 6 7 8 9
Ákos Tisza – Ádám Csikós – Ádám Simon – Gabriella Gulyás - András Jávor – Levente Czeglédi * Institute of Animal Science, Biotechnology and Nature Conservation, University of Debrecen, 138. Boszormenyi Street, 4032 Debrecen. Hungary
RI PT
*Corresponding author. Tel. +36 52508444/88199; e-mail address: [email protected] (L. Czeglédi)
Abstract
In the last decades, animal species identification became more important to prevent food adulteration.
11
Here, we demonstrate the identification of seven poultry species, chicken, guinea fowl, pheasant,
12
turkey, goose, duck and muscovy duck, through the use of the polymerase chain reaction-single
13
strand conformation polymorphism (PCR-SSCP) and capillary electrophoresis-single strand
14
conformation polymorphism (CE-SSCP) methods. DNA were isolated from poultry meat and meat
15
products and were amplified with universal primers, designed for the mitochondrial 12S rRNA.
16
Species-specific patterns and the reliable detection limit were identified as 0.5% for PCR and CE
17
applications. Analyses of commercially available poultry products revealed fraud, as 6 of 36 contained
18
undeclared species. The above-mentioned techniques are sensitive, reproducible and reliable for
19
poultry species identification from foodstuffs.
21
Highlights
EP
20
TE D
M AN U
SC
10
• PCR-SSCP and CE-SSCP methods were developed for poultry species identification
22
in raw meat and processed meat products as well.
AC C
23 24
• Detection limit of the assays was 0.5%.
25
• Fraud was revealed, 6 of 36 meat products contained undeclared species.
26
• Assays are sensitive, reproducible and reliable.
27
1
Abbreviations: PCR–SSCP - polymerase chain reaction-single strand conformation polymorphism; CE–SSCP - capillary electrophoresis-
single strand conformation polymorphism; IEF - isoelectric focusing; PAGE - polyacrylamide gel electrophoresis; ELISA - enzyme-linked immunosorbent assay; RAPD–PCR - random amplified polymorphic DNA; RFLP - restriction fragment length polymorphism; TGGE temperature gradient gel electrophoresis; DGGE - denaturing gradient gel electrophoresis; 6-FAM - 6-carboxyfluorescein
1
ACCEPTED MANUSCRIPT 28
Keywords: species identification; PCR; SSCP; CE-SSCP; poultry species
29 30
1. Introduction
31 Identification of animal species in foodstuffs has become a substantial issue in the last
33
decades, in order to prevent substitutions and admixtures in animal products for religious
34
reasons, as well as to meet health and government regulations (Meyer et al., 1995; Arslan et
35
al., 2006; Mane et al., 2006). Procedures for food labeling have been legislated by the
36
European Union, which ensure that consumers receive truthful and important information
37
about the quality of animal products. There are several methods for identifying species in
38
foodstuffs, including analysis of fatty acid, protein or DNA. Fatty acid detection methods are
39
usually used for identification in dairy products. Protein based methods, such as isoelectric
40
focusing (IEF) (King & Kurth, 1982), polyacrylamide gel electrophoresis (PAGE) (Craig et
41
al., 1995), enzyme-linked immunosorbent assays (ELISA) (Chen & Hsieh, 2000) and Western
42
blot are time consuming, expensive and inaccurate methods, due to the processing of meat
43
products and tissue dependency. DNA-based methods are more reliable, because of high
44
specificity, sensitivity, rapidity and cost effectivity (Bottero et al., 2003b; Dalmasso et al.,
45
2004; Calvo et al., 2001b). Recently, mitochondrial DNA (mtDNA) investigations have
46
become more frequent, due to its structure (no introns in the sequence), compared to nuclear
47
DNA. The inheritance of mtDNA is maternal, and because of the lack of recombination
48
events and its conservative sequences, it is advantageous for DNA-based experiments (Rokas
49
et al., 2003). The average number of mitochondria is about 1000 mitochondria/cell; therefore,
50
the amplification procedures are easily feasible (Kocher et al., 1989; Greenwood &
51
1999). Several different mitochondrial regions are usually amplified for meat species
52
identification, such as the mitochondrial D-loop region (Montiel-Sosa et al., 2000), 12S rRNA
53
(Stamoulis et al., 2010; Rojas et al., 2012), 16S rRNA (Borgo et al., 1996; Bottero et al.,
AC C
EP
TE D
M AN U
SC
RI PT
32
2
Pääbo,
ACCEPTED MANUSCRIPT 2003b; Dalmasso et al., 2004) and cytochrome b (Matsunaga et al., 1999; Girish et al., 2004).
55
In the last three decades, several polymerase chain reaction (PCR)-based methods were
56
developed (Mullis & Faloona, 1987). The most commonly used techniques for species
57
identification are species-specific PCR (Man et al., 2007; Arslan et al., 2006), multiplex PCR
58
(Dalmasso et al., 2004; Matsunaga et al., 1999), random amplified polymorphic DNA
59
(RAPD–PCR) (Calvo et al., 2001a), DNA hybridization (Ebbehøj & Thomsen, 1991a;
60
Ebbehøj & Thomsen,1991b; Hunt et al., 1997), PCR - restriction fragment length
61
polymorphism (PCR-RFLP) analysis (Fajardo et al., 2006) and real-time PCR (Sawyer et al.,
62
2003; Zhang et al., 2007), mutation scanning methods, such as temperature gradient gel
63
electrophoresis (TGGE), denaturing gradient gel electrophoresis (DGGE) and single strand
64
conformation polymorphism (SSCP) perform unique methods (Gasser, R. B., 1997; Cotton,
65
R. G., 1997). SSCP is an appropriate tool for fraud detection, due to its low cost and
66
sensitivity, which is based on the electrophoretic mobility of the single-stranded DNA
67
molecule under denaturing conditions in a polyacrylamide gel, where the mobility depends on
68
the DNA conformation (Orita et al., 1989; Hayashi K., 1991). Recently, the PCR-based
69
capillary electrophoresis (CE) applications have begun to gain more interest, owing to their
70
advantages, such as automation, higher resolution, faster speed and reproducibility. Presently,
71
CE based analytical methods are more relevant for animal species identification, especially
72
meat and meat products, fish and seafood products (Rodríguez-Ramírez et al., 2011).
SC
M AN U
TE D
EP
AC C
73
RI PT
54
74
The aims of this study were to develop a simple, sensitive PCR-SSCP protocol and a fast and
75
sensitive CE-SSCP method for the identification of poultry species in meat and meat
76
products.
77 78 3
ACCEPTED MANUSCRIPT 79
2. Materials and methods
80 81
2.1 Sample preparation
82 Muscle tissues were collected from chicken, guinea fowl, pheasant, turkey, goose, duck and
84
muscovy duck. 3 non-relative individuals per species were included in the study. Bird species
85
were confirmed by visual inspection. Poultry products were purchased in department stores.
86
Each sample was stored at -20 °C until further analysis. DNA was prepared from 70 mg of
87
muscle or meat products by phenol-chloroform extraction method as described by De et al.
88
(2011). Concentration and quality of DNA samples was determined using NanoDrop 1000
89
spectrophotometer (Thermo Fischer Scientific, USA).
M AN U
SC
RI PT
83
90 91
93
2.2 Primer design
TE D
92
Nucleotide sequences of 12S rRNA mitochondrial gene of chicken (GenBank: FJ610339.1),
95
guinea fowl (GenBank: FN675566.1), pheasant (GenBank: U83742.1), turkey (GenBank:
96
AJ490508.1), goose (GenBank: JN695752.1), duck (GenBank: JN695762.1) and muscovy
97
duck (GenBank: AM902523.1) were downloaded from the NCBI GenBank database.
98
Nucleotide sequences were aligned using the CLUSTAL OMEGA algorithm of European
99
Bioinformatics Institute (EBI) (Figure 1). The phylogenetic trees of eight poultry species
100
were generated using the amplified region of 12S rRNA using the neighbor-joining method of
101
CLUSTALW2 PHYLOGENY algorithm of EBI (Figure 2). The universal forward and
102
reverse primers produce 277 bp amplicons for pheasant and turkey, 278 bp amplicons for
103
chicken, guinea fowl and goose, 280 bp and 281 bp amplicons for duck and muscovy duck,
AC C
EP
94
4
ACCEPTED MANUSCRIPT 104
respectively. Primers were tested by Oligoanalyzer software (Integrated DNA Technologies,
105
Inc.) for hairpin, self-dimer and hetero-dimer structures.
153 154
155
ACTCTAAGGACTTGGCGGTG.............................A.......... .................................................A...C...... .................................................A.......... .......................T.........................G.......... .................................................AC......... .......................T.........................G........G. .......................T.........................G..........
Chicken Guinea fowl Pheasant Turkey Goose Duck Muscovy duck
FJ610339.1| FN675566.1| U83742.1| AJ490508.1| JN695752.1| JN695762.1| AM902523.1|
........T...................-.G............................. .....................T......-............................... ............................-............................... .....................T......-............................... ...C....TA..................-............................... .........A........G.........A.G............................. C........A..................-..A............................
Chicken Guinea fowl Pheasant Turkey Goose Duck Muscovy duck
FJ610339.1| FN675566.1| U83742.1| AJ490508.1| JN695752.1| JN695762.1| AM902523.1|
.....T.....A..A............T..----ATAGC...T......T.......... ....AC.TGA.AGCGCA.CAG-....-CTC----AACAGT....A....C.......... ....AA.TGA.AG..CA.CAGTGAGC-T..----ACAGT..A..A...GC.......... ....AA.....A..AT...T......-CTC----AATAGT....A....C.......... .....G.....G............GA.A...T..C---..-........T.......... .....G.....G...G........G..G......C---..-........T.......... .....G.....G...............G......C---...........T..........
Chicken Guinea fowl Pheasant Turkey Goose Duck Muscovy duck
FJ610339.1| FN675566.1| U83742.1| AJ490508.1| JN695752.1| JN695762.1| AM902523.1|
...........T.........G......................CA.....C.A.--CGA ...........C...A...........................GCA.....C.CTCACGA ...........C................................TA.....T.A.--CGA ...........C...A............................CA.....C.G.--CGA ...........T....A.....................CC...TTCATAG--GGCA.ACG ...........T.....AC...................CC...TGCA-T.GGGC.A.ACG ...........C......C...................CC...CACACT.GGGC.G.ACG
Chicken Guinea fowl Pheasant Turkey Goose Duck Muscovy duck
FJ610339.1| FN675566.1| U83742.1| AJ490508.1| JN695752.1| JN695762.1| AM902523.1|
.....G.CG.GA..C......T.A.AAGGAGGATTTAGCAGTAAA .....AGCA.GA..C.T....T.A..................... .....G.CG.GA..C...T..T.G..................... .....GGCG.GA..CT.....T.G..................... G....A.GCGTG..A..A.TTC.G.....C............... G....A.GTATG..A.T..TTC.A..................... .....A.GCATG..A.T..TTC.G.....................
TE D
M AN U
SC
RI PT
FJ610339.1| FN675566.1| U83742.1| AJ490508.1| JN695752.1| JN695762.1| AM902523.1|
Figure 1. Multiple sequence alignment for 12S rRNA of seven poultry species
EP
152
Chicken Guinea fowl Pheasant Turkey Goose Duck Muscovy duck
AC C
106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130 131 132 133 134 135 136 137 138 139 140 141 142 143 144 145 146 147 148 149 150 151
156
Figure 2. Phylogenetic tree of seven poultry species based on 12S rRNA target sequence Phylogenetic tree was generated using the
157
neighbor-joining method by CLUSTALW2 PHYLOGENY algorithm of EBI
158 159
5
ACCEPTED MANUSCRIPT 160
2.3 Polymerase chain reaction with universal primers
161 The PCR amplification of DNA from meat samples was performed in 30 µl volume
163
containing 1x Dream Taq Buffer (Fermentas), 200 µM dNTP mix (Fermentas), 4 mM MgCl2
164
(Promega), 0.1 µM forward primer (5’-ACTCTAAGGACTTGGCGGTG-3’) (Sigma-
165
Aldrich), 0.1 µM reverse primer (5’-TTTACTGCTAAATCCTCCTT-3’) (Sigma-Aldrich),
166
1.5 U Dream Taq polymerase (Fermentas) and 150 ng DNA template. PCR was carried out in
167
a thermal cycler PTC-200 (Bio-Rad, USA). Amplification protocol was as follows: first step
168
is denaturation at 95 °C for 1.5 min, followed by 35 cycles consisting of denaturation at 95 °C
169
for 30 sec, primer annealing at 60 °C for 30 sec and extension at 72 °C for 30 sec. The final
170
extension step was 5 min at 72 °C. Amplified PCR products were stained by Ethidium
171
Bromide (Bio-Rad, USA) and were analyzed by electrophoresis in 2% agarose gel (Lonza) for
172
1 h with 10 V/cm in TAE (Tris-Acetate-EDTA, pH:8) (Lonza) buffer.
173
According to the CE application, primers were labeled with fluorescent dyes (Life
174
Technologies). Forward primer was 5’-labeled with 6-carboxyfluorescein (6-FAM) (5’-
175
ACTCTAAGGACTTGGCGGTG-3’). Reverse primer was 5’-labeled with VIC (5’-
176
TTTACTGCTAAATCCTCCTT-3’). The PCR mixtures consisted of 1x DreamTaq Buffer
177
(Fermentas), 4 mM MgCl2 (Promega), 200 µM of each dNTP (Fermentas), 0.1 µM of each
178
labeled primers (Sigma-Aldrich), 0.05 U/µl of DreamTaq DNA Polymerase (Fermentas) and
179
150 ng of template DNA. The steps of the PCR reaction were the same, as described above.
181
SC
M AN U
TE D
EP
AC C
180
RI PT
162
2.4 PCR-single strand conformation polymorphism (PCR-SSCP)
182 183
Amplified PCR products were diluted in denaturing solution containing 90 v/v% formamide-
184
dye (8 v/v% bromophenol blue stain (Sigma-Aldrich); 92 v/v% formamide). The solution was
6
ACCEPTED MANUSCRIPT heat-denatured at 95 °C for 5 min and chilled immediately on ice. Denatured DNA fragments
186
were loaded onto a 10% acrylamide:bis-acrylamide (37.5:1) non-denaturing polyacrylamide
187
gel (20 cm × 16 cm × 0.75 mm). Polyacrylamide gel electrophoresis was performed at 4 °C
188
for 8 h with 25 V/cm on a Protean II xi Cell vertical format electrophoresis system (Bio-Rad,
189
USA) with a Power Pac Universal Power Supply (Bio-Rad, USA).
190
After electrophoresis, the polyacrylamide gel was stained using the silver staining method
191
according to Merril et al. (1984) and documented with the Uvipro Platinum (Uvitec) gel
192
documentation system.
SC
RI PT
185
193
2.5 Sample preparation and conditions of capillary electrophoresis-single strand
195
conformation polymorphism (CE-SSCP)
M AN U
194
196
CE was performed on an ABI Prism 310 Genetic Analyzer (Life Technologies), equipped
198
with an argon-ion laser, light emitted at 488 – 514 nm. Samples were electro-kinetically-
199
injected at 15 kV for 10 sec to a 47 cm (effective length: 30 cm) long diameter 50 µm (Life
200
Technologies) capillary filled with 15 wt% solution of Pluronic F108 polymer (Sigma-
201
Aldrich), containing 0.7x Genetic Analyzer buffer (Life Technologies). Electrophoresis was
202
performed at 35 °C with 15 kV. Signal detection was used in all separations between 525-650
203
nm wavelengths. Results were collected using the Data Collection Software Version 3.1.0
204
program, and were analyzed with GeneMapper® 3.7 (Life Technologies) software. To correct
205
run-to-run variations, electropherograms were normalized by fixing the positions of peaks
206
produced by GeneScanTM 500 LIZTM dye Size Standard (Life Technologies). Samples were
207
prepared for capillary electrophoresis analysis as follows: 0.5 µl PCR product + 0.5 µl 500
208
LIZTM dye + 9 µl of deionized Hi-Di formamide (Life Technologies) were mixed, then
209
denatured at 95°C for 3 minutes followed by quick cooling on ice. Electrophoresis was
AC C
EP
TE D
197
7
ACCEPTED MANUSCRIPT 210
performed at 35 °C, using 15 kV electrophoresis voltage with 5 sec injection time and 40 min
211
electrophoresis running time.
212 213
2.6 DNA sequencing
RI PT
214 The amplified fragments of 12S rRNA gene were purified using the PCR Advanced™ PCR
216
Clean Up System (Viogene, Taiwan) according to the manufacturer’s instructions. The
217
purified PCR amplicons were sequenced by Macrogen Europe Inc. in Amsterdam, The
218
Netherlands. Obtained sequence data were aligned with sequences from the NCBI GenBank
219
database.
220 221
3. Results
222
224
3.1 Polymerase chain reaction
TE D
223
M AN U
SC
215
Primers were designed to amplify a 277-285 bp region of mitochondrial 12S rRNA of poultry
226
species. Following PCR reaction, amplicons were separated on agarose gel using 50 bp
227
GeneRulerTM DNA ladder (Life Technologies). PCR products of each species were of sharp,
228
good quality and no artifacts were detected at PCR with either unlabeled nor labeled primers.
230 231
AC C
229
EP
225
3.2 PCR-single strand conformation polymorphism (PCR-SSCP)
232 233
In this study, PCR-SSCP and CE-SSCP were tested using the DNA of seven poultry species,
234
chicken, turkey, duck, muscovy duck, goose, guinea fowl and pheasant and a mixture of
8
ACCEPTED MANUSCRIPT chicken and duck DNA samples. The applicability of these methods was tested using meat
236
products.
237
Double-stranded PCR amplicons were transformed into single-stranded conformers with
238
unique secondary structure after denaturation. In the case of the PCR-SSCP method, single-
239
stranded DNA molecules were separated on polyacrylamide gel, in contrast to CE-SSCP,
240
where running of denaturated amplicons took place in a polymer matrix. Species-specific
241
conformations were visualized by silver staining (Merril et al., 1984). With SSCP analysis,
242
each poultry species had distinct patterns and could be differentiated from each other (Figure
243
3). Polyacrylamide gel electrophoresis resulted in no false positives.
244
Chicken-duck DNA mixtures were tested, to determine the sensitivity of this method. Duck
245
DNA samples contained chicken DNA in 20; 10; 5; 1; 0.5%, respectively (Figure 4). Positive
246
control samples contained chicken and duck DNA in 100%. The detection threshold of PCR-
247
SSCP method was 0.5% presence of chicken DNA in the chicken-duck DNA mixture (Table
248
1).
249
The applicability of the PCR-SSCP method was tested on 36 commercial meat products, such
250
as sausages, salamis and frankfurters, as shown in Table 2. Chicken, turkey, duck and goose
251
DNA were used as positive controls on polyacrylamide gel. Species-specific patterns were
252
obtained in these products and the presence of undeclared species was detected in six cases,
253
that is 16.67% of all the analyzed samples. 35.7% of indicated turkey meat products (5 of 14
254
meats) contained turkey and chicken DNA. 12.5% of meat products labeled as chicken meat
255
(1 of 8 meats) contained turkey and chicken DNA. 83.4% of all meat products, 30 of 36,
256
exclusively contained indicated species; consequently, fraud was not detected by PCR-SSCP
257
(Table 2).
AC C
EP
TE D
M AN U
SC
RI PT
235
9
RI PT
ACCEPTED MANUSCRIPT
258
SC
Figure 3. PCR-SSCP pattern of 12S rRNA of seven poultry species. 1.: duck; 2.: muscovy duck; 3.: chicken; 4.: goose; 5.: guinea fowl; 6.: pheasant; 7.: turkey.
Figure 4. Electrophoretic analysis of PCR-SSCP from chicken and duck meat DNA. 1.: chicken; 2.: duck; 3, 4, 5, 6 and 7 DNA mixture of chicken and duck DNA containing 20, 10, 5, 1, 0.5% of chicken DNA, respectively. Table 1. Determination of detection limit of PCR-SSCP and CE-SSCP methods analyzing 10 DNA mixtures containing 0.5% and 99.5%, and 10 DNA mixtures containing 0.1% and 99.9% chicken and duck DNA, respectively.
AC C
263 264 265 266 267 268
EP
262
TE D
M AN U
259 260 261
Number of detectable chicken specific DNA band by PCR-SSCP/ all samples Number of detectable chicken specific DNA band by CE-SSCP/ all samples
0.5% chicken and 99.5% duck
0.1% chicken and 99.9% duck
10/10
0/10
10/10
0/10
269 270
3.3 Capillary electrophoresis-single strand conformation polymorphism (CE-SSCP)
271
10
ACCEPTED MANUSCRIPT PCR products were heat denatured and prepared for CE-SSCP analysis. Fluorescently labeled
273
primers were used to detect the unique single strand conformers. Seven poultry species were
274
distinguished from each other, which demonstrates that our fluorescently labeled universal
275
primers are capable of use for pattern recognition. Two single strand conformers appeared for
276
each poultry species. Species-specific patterns are shown on Figure 5.
277
The sensitivity of the CE-SSCP method for analyzing the chicken-duck DNA mixture,
278
containing decreasing concentrations of chicken DNA, was determined in the same way as
279
was set for PCR-SSCP (Figure 6). The detection threshold of chicken was 0.5% DNA in the
280
chicken-duck DNA mixture, similar to the PCR-SSCP method (Table 1).
281
The applicability of the CE-SSCP was tested on the same commercial meat products used in
282
the PCR-SSCP analyses, and the same chicken, turkey, duck and goose positive control
283
samples were used in the CE-SSCP analysis, as well. After CE, species-specific patterns were
284
obtained and the presence of undeclared species were detected in six products, similarly to the
285
findings when using PCR-SSCP, which is 16.67% of commercial products included in this
286
study. All of the 6 fraudulent findings revealed the presence of chicken, as this was the non-
287
labeled species in the “poultry-duck” meat product. The presence of a PCR conformer
288
reflecting turkey was identified, in contrast to PCR-SSCP, where only duck and chicken
289
patterns were observed. We found an additional VIC labeled conformer in the same poultry
290
meat product, but it was not identical to any of the 7 bird species. No fraud was detected in
291
the rest of meat products using the CE-SSCP method (Table 2).
SC
M AN U
TE D
EP
AC C
292
RI PT
272
293 294 295
3.4 DNA sequencing
296
11
ACCEPTED MANUSCRIPT We performed sequence alignment (EBI, CLUSTAL OMEGA algorithm) with sequences
298
from the NCBI GenBank database and sequenced DNA regions from the meat products. We
299
established that one product, labeled as turkey, contained turkey and also chicken DNA.
300
Thymine (turkey) and cytosine (chicken) bases were found at the 56th position of the
301
sequenced 12S rRNA amplicons. The presence of turkey and chicken DNA were verified with
302
PCR-SSCP and CE-SSCP methods as well (Figure 7).
303
← VIC
data points
TE D
RFU
B Numida meleagris (Guinea fowl)
6-FAM →
304
SC
← 6-FAM
M AN U
RFU
A Gallus gallus domestica (Chicken)
← VIC
EP
data points
305
AC C
RFU
C Phasianus colchicus (Pheasant)
6-FAM →
RI PT
297
← VIC
data points
12
ACCEPTED MANUSCRIPT
RFU
D Meleagris galoppavo (Turkey)
306
← VIC
RI PT
6-FAM →
data points
← VIC
307
M AN U
6-FAM →
SC
RFU
E Anser anser domestica (Goose)
F Anas platyrhynchos domestica (Duck)
TE D
RFU
data points
6-FAM →
308
← VIC
EP
data points
AC C
RFU
G Cairina moschata (Muscovy duck)
6-FAM →
309
← VIC
data points
13
ACCEPTED MANUSCRIPT
310
data points
Figure 5. Species-specific patterns of 12S rRNA of seven poultry species on capillary electrophoresis electropherograms: Chicken (A), Guinea fowl (B), Pheasant (C), Turkey (D), Goose (E), Duck (F), Muscovy duck (G), No Template Control (H). GeneScanTM 500 LIZTM dye Size Standard is shown as reference peaks (pale gray), 6-FAM and VIC labeled strands are shown as gray and dark gray peaks, respectively. Y- and X-axis shows relative fluorescence unit (RFU) and data points (1 data point is equal with 220 msec migration time), respectively.
SC
311 312 313 314 315 316
RI PT
RFU
H No Template Control
M AN U
RFU
A Chicken control C→ 6-FAM
CVIC
317 318
→
data points
RFU
TE D
B Duck control D→ 6-FAM
321 322
AC C
RFU
C 80% Duck, 20% Chicken
data points
EP
319 320
C→ 6-FAM
D→ 6-FAM
DVIC
CVIC
DVIC
→
→
data points
RFU
D 90% Duck, 10% Chicken
323 324
C→ 6-FAM
D→ 6-FAM
CVIC
DVIC
→
data points
14
→
→
ACCEPTED MANUSCRIPT
RFU
E 95% Duck, 5% Chicken C→ 6-FAM
D→ 6-FAM
325 326
CVIC
DVIC
→
→
CD- → 6-FAM 6-FAM ↓
327 328
→
M AN U
RFU
CD- → 6-FAM 6-FAM ↓
329 330
CVIC ↓
DVIC
→
data points
TE D
RFU
H No Template Control
EP
data points
Figure 6. Mixture of duck and chicken 12S rRNA samples. 100% Chicken; C (A), 100% Duck; D (B). Percentage of the DNA mixtures: 80% Duck, 20% Chicken (C), 90% Duck, 10% Chicken (D), 95% Duck, 5% Chicken (E), 99% Duck, 1% Chicken (F), 99.5% Duck, 0.5% Chicken (G), No Template Control (H). Y- and X-axis shows relative fluorescence unit (RFU) and data points (1 data point is equal with 220 msec migration time), respectively.
AC C
339
DVIC
data points G 99.5% Duck, 0.5% Chicken
331 332 333 334 335 336 337 338
CVIC ↓
SC
RFU
F 99% Duck, 1% Chicken
RI PT
data points
15
ACCEPTED MANUSCRIPT A
B
C
T
RI PT
Ch
340
Figure 7. Meat product was labeled as made of turkey meat, sequencing, PCR-SSCP and CE-SSCP proved the fraud, as it contained chicken DNA as well. The presence of turkey (Thymine) and chicken (Cytosine) DNA were confirmed by DNA sequencing (A); PCR-SSCP, from left turkey control (T), chicken control (Ch), meat product (M) (B); and CE-SSCP, turkey (T), chicken (Ch) (C).
SC
341 342 343 344 345
347
Table 2. Identification of species in commercial meat products with PCR-SSCP and CE-SSCP methods.
Label (species)
Number of meat products
9
Chicken
8
Duck, poultry
349
EP
AC C
Poultry
Detected species by
PCR-SSCP method
CE-SSCP method
7 chicken
1 chicken+turkey
14
Turkey, chicken, poultry Turkey, poultry
Detected species by
9 turkey+chicken
TE D
Turkey, chicken
Turkey
348
M AN U
346
9 turkey 5 turkey+chicken
1
1 turkey+chicken
2
2 turkey+chicken
1
1 chicken
1
1 chicken+duck
1 turkey+chicken+duck
4. Discussion
350 351
In our study, we were able to identify seven poultry species by polymerase chain reaction-
352
single strand conformation polymorphism (PCR-SSCP) and capillary electrophoresis-single
353
strand conformation polymorphism (CE-SSCP) methods, applied to analyze commercially 16
ACCEPTED MANUSCRIPT available poultry products. In the last decades, the importance of identification of food species
355
and the composition of foods has increased. Species identification DNA methods are based on
356
nuclear and mtDNA markers. In meat species identification, mtDNA regions are frequently
357
used. Design of universal primers for the simultaneous amplification of poultry species played
358
a critical role. Commonly used mitochondrial regions have strong conservative sequences,
359
despite the diversity of species, which are capable for use in the design of universal primers,
360
and also have variable regions, which can be used to differentiate species (Horreo et al.,
361
2013). In the literature, one of the most commonly used mitochondrial regions is the 12S
362
rRNA (Stamoulis et al., 2010; Rojas et al., 2012). Among the PCR-based species detection
363
methods, SSCP is a cost effective and rapid technique (Orita et al., 1989; Hayashi K., 1991)
364
and has the ability to detect single nucleotide polymorphisms (Oohara I., 1997). Due to this
365
advantage, the SSCP method can be effective and appropriate for poultry species
366
identification. Studies using CE-SSCP focused on medical fields, such as pathogen
367
identification, use 16S rRNA for the separation of microorganisms (Chung et al., 2007; Shin
368
et al., 2010). CE-based techniques were developed for food authentication in the last decades,
369
such as in fish (Dooley et al., 2005), and in meat and seafood (Rodríguez-Ramírez et al.,
370
2011).
371
In our work, we compared PCR-based SSCP and CE-SSCP methods with the identification of
372
seven poultry species (Gallus gallus domestica, Numida meleagris, Phasianus colchicus,
373
Meleagris galoppavo, Anser anser domestica, Anas platyrhynchos domestica, Cairina
374
moschata). Designed 12S rRNA primers successfully amplified the poultry DNA in each
375
species. DNA patterns were distinguished successfully using both applications. Sensitivity of
376
methods was tested with DNA mixtures using duck and chicken. The detection limit of
377
chicken DNA in the duck-chicken mixture was 0.5% for PCR-SSCP and CE-SSCP as well,
378
which is equal to 0.75 ng chicken DNA, as the total DNA were 150 ng in the PCR reaction. In
AC C
EP
TE D
M AN U
SC
RI PT
354
17
ACCEPTED MANUSCRIPT this case, the sensitivity of both methods was the same, unlike in some other studies. Ru et al.
380
(2000) have described that the accuracy of CE-SSCP in the detection of colon tumor is better,
381
than conventional SSCP. Andersen et al., (2003) have reported, that capillary array
382
electrophoresis-SSCP (CAE-SSCP) has increased sensitivity, compared to traditional SSCP.
383
López-Calleja et al. (2007) detected 1% cows’ milk in sheep’s and goats’ milk cheeses,
384
Colgan et al. (2001) proved the presence of 0.3% bovine and ovine species and the presence
385
of 1% of porcine species in meat and bone meal with species specific PCR. Rodríguez et al.
386
(2005) have detected pork in pork-beef DNA mixtures in the range of 0.5-5% with real-time
387
PCR assay. Another study referred that 2% cow milk is detectable in buffalo milk (Dalmasso
388
et al., 2011) and with species-specific real-time PCR, 1% of target species is able to be
389
detected in the mixture (Cammà et al., 2012). In multiplex PCR reaction, the detection limits
390
of goats’, sheep’s and cows’ milk were 0.5% in dairy products (Bottero et al., 2003a). With
391
the PCR-RFLP method, sensitivity was 1% for pig presence among the studied species (Partis
392
et al., 2000). Species-specific PCR techniques have the most effective sensitivity, where the
393
detection limit is even 0.25 ng DNA for all meat samples (Matsunaga et al., 1999). The
394
sensitivity threshold is 0.1 % for the DNA detection of cow in water buffalo milk and
395
mozzarella cheese (López-Calleja et al., 2005) and, in the case of ruminant species, in raw and
396
heat treated foodstuffs (Martín et al., 2007). Compared with these studies, sensitivity of PCR-
397
SSCP due to the silver staining method is very high, around 1 pg/mm2 (Bassam et al., 1991).
398
CE-SSCP has the same sensitivity, because of laser-induced detection (Albin et al., 1991).
399
These detection limit values are suitable for fraud detection.
400
In our study, 36 commercially available poultry products were tested and 6 of them contained
401
undeclared species.
AC C
EP
TE D
M AN U
SC
RI PT
379
402 403
18
ACCEPTED MANUSCRIPT 404
5. Conclusions
405 In summary, a polymerase chain reaction-single strand conformation polymorphism (PCR-
407
SSCP) and capillary electrophoresis-single strand conformation polymorphism (CE-SSCP)
408
methods were developed to identify seven poultry species. A reliable detection limit was 0.5%
409
DNA for both applications, which reflect 0.75 ng DNA. 36 commercially available poultry
410
products were analyzed using the PCR-SSCP and CE-SSCP techniques, on the bases of which
411
we identified 6 products with undeclared species. In conclusion, the sensitivity,
412
reproducibility and reliability of PCR-SSCP and CE-SSCP are close to each other, cost
413
efficiency is better in the case of PCR-SSCP. On the other hand, the latter is more labor-
414
intensive and the environmental impact is higher, than for CE-SSCP. Both methods proved to
415
be appropriate tools for poultry species identification in raw meat and processed meat
416
products.
M AN U
SC
RI PT
406
TE D
417 Acknowledgements
419
This work was supported by the TÁMOP-4.1.1.C-12/1/KONV-2012-0014 project, Hungary.
420 421
423
References
AC C
422
EP
418
424 425
Albin, M., Weinberger, R., Sapp, E., & Moring, S. (1991). Fluorescence detection in capillary electrophoresis: evaluation of derivatizing reagents and techniques. Analytical Chemistry, 63(5), 417-422.
426 427 428
Andersen, P. S., Jespersgaard, C., Vuust, J., Christiansen, M., & Larsen, L. A. (2003). High‐throughput single strand conformation polymorphism mutation detection by automated capillary array electrophoresis: validation of the method. Human mutation, 21(2), 116-122.
429 430
Arslan, A., Ilhak, O. I., & Calicioglu, M. (2006). Effect of method of cooking on identification of heat processed beef using polymerase chain reaction (PCR) technique. Meat Science, 72(2), 326-330.
431 432
Bassam, B. J., Caetano-Anollés, G., & Gresshoff, P. M. (1991). Fast and sensitive silver staining of DNA in polyacrylamide gels. Analytical Biochemistry, 196(1), 80-83.
19
ACCEPTED MANUSCRIPT Borgo, R., Souty-Grosset C., Bouchon, D., & Gomot, L. (1996). PCR‐RFLP Analysis of Mitochondrial DNA for Identification of Snail Meat Species. Journal of Food Science, 61(1), 1-4.
435 436 437
Bottero, M., Civera, T., Nucera, D., Rosati, S., Sacchi, P., & Turi, R. (2003a). A multiplex polymerase chain reaction for the identification of cows’, goats’ and sheep's milk in dairy products. International Dairy Journal, 13(4), 277-282.
438 439 440
Bottero, M., Dalmasso, A., Nucera, D., Turi, R., Rosati, S., Squadrone, S., Goria, M., & Civera, T. (2003b). Development of a PCR assay for the detection of animal tissues in ruminant feeds. Journal of Food Protection®, 66(12), 2307-2312.
441 442
Calvo, J. H., Zaragoza, P., & Osta, R. (2001a). Random amplified polymorphic DNA fingerprints for identification of species in poultry pate. Poultry science, 80(4), 522-524.
443 444 445
Calvo, J., Zaragoza, P., & Osta, R. (2001b). Technical note: A quick and more sensitive method to identify pork in processed and unprocessed food by PCR amplification of a new specific DNA fragment. JOURNAL OF ANIMAL SCIENCE-MENASHA THEN ALBANY THEN CHAMPAIGN ILLINOIS-, 79(8), 2108-2112.
446 447
Cammà, C., Di Domenico, M., & Monaco, F. (2012). Development and validation of fast real-time PCR assays for species identification in raw and cooked meat mixtures. Food Control, 23(2), 400-404.
448 449
Chen, F., & Hsieh, Y. (2000). Detection of pork in heat-processed meat products by monoclonal antibody-based ELISA. Journal of AOAC International, 83(1), 79-85.
450 451 452
Chung, J. H., Park, Y. S., Kim, J., Shin, G. W., Nam, M. H., Oh, M., Kim, C. W., Jung, G. Y., & Hyun Park, J. (2007). Parallel analysis of antimicrobial activities in microbial community by SSCP based on CE. Electrophoresis, 28(14), 2416-2423.
453 454
Colgan, S., O’brien, L., Maher, M., Shilton, N., McDonnell, K., & Ward, S. (2001). Development of a DNAbased assay for species identification in meat and bone meal. Food Research International, 34(5), 409-414.
455
Cotton, R. G. (1997). Slowly but surely towards better scanning for mutations. Trends in genetics, 13(2), 43-46.
456 457
Craig, A., Ritchie, A., & Mackie, I. (1995). Determining the authenticity of raw reformed breaded scampi (Nephrops norvegicus) by electrophoretic techniques. Food Chemistry, 52(4), 451-454.
458 459
Dalmasso, A., Civera, T., La Neve, F., & Bottero, M. T. (2011). Simultaneous detection of cow and buffalo milk in mozzarella cheese by real-time PCR assay. Food Chemistry, 124(1), 362-366.
460 461
Dalmasso, A., Fontanella, E., Piatti, P., Civera, T., Rosati, S., & Bottero, M. (2004). A multiplex PCR assay for the identification of animal species in feedstuffs. Molecular and cellular probes, 18(2), 81-87.
462 463 464
De, S., Brahma, B., Polley, S., Mukherjee, A., Banerjee, D., Gohaina, M., Singh, K. P., Singh, R., Datta, T. K., & Goswami, S. L. (2011). Simplex and duplex PCR assays for species specific identification of cattle and buffalo milk and cheese. Food Control, 22(5), 690-696.
465 466
Dooley, J. J., Sage, H. D., Brown, H. M., & Garrett, S. D. (2005). Improved fish species identification by use of lab-on-a-chip technology. Food Control, 16(7), 601-607.
467 468
Ebbehøj, K. F., & Thomsen, P. D. (1991a). Species differentiation of heated meat products by DNA hybridization. Meat Science, 30(3), 221-234.
469 470
Ebbehøj, K. F., & Thomsen, P. D. (1991b). Differentiation of closely related species by DNA hybridization. Meat Science, 30(4), 359-366.
471 472
Fajardo, V., González, I., López-Calleja, I., Martín, I., Hernández, P. E., García, T., & Martín R. (2006). PCRRFLP authentication of meats from red deer (Cervus elaphus), fallow deer (Dama dama), roe deer (Capreolus
AC C
EP
TE D
M AN U
SC
RI PT
433 434
20
ACCEPTED MANUSCRIPT capreolus), cattle (Bos taurus), sheep (Ovis aries), and goat (Capra hircus). Journal of Agricultural and Food Chemistry, 54(4), 1144-1150.
475 476
Gasser, R. B. (1997). Mutation scanning methods for the analysis of parasite genes. International journal for parasitology, 27(12), 1449-1463.
477 478
Girish, P., Anjaneyulu, A., Viswas, K., Anand, M., Rajkumar, N., Shivakumar, B., & Bhaskar, S. (2004). Sequence analysis of mitochondrial 12S rRNA gene can identify meat species. Meat Science, 66(3), 551-556.
479 480
Greenwood, A. D., & Pääbo, S. (1999). Nuclear insertion sequences of mitochondrial DNA predominate in hair but not in blood of elephants. Molecular ecology, 8(1), 133-137.
481 482
Hayashi, K. (1991). PCR-SSCP: a simple and sensitive method for detection of mutations in the genomic DNA. PCR methods and applications, 1(1), 34-38.
483 484 485
Horreo, J. L., Ardura, A., Pola, I. G., Martinez, J. L., & Garcia‐Vazquez, E. (2013). Universal primers for species authentication of animal foodstuff in a single polymerase chain reaction. Journal of the science of food and agriculture, 93(2), 354-361.
486 487
Hunt, D. J., Parkes, H. C. & Lumley, I. D. (1997). Identification of the species of origin of raw and cooked meat products using oligonucleotide probes. Food Chemistry, 60(3), 437-442.
488 489
King, N., & Kurth, L. (1982). Analysis of raw beef samples for adulterant meat species by enzyme‐staining of isoelectric focusing gels. Journal of Food Science, 47(5), 1608-1612.
490 491 492
Kocher, T. D., Thomas, W. K., Meyer, A., Edwards, S. V., Paabo, S., Villablanca, F. X., & Wilson, A. C. (1989). Dynamics of mitochondrial DNA evolution in animals: amplification and sequencing with conserved primers. Proceedings of the National Academy of Sciences of the United States of America, 86(16), 6196-6200.
493 494 495
López-Calleja, I. M., González, I., Fajardo, V., Hernandez, P. E., García, T., & Martín, R. (2007). Application of an indirect ELISA and a PCR technique for detection of cows’ milk in sheep's and goats’ milk cheeses. International Dairy Journal, 17(1), 87-93.
496 497 498
López-Calleja, I., Alonso, I. G., Fajardo, V., Rodríguez, M., Hernández, P., García, T., & Martín, R. (2005). PCR detection of cows’ milk in water buffalo milk and mozzarella cheese. International Dairy Journal, 15(11), 1122-1129.
499 500
Man, Y. C., Aida, A., Raha, A., & Son, R. (2007). Identification of pork derivatives in food products by speciesspecific polymerase chain reaction (PCR) for halal verification. Food Control, 18(7), 885-889.
501 502
Mane, B., Tanwar, V., Girish, P., & Dixit, V. (2006). Identification of species origin of meat by RAPD–PCR technique. Journal of Veterinary Public Health, 4(2), 87-90.
503 504
Martín, I., García, T., Fajardo, V., López-Calleja, I., Hernández, P. E., González, I., & Martín, R. (2007). Species-specific PCR for the identification of ruminant species in feedstuffs. Meat Science, 75(1), 120-127.
505 506 507
Matsunaga, T., Chikuni, K., Tanabe, R., Muroya, S., Shibata, K., Yamada, J., & Shinmura, Y. (1999). A quick and simple method for the identification of meat species and meat products by PCR assay. Meat Science, 51(2), 143-148.
508 509
Merril, C. R., Goldman, D., & Van Keuren, M. L. (1984). [30] Gel protein stains: Silver stain. Methods in enzymology, 104, 441-447.
510 511 512
Meyer, R., Hofelein, C., Luthy, J., & Candrian, U. (1995). Polymerase chain reaction-restriction fragment length polymorphism analysis: a simple method for species identification in food. Journal of AOAC International, 78(6), 1542-1551.
AC C
EP
TE D
M AN U
SC
RI PT
473 474
21
ACCEPTED MANUSCRIPT Montiel-Sosa, J., Ruiz-Pesini, E., Montoya, J., Roncales, P., López-Pérez, M., & Pérez-Martos, A. (2000). Direct and highly species-specific detection of pork meat and fat in meat products by PCR amplification of mitochondrial DNA. Journal of Agricultural and Food Chemistry, 48(7), 2829-2832.
516 517
Mullis, K. B., Faloona, F. A. (1987) Specific synthesis of DNA in vitro via a polymerase-catalyzed chain reaction. Methods Enzymol. 155, 335-50
518 519 520
Oohara, I. (1997). Detection of single strand conformation polymorphisms (SSCPs) on mitochondrial DNA fragments between two domesticated strains of rainbow trout Oncorhynchus mykiss. Fisheries science, 63(1), 151-152.
521 522
Orita, M., Suzuki, Y., Sekiya, T., & Hayashi, K. (1989). Rapid and sensitive detection of point mutations and DNA polymorphisms using the polymerase chain reaction. Genomics, 5(4), 874-879.
523 524
Partis, L., Croan, D, Guo, Z., Clark, R., Coldham, T., & Murby, J. (2000). Evaluation of a DNA fingerprinting method for determining the species origin of meats. Meat Science, 54(4), 369-376.
525 526
Rodríguez, M. A., García, T., González, I., Hernández, P. E., & Martín, R. (2005). TaqMan real-time PCR for the detection and quantitation of pork in meat mixtures. Meat Science, 70(1), 113-120.
527 528 529
Rodríguez-Ramírez, R., González-Córdova, A. F., & Vallejo-Cordoba, B. (2011). Review: Authentication and traceability of foods from animal origin by polymerase chain reaction-based capillary electrophoresis. Analytica Chimica Acta, 685(2), 120-126.
530 531 532
Rojas, M., González, I., García, T., Hernández, P. E., & Martín, R. (2012). Authentication of meat and commercial meat products from common pigeon (Columba livia) woodpigeon (Columba palumbus) and stock pigeon (Columba oenas) using a TaqMan® real-time PCR assay. Food Control, 23(2), 369-376.
533 534
Rokas, A., Ladoukakis, E., & Zouros, E. (2003). Animal mitochondrial DNA recombination revisited. Trends in Ecology & Evolution, 18(8), 411-417.
535 536 537
Ru, Q., Jing, H., Luo, G., & Huang, Q. (2000). Single-strand conformation polymorphism analysis to detect the p53 mutation in colon tumor samples by capillary electrophoresis. Journal of Chromatography A, 894(1), 171177.
538 539
Sawyer, J., Wood, C., Shanahan, D., Gout, S., & McDowell, D. (2003). Real-time PCR for quantitative meat species testing. Food Control, 14(8), 579-583.
540 541
Shin, G. W., Hwang, H. S., Oh, M., Doh, J., & Jung, G. Y. (2010). Simultaneous quantitative detection of 12 pathogens using high‐resolution CE‐SSCP. Electrophoresis, 31(14), 2405-2410.
542 543
Stamoulis, P., Stamatis, C., Sarafidou, T., & Mamuris, Z. (2010). Development and application of molecular markers for poultry meat identification in food chain. Food Control, 21(7), 1061-1065.
544 545 546
Zhang, C., Fowler, M. R., Scott, N. W., Lawson, G., & Slater, A. (2007). A TaqMan real-time PCR system for the identification and quantification of bovine DNA in meats, milks and cheeses. Food Control, 18(9), 11491158.
AC C
EP
TE D
M AN U
SC
RI PT
513 514 515
22