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FRIN-05769; No of Pages 6

Food Research International xxx (2015) xxx–xxx

Contents lists available at ScienceDirect

Food Research International

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

Potential DNA markers as a rapid tracing tool for animal adulterants in


vegetarian food
Xiao Mi 1, Jie Yang 1, Lichao Cao, Xiaofeng Wei, Ying Zhu, Qiyuan Li, Xiaopan Liu, Xuheng He,
Qiuyan Liao, Zhixiang Yan ⁎
Beijing Genomics Institute (BGI)—Shenzhen, Beishan Industrial Zone, Yantian District, Shenzhen 518083, China

a r t i c l e i n f o a b s t r a c t

Article history: DNA-based methods are rapid, cost-effective and broadly applicable approaches for food authentication. Recent-
Received 11 July 2014 ly, the requirements for food safety and food integrity have increased with improved quality of life. Methodolo-
Received in revised form 31 March 2015 gies regarding food authentication based on DNA analysis are more commonly being used. With the increasing
Accepted 2 April 2015
number of vegetarians, searching for markers for blind identification across kingdom species, such as an ingredi-
Available online xxxx
ent of animal origin in vegetarian food, would be valuable and attractive. Using bioinformatic analysis of an
Keywords:
existing data source composed of 481 ultraconserved sequences, we selected 6 new candidate DNA segments
DNA marker that exist in most vertebrates but that do not exist in plants. Then, primers were designed for all of the candidate
Vegetarian adulteration DNA markers, and DNA samples isolated from cow, pig, chicken, duck, soy bean, rice, pepper, wheat, sunflower
Animal ingredient and colza were amplified using each primer pair. None of the plant DNA samples generated a PCR product,
Food safety while the DNA samples of animal origin were amplified successfully using 5 of the candidate segment primers;
the 6th segment primer failed to amplify the DNA and was discarded. Moreover, a simulation experiment con-
taining a plant product contaminated by an animal component indicated that the candidate DNA markers can
be used for the rapid detection of animal adulterants in vegetarian products with a promising 5% detection
limit. The identified candidate DNA markers for the blind identification of animal adulteration in vegetarian
food may be highly desirable in the vegetarian food market, and these markers may facilitate the study of molec-
ular technology for food authentication.
© 2015 Elsevier Ltd. All rights reserved.

1. Introduction necessary for food safety. In recent years, the vegetarian population has
increased throughout the world due to religious practices or health rea-
Food quality and safety are always a primary focus of the food indus- sons. With an increasing demand for vegetarian foods in the market,
try. With improvements in the quality of life, the food industry has pre- some vegetarian products have been mixed with animal ingredients
sented unprecedented progress and prosperity, resulting in an endless to improve the texture and taste or to reduce the cost. Even more
stream of various new food products that are emerging. Meanwhile, disturbing, some animal ingredients have imposed some deleterious,
food problems have drawn increasing attention due to various inaccu- poisonous, allergic or health risks, such as gutter oil (made from
rate descriptions and fraud, such as olive oil mixed with other cheaper recycled kitchen oil) disguised as vegetable oil (Moore, Spink, & Lipp,
oils (Aparicio, Morales, Aparicio-Ruiz, Tena, & García-González, 2013; 2012). However, current DNA markers are primarily used as species-
Kumar, Kahlon, & Chaudhary, 2011), beef mingled with horseflesh or specific markers for authenticating a plant species or for identifying an
pork (Matsunaga et al., 1999), wild meat adulterated with domesticated animal species within one kingdom. For example, the marker of cyto-
meat (Fajardo et al., 2007), market substitution of seafood (Barbuto chrome oxidase sub-unit I (COI) or cytochrome b (cytb) is used to iden-
et al., 2010; Wong & Hanner, 2008), and vegetarian food contaminated tify animal species (Hebert, Ratnasingham, & de Waard, 2003), and the
with animal components (Cheng, Shi, Lin, Chou, & Huang, 2012; markers of chloroplast gene RuBisCO large subunit (rbcL) and
Wallace et al., 2012). The fraudulent inaccurate description of food con- megakaryocyte-associated tyrosine kinase (matK) are combined to dis-
tents, which is a widespread problem, has a negative effect on consumer tinguish plants (Bruni et al., 2015). Differentiating species subtly is cer-
confidence in food products. Thus, a reliable food authentication tool is tainly useful (Hollingsworth et al., 2009); however, because these
approaches are highly species-specific, they require access to the correct
⁎ Corresponding author. Tel./fax: +86 755 25278192.
DNA sequences of the organisms, and their application is often limited
E-mail address: yanzhixiang@genomics.cn (Z. Yan). to a single taxon or to closely related taxa (Galimberti et al., 2013). A
1
These authors contributed equally to this study. subtle differentiation method is not feasible when food is admixed

http://dx.doi.org/10.1016/j.foodres.2015.04.007
0963-9969/© 2015 Elsevier Ltd. All rights reserved.

Please cite this article as: Mi, X., et al., Potential DNA markers as a rapid tracing tool for animal adulterants in vegetarian food, Food Research In-
ternational (2015), http://dx.doi.org/10.1016/j.foodres.2015.04.007
2 X. Mi et al. / Food Research International xxx (2015) xxx–xxx

with unknown cross-kingdom ingredients; however, a DNA marker The OneKP project, which was announced in November 2008, has se-
that can locate a class or category of species can be more effective for quenced the transcriptomes of approximately 1000 plants, including
cross-kingdom species identification. algae, terrestrial and aquatic plants (Matasci et al., 2014). Sequences
Over the years, the application of methodologies for food authentica- with no hits in OneKP database were selected and put into NCBI to per-
tion based on DNA analysis has attracted a growing interest. Most of the form a BLASTn alignment with the genomes of cow, pig, sheep, chicken,
DNA-based methods rely on the highly specific amplification of one or zebrafish and rat. Pigs, cows, sheep and poultry are the four primary
more DNA fragments using conventional PCR and real-time PCR types of animals used for meat production worldwide (Warriss, 2010),
(Costa, Mafra, & Oliveira, 2012). Conventional PCR provides an “on/off and zebrafish and rats are model organisms. Therefore, these animals
answer” but does not provide quantitation. Otherwise, polymerase were selected as representative animals. Sequences with high identity
chain reaction denaturing gradient gel electrophoresis (PCR-DGGE) is among the aforementioned animals were recognized as the candidate
a recent DNA-based technique used for tracing foodstuffs (El Sheikha DNA markers.
& Montet, 2012). With the emergence of next-generation sequencing
(NGS), the genomes of many species have been sequenced. Abundant 2.2. Materials and DNA extraction
homologous genes (paralogous genes and orthologous genes) have
been found simultaneously (Kusaba, Honda, & Kano‐Murakami, 2001; Rice, wheat and wheat products are traditional staple foods of the
Mural et al., 2002; Yeh, Lim, & Burge, 2001). With the development of Chinese people. Additionally, soybean, colza, corn and sunflower are
DNA technology, DNA-based methods have become widely used in tax- the primary sources of vegetable oil production in the world, and red
onomy, species identification, food and environment detection, and an- pepper is an important condiment in the diet of many countries. We
imal protection. Organisms frequently have ultraconserved sequences chose the above species to represent plants. The seeds of rice, wheat,
(Elgar & Vavouri, 2008; McEwen et al., 2006; Venkatesh et al., 2006). corn, pepper, soybean, colza and sunflower were procured from online
In 2004, Bejerano (Bejerano et al., 2004) reported 481 segments (longer stores in November 2013. Meanwhile, pork, beef, chicken and duck
than 200 bp) that are absolutely conserved between orthologous re- were purchased from a local supermarket. The DNA of the plant
gions of the human, rat, and mouse genomes, with 100% identity and seeds was extracted using a Hi Pure Plant DNA Mini Kit (Magen,
no insertions or deletions. Furthermore, nearly all of these segments #D3161-02), and DNA of the animal tissues was extracted using a
are also conserved in the chicken and dog genomes with an average of HiPure Tissue DNA Mini Kit (Magen, #D3121-02) according to the
95% and 99% identity, respectively. If different species have their own manufacturer's instructions. The extracted DNA samples were stored
conserved sequences at different levels, then DNA markers for the iden- at −20 °C.
tification of the kingdom, phylum, and class are feasible. Conserved DNA
markers for a category of species will be convenient and effective for 2.3. PCR amplification
cross-kingdom, cross-phylum and cross-class identification.
The aim of the present study was to explore candidate DNA markers Experimental validation is required to ascertain whether the candi-
for the authentication of vegetarian food using bioinformatic analysis. date DNA markers are able to distinguish animal and plant specimens.
These segments can be used as DNA markers for tracing animal adulter- A control group was designed to avoid errors in operation or inhibitory
ants in vegetarian products. PCR enzymes (Table 2). RbcL sequences of rice, wheat, corn, pepper,
soybean, colza, and sunflower and cytb sequences of pig, cow, chicken,
2. Materials and methods and duck were obtained from the NCBI genome database. The primers
for each sequence were designed using Primer 5.0 software (Table 1).
2.1. Generation of candidate DNA markers Similarly, the primers for the 6 candidate DNA markers were also
designed. All of the primers were synthesized by Beijing Liuhe Huada
In total, 481 ultraconserved segments of vertebrates were collected Genomics Technology Company.
from the Internet according to the study by Bejerano et al. (2004). We The genomic DNA samples from each plant and animal were ampli-
used PERL to obtain information regarding the segments, including fied first with the corresponding primers of rbcL and cytb, respectively,
their original locations and genome reference sequences. The 481 se- and then all of the samples were amplified using the primers of the 6
quences were aligned with the 1000 Plant Genomes Project (OneKP) segments. The 25 μL PCR reaction system included 3 μL DNA template
database (http://www.bioinfodata.org/Blast4OneKP/) using BLASTn. (approximately 50 ng/μL), 0.125 μL DNA polymerase (TaKaRa® ExTaq)

Table 1
Sequences of the 6 candidate DNA markers.

Seq Origin Length Sequence


name ID (bp)

seq1 uc.403 206 GCATGCGATAATCCCCTCTGGATGCTGGCTTGATCAGATGTTGGCTTTGTAATTAGACGGGCAGAAAATCATTATTTCATGTTCAAATAGAAAATGAGGTTGGTGGGA


AGTTAATTTCTCTCCGCTCTGTGAAGCGTAGACAAGAATTTAATGATTTAATTACAGTTGTAAGCCCTTTCCATGAGACTTAAATTGAGCTGAGAATA
seq2 uc.61 326 TCACTGTGCCATTTTTTCATGTGTTTCTCCAGGGTACTGTACACGCTAAAAGGCATCTTACAAATTTCACATTTGTAAACGTCCTTCCCCACCTGGCCATGCGTTTTCAT
GTGCCTGGTGAGCTTGCTACTCTGGGCACAGGCATAGTTGCACAGCTCGCATTTATAAGGCCTTTCGCCCGTGTGGCTTCTCCTGTGGACAGTGAGATTGCTACAGT
TCTTGAAGACTTTCCCACAGTACTCACAAGTGTCGCTGCGTCTGCCCTCTTTTGAGCTGGGCCTGCCCGGGCCCGGACCACTAATATGGGGCGTGCTCCCTCCACTTCC
seq3 uc.211 291 TCTCTGAATGGCAGAAGTCTGTTCTGCTCAAGCACAAAGTATACACAGATGTCTATATTACATTCTTATAGGTTTGACAGCAACTGCATGTTGTATCTATAAACTGTCCT
TTAGCAGTGACACTTCCTCAATGATGATAAGCTAATTTATGCAACTAAATCTCCTTTGTGCAGAAATGAAAGCTAATTGAGTCATTACAAAGTAATTCAGGAAGGAAT
ACCTTGTATAAATTGGCTTACTTTATAAATCCTTCTCAAGTGGTTTTCATGCATCCTAAATGGGACTAAGCAG
seq4 uc.294 444 CGAGATGAAATTGAGACATGGAAGAATTTATTGCCCAGAAAATTCCATTCTGCTATCTGATTCAAAAAGTCCAGTCCCCCCAGCTTCGGAAAATCTATTTTCCACATTTT
AATACCCTGCAGAACAGTCCTCATAACTCATCCGAGTGTGTTAAGCACAGTTTTATTAGATCTGAAACAAATTTTGGTGGGGAGATACTATAGGTCATTAACCATGGA
GTAATTTTATCCTTGTTTCCCTAATGATGCCATAATGGCGAGCGAATTTCTTAACTAAAGACCAAAGAACATTTTGAAGGTCAGTTTCATCTGTGAGCTCCTTCAAGCG
CTTCTCAGAGAAGATTGGAAAACTCGCCGATTTTTTGAAGAGTCATTAATAATGTGAAAGCTGAAAGCACCCTCCATTTGCGTTCCTGCTTTTTACCTTTTAATTTTATA
TCGTCCC
seq5 uc.430 213 GTGCTCCTGATTTCCCCTGGGCCTTTCTGCCTGGTAATAAGCCTTCTTTAATCTGGCAGGCCCCTTGGTGAAATCAGCACCACGGACAGTGATATTGATTGCTTCTGAGA
AACCATCACAGCTGCTCAACCAAATTACCTTTGATATGTGACCTTTAATGTAATTACTGCAACATCAAAGTATTTCTATAAATTATTAAAGAACTAATACACT
seq6 uc.274 327 ACAAAACAGATGCTGTTTAAGAAAAGGGGGAACATAATTTTGTGGGCAATGAATTAAGTGTTTTTGTGGCCCTCTCATCCGTAGCTAGGAGCAGTTTGTGGACCGCGTCT
GTGAACGCGGCTCATAATTGTTTTTCACACATAAGTTATGCAAATGAGCTTTTATGGCAACTGGCATAACAATTAGCATCCTCCAGCAATATTTTAGCAGGTTAATTG
CAAAATTTCTAAATTGTACATCTGACTTGTTAATTAGGCATGACAGAGGTGGTAAAATAGTTATCTTCAGGCAGTGGCAGCCAGGAGCTGCTTGAAATGCAAAGAGCAA

Please cite this article as: Mi, X., et al., Potential DNA markers as a rapid tracing tool for animal adulterants in vegetarian food, Food Research In-
ternational (2015), http://dx.doi.org/10.1016/j.foodres.2015.04.007
X. Mi et al. / Food Research International xxx (2015) xxx–xxx 3

Table 2
Primers of the quality control group.

RefseqID (gene) Species Length Primer-F Primer-R

refseq06 (rbcL) Pepper, soybean, colza, sunflower 195 bp GTCACCACAA ACAGAGACTA CATGTACCAG TAGAAGATTC
refseq08 (rbcL) Rice, wheat, corn 433 bp AATCTTCTACTGGTACATGGAC TCATCATCTTTGGTAAAATCAAG
refseq05(cytb) Pig 247 bp CACGATTCTTCGCCTTCC GGGGTGTAGTTGTCTGGGT
refseq03(cytb) Cow 270 bp TCATCCGATACATACACGC ATCCGCCTCAGATTCATT
refseq04(cytb) Duck 159 bp AGGCTTCTACTACGGCTCC AGGGCTGAAAATAAGTTGGT
refseq01(cytb) chicken 301 bp GGAATCTCCACGCAAACG GCGAAGAATCGGGTAAGG

(4 U/μL), 2.5 μL 10 × Ex Taq Buffer (Mg2 + Plus), 2 μL dNTP mixture no hits to plant genome database OneKP were obtained after alignment
(2.5 mM), 1 μL forward primer (10 μmol), 1 μL reverse primer with the 481 ultraconserved segments of the OneKP database. Further
(10 μmol) and 20.375 μL ddH2O. The reaction conditions consisted of NCBI blast alignment with the genomes of several animals resulted in
95 °C for 5 min; followed by 25 cycles of 98 °C for 10 s, 60 °C for 30 s 6 high identity segments, which were selected as candidate DNA
and 72 °C for 60 s; and a final extension at 72 °C for 10 min. All of the markers and labeled seq01, seq02, seq03, seq04, seq05, and seq06 in
reactions ended at 4 °C. The PCR products were visualized on 2% agarose due order (Table 1). We predicted that these candidate DNA segments
gels. The PCR products with positive results in all of the animal origin could rapidly detect whether an unknown vegetarian sample contained
genomic DNA samples were purified using a QIAquick PCR Purification an ingredient of animal origin.
Kit (Qiagen) and bi-directionally sequenced using an ABI 3730XL se-
quencer. The sequences of the controls were compared with the original
theoretical base information obtained by bioinformatic analysis using 3.2. PCR amplification
DNAMAN software. Meanwhile, the PCR products from identical
primers were used to perform sequence alignment using ClustalW The genomic DNA from all of the samples of the control group was
software. successfully amplified via PCR (Fig. 1). In the candidate DNA marker
test (Table 3), the DNA from cow, pig, chicken and duck samples was
2.4. PCR validation of adulterated plant products successfully amplified using the primers of all of the candidate markers,
except for seq05 (Fig. 2), which was discarded from further analysis. No
We performed a simulation test to validate whether the identified amplification product was generated from the templates of any of the
segments could be applied to trace animal product in adulterated plant specimens. The gel electrophoresis results demonstrated that
plant products. With the same materials listed above, we prepared a the sizes of all of the positive PCR products were consistent with the cor-
plant mixture (rice:wheat; 1:1) and an animal mixture (pork:beef; responding controls and with the expected values.
1:1) by homogenizing the products in different weight ratios. DNA
was extracted from the plant samples adulterated with meat in ratios
of 0.5%, 1%, 5%, 10% and 20% using a HiPure Plant DNA Mini Kit 3.3. Sequencing analysis
(Magen). Then, PCR amplification was performed with the seq1, seq2,
seq3, seq4 and seq6 primers using the above-described reaction According to the gel electrophoresis results, the products related to
conditions. seq1, seq2, seq3, seq4, seq6 and the controls were sequenced and
aligned. For the alignment outcome related to the human genome, the
3. Results consistency rate between the theoretical and practical PCR products of
the 5 candidate DNA markers was as follows (Table 4): seq1: 98.22%;
3.1. Screening candidate DNA markers using bioinformatic analysis seq2: 98.39%; seq3: 100.00%; seq4: 99.76%; and seq6: 97.91%. The dif-
ferences in the PCR products corresponding to each DNA marker
Using bioinformatic procedures, we aimed to identify conserved se- among the different species are shown in Table 5. The PCR products of
quences present in animals but not present in plants as candidate DNA each segment of pig, cow, chicken and duck displayed little difference,
markers for vegetarian food authentication. In total, 65 sequences with thus reflecting that these 5 segments are highly homologous in those
animals and are capable of tracking nucleic acids of animal origin.
The DNA segments of seq2, seq3, seq6 are within the BCL11A, SKAP2
and PBX3 genes, respectively, while the gene locations of seq1 and seq4
are unknown. BCL11A (Liu et al., 2003) encodes a C2H2 type zinc-finger
protein, which is likely associated with leukemia, and SKAP2 (Albizua
et al., 2011) encodes a protein that belongs to the src family kinases.

Table 3
Primers of the DNA markers.

DNA Length Primer-F Primer-R


barcode
ID

seq01 167 bp CCCTCTGGATGCTGGCTTGA ATGGAAAGGGCTTACAACTG


Fig. 1. Genomic DNA amplification using a 2.0% agarose gel for quality control. Samples:
seq02 248 bp TTCTCCAGGGTACTGTACACGC CTCAAAAGAGGGCAGACGCA
L1—chicken liver (primer ID: refseq01); L2—bovine (primer ID: refseq03); L3—duck
seq03 260 bp GAAGTCTGTTCTGCTCAAGC GGATGCATGAAAACCACTTG
(primer ID: refseq04); L4—pork (primer ID: refseq05); L5—pepper (primer ID: refseq06);
seq04 409 bp CATGGAAGAATTTATTGCCC AAGGTAAAAAGCAGGAACGC
L6—rape (primer ID: refseq06); L7—soybean (primer ID: refseq06); L8—sunflower
seq05 164 bp TTTCTGCCTGGTAATAAGCC AGAAATACTTTGATGTTGC
(primer ID: refseq06); L9—corn (primer ID: refseq08); L10—wheat (primer ID: refseq08);
seq06 286 bp TGCTGTTTAAGAAAAGGGGG TGCCACTGCCTGAAGATAAC
and L11—rice (primer ID: refseq08).

Please cite this article as: Mi, X., et al., Potential DNA markers as a rapid tracing tool for animal adulterants in vegetarian food, Food Research In-
ternational (2015), http://dx.doi.org/10.1016/j.foodres.2015.04.007
4 X. Mi et al. / Food Research International xxx (2015) xxx–xxx

Fig. 2. Electrophoretic analysis of the PCR amplification products from plant and animal samples. Each graph represents one primer pair. Samples in (a) to (f) are listed in the same order as
L1—pepper, L2—rape, L3—soybean, L4—sunflower, L5—corn, L6—wheat, L7—rice, L8—marker, L9—chicken, L10—bovine, L11—duck, L12—pig, L13—H2O (as a negative control), and
L14—human (as the positive control). (a) Primer ID: seq 01; (b) primer ID: seq 02; (c) primer ID: seq03; (d) primer ID: seq04; and (e) primer ID: seq06.

3.4. Simulation test of vegetarian product authentication Weston, 2014; Wallace et al., 2012). DNA markers are a fast detection
method that offers a specific and sensitive alternative for species origin
A testable model was constructed by mixing animal ingredients into identification. DNA markers that are independent of environmental
vegetarian materials in a proportion of 0.5%, 1%, 5%, 10% and 20% to con- conditions have been widely applied in food authenticity and the trace-
firm that the candidate DNA markers could be used to detect meat adul- ability of variety/type composition of complex food matrices for a long
teration in vegetarian food. The animal ingredient adulterated in time (Martins-Lopes, Gomes, Santos, & Guedes-Pinto, 2008). In the
vegetarian materials could be detected with the use of the primers of present study, we screened novel DNA markers using bioinformatic
seq1 and a 5% ratio; similar results were found with the primers of analysis. Intriguingly, these markers did not trace a single and unequiv-
seq2, seq3, seq4, and seq6 (Fig. 3). However, this result does not mean ocal species at the species level without sufficient variability to distin-
that the limit of detection (LOD) is only 5% because we have not per- guish even closely related species. In contrast, these markers are
formed a subdivided gradient test. Additionally, the sensitivity of this highly homologous in vertebrate animals but do not exist in plants.
measurement could be further improved by optimizing the methods Therefore, these novel markers are more powerful and efficient in the
of DNA extraction and the PCR reaction conditions. Nevertheless, identification of meat adulteration in vegetarian food at the cross-
these 5 segments are able to detect animal adulterants in vegetarian kingdom level.
products. With increasing concerns regarding adulteration in vegetarian food,
reliable detection methods for food authenticity are urgently needed to
protect customers (Madesis et al., 2014). This study demonstrates an ef-
4. Discussion fective method to establish DNA marker feasibility based on bioinfor-
matic analysis and data mining. Furthermore, we identified 6 novel
Authentication testing and adulterant detection of food products are DNA segments that can be used to detect common animal components
essential to ensure consumer protection against fraud. DNA-based in vegetarian materials rapidly. The PCR amplification of several repre-
methods for food authenticity have made great progress with the use sentative animals and plants demonstrated that primers based on 5 of
of PCR methods, high resolution melting (HRM) analysis, capillary elec- the 6 selected segments were able to amplify the animal samples but
trophoresis, microarrays and NGS (Madesis, Ganopoulos, Sakaridis, not the plant samples. Although the number of samples was limited,
Argiriou, & Tsaftaris, 2014). The amplification of DNA fragments by the samples were all typical edible plants and animals. Therefore, we
PCR followed by agarose gel electrophoresis for fragment size verifica- suggest that these 5 segments can be used as DNA markers due to
tion is the simplest methodology, is extremely sensitive and is often their easy amplification and ability to distinguish between animals
faster than other technologies. Robust DNA-based methods, including and plant samples. Although one of the primers failed to amplify the
DNA markers, now exist for the accurate identification of species DNA, we suggest that the 5 effective DNA markers are sufficient to de-
through the analysis of the variability within a standard region of the tect animal components in vegetarian food accurately. Furthermore,
genome at the species level (Alpen, Gopurenko, Wu, Lepschi, & we mixed the animal ingredients into vegetarian materials in different
proportions and used the primers of these markers to provide validation
and a preliminary estimate of the detection efficiency of the 5 DNA
Table 4 markers. The sensitivity of detection appears to be extremely limited,
Theoretical human results vs. actual human results. at only ≥5%; however, this preliminary test was primarily used for qual-
itative verification. In the future, a set of blind tests to validate the reli-
Sequence PCR product Mismatch Difference
no. length ability of these markers should be performed. Additionally, the LOD
(theoretical) should be improved by optimizing the DNA extraction and detection
seq01 167 bp 3 1./C;37A/T;38T/A methods based on these markers.
seq02 248 bp 4 32T/C;33C/T;34T/C;35T/C Significantly, DNA markers can be obtained effectively by bioinfor-
seq03 260 bp 0 – matic analysis. The success in this work indicates that bioinformatics
seq04 409 bp 1 11T/A can work well in combination with traditional biology, thus assisting bi-
seq06 286 bp 6 32G/T;33T/G;34G/T;37C/G;38A/C;43A/
ological research and making better use of data. This DNA marker

Please cite this article as: Mi, X., et al., Potential DNA markers as a rapid tracing tool for animal adulterants in vegetarian food, Food Research In-
ternational (2015), http://dx.doi.org/10.1016/j.foodres.2015.04.007
X. Mi et al. / Food Research International xxx (2015) xxx–xxx 5

Table 5
The differences in the PCR products corresponding to the same DNA marker in different species.

Sequence Species vs Product Mismatch Difference


no. human (bp)

seq01 Pig 168 2 39T/A;40A/T


Cow 168 2 39T/A;40A/T
Chicken 168 2 39T/A;40A/T
Duck 168 3 39T/A;40A/T;46G/T
Seq02 Pig 249 4 32C/T;33T/C;34C/T;35C/T
Cow 249 4 32C/T;33T/C;34C/T;35C/T
Chicken 249 20 32C/T;33T/C;34C/T;35C/T;43T/C;46A/G;63C/T;72G/A;78C/T;92T/G;114A/G;120A/G;129C/A;135G/A;138T/C;144A/G;
149T/C;150T/C;165T/C;167T/G;168C/T;196G/A
Duck 248 21 1T/.;32C/T;33T/C;34C/T;35C/T;43T/C;46A/G;63C/T;72G/A;78C/T;92T/G;114A/G;120A/G;129C/A;135G/A;138T/C;144A/G;
149T/C;150T/C;165T/C;167T/G;168C/T;196G/A
seq03 Pig 258 3 1G/.;2A/.;57T/G
Cow 260 1 148T/C;
Chicken 260 2 11./T;104A/T;
Duck 261 0 –
seq04 Pig 409 1 12A/T;
Cow 408 1 400C/.
Chicken 413 9 6./G;49A/G;319A/G;380C/A;381A/G;383C/T;387./T;383./C;389./C;
Duck 411 15 6./A;12A/.;49A/G;319A/G;367T/.;373C/T;374T/G;375G/A;378A/G; 380C/A;381A/G;383C/T;387./T;383./C;389./C;
Seq06 Pig 286 4 39T/A;40./T;41./G;43G/A;
Cow 287 5 39T/A;40./T;41./G;42./A; 43G/A;
Chicken 286 5 39T/A;40./T;41./G;43G/A;110G/C;
Duck 286 5 39T/A;40./T;41./G;43G/A;110G/C;

methodology can be extended to species in other categories, such as de- of processed foods. HRM, which measures the rate of double-stranded
tecting virus-infected plants and microorganism-contaminated animal DNA dissociation to single-stranded DNA with increasing temperature,
foods. Fresh food products without any processing are certainly suitable is a promising technique that has been widely used in food authentica-
for any DNA-based molecular analysis method; however, because most tion (Faria, Magalhães, Nunes, & Oliveira, 2013; Ganopoulos, Bazakos,
foodstuffs are processed to some extent, DNA is usually altered and Madesis, Kalaitzis, & Tsaftaris, 2013). The rapid development and
fragmented (Madesis et al., 2014). DNA-based analytical methods are plummeting cost of NGS allow one to accurately distinguish raw mate-
limited by the stability of the DNA due to environmental conditions, rials in processed foods where DNA, or at least small DNA fragments, re-
farming and production techniques used during the processing of cer- main present using DNA markers (Ortola-Vidal, Schnerr, Rojmyr,
tain food products (Galimberti et al., 2013). Therefore, screening for ac- Lysholm, & Knight, 2007). The identified DNA markers deserve to be
curate DNA markers is the first hurdle. The novel markers presented in further studied for their potential application in vegetarian food
this work were successful at differentiating animal and plant raw mate- authentication.
rials in our validation experiment using conventional PCR. In addition to
PCR, abundant DNA-based applications have been used to establish food
authenticity accurately. Some of these methods, such as HRM and NGS, 5. Conclusion
are reliable for small amplification products, which permit the analysis
In conclusion, we identified 6 DNA markers using bioinformatic
analysis. Five of the markers displayed the ability to detect ingredients
of animal origin admixed in vegetarian products. The identified 5
novel DNA markers can be applied for the reliable food authentication
of vegetarian foods using powerful DNA-based methods with these
markers. Our study provides the groundwork for further studies of mo-
lecular technology in detecting food fraud and opens a new perspective
for the exploration of molecular methods for food safety.

References
Albizua, E., Gallardo, M., Barrio, S., Rapado, I., Jimenez, A., Ayala, R., et al. (2011). Differen-
tial expression of JAK2 and Src kinase genes in response to hydroxyurea treatment in
polycythemia vera and essential thrombocythemia. Annals of Hematology, 90(8),
939–946.
Alpen, K., Gopurenko, D., Wu, H., Lepschi, B. J., & Weston, L. A. (2014). The development of
a DNA barcode system for species identification of Conyza spp. (fleabane). In M.
Baker (Ed.), Paper presented at the Proceedings of the 19th Australasian Weeds
Conference.
Aparicio, R., Morales, M. T., Aparicio-Ruiz, R., Tena, N., & García-González, D. L. (2013).
Authenticity of olive oil: Mapping and comparing official methods and promising
alternatives. Food Research International, 54(2), 2025–2038.
Barbuto, M., Galimberti, A., Ferri, E., Labra, M., Malandra, R., Galli, P., et al. (2010). DNA
barcoding reveals fraudulent substitutions in shark seafood products: The Italian
case of “palombo”(Mustelus spp.). Food Research International, 43(1), 376–381.
Fig. 3. The PCR detection of adulterated plant samples containing 0%, 0.5%, 1%, 5%, 10% and Bejerano, G., Pheasant, M., Makunin, I., Stephen, S., Kent, W. J., Mattick, J. S., et al. (2004).
20% meat product. The mixed meat sample contained pork and bovine products at a ratio Ultraconserved elements in the human genome. Science, 304(5675), 1321–1325.
of 1:1, and the plant sample contained rice and wheat at a ratio of 1:1. (a) Primer ID: Bruni, I., Galimberti, A., Caridi, L., Scaccabarozzi, D., De Mattia, F., Casiraghi, M., et al.
seq01; (b) primer ID: seq02; (c) primer ID: seq 03; (d) primer ID: seq 04; and (2015). A DNA barcoding approach to identify plant species in multiflower honey.
(e) primer ID: seq05. Food Chemistry, 170, 308–315.

Please cite this article as: Mi, X., et al., Potential DNA markers as a rapid tracing tool for animal adulterants in vegetarian food, Food Research In-
ternational (2015), http://dx.doi.org/10.1016/j.foodres.2015.04.007
6 X. Mi et al. / Food Research International xxx (2015) xxx–xxx

Cheng, C. -Y., Shi, Y. -C., Lin, S. -R., Chou, C. -C., & Huang, C. -C. (2012). Use of real-time PCR Madesis, P., Ganopoulos, I., Sakaridis, I., Argiriou, A., & Tsaftaris, A. (2014). Advances of
to detect surimi adulteration in vegetarian foods. Journal of Marine Science and DNA-based methods for tracing the botanical origin of food products. Food Research
Technology, 20(5), 570–574. International, 60, 163–172.
Costa, J., Mafra, I., & Oliveira, M. (2012). Advances in vegetable oil authentication by DNA- Martins-Lopes, P., Gomes, S., Santos, E., & Guedes-Pinto, H. (2008). DNA markers for
based markers. Trends in Food Science & Technology, 26(1), 43–55. Portuguese olive oil fingerprinting. Journal of Agricultural and Food Chemistry,
El Sheikha, A., & Montet, D. (2012). The determination of geographical origin of foodstuffs 56(24), 11786–11791.
by using innovative biological bar-code. Journal of Life Sciences, 6, 1334–1342. Matasci, N., Hung, L. -H., Yan, Z., Carpenter, E. J., Wickett, N. J., Mirarab, S., et al. (2014).
Elgar, G., & Vavouri, T. (2008). Tuning in to the signals: Noncoding sequence conservation Data access for the 1,000 Plants (1KP) project. GigaScience, 3(1), 17.
in vertebrate genomes. Trends in Genetics, 24(7), 344–352. Matsunaga, T., Chikuni, K., Tanabe, R., Muroya, S., Shibata, K., Yamada, J., et al. (1999). A
Fajardo, V., González, I., López-Calleja, I., Martín, I., Rojas, M., Hernández, P., et al. (2007). quick and simple method for the identification of meat species and meat products
Identification of meats from red deer (Cervus elaphus), fallow deer (Dama dama), and by PCR assay. Meat Science, 51(2), 143–148.
roe deer (Capreolus capreolus) using polymerase chain reaction targeting specific McEwen, G. K., Woolfe, A., Goode, D., Vavouri, T., Callaway, H., & Elgar, G. (2006). Ancient
sequences from the mitochondrial 12S rRNA gene. Meat Science, 76(2), 234–240. duplicated conserved noncoding elements in vertebrates: A genomic and functional
Faria, M., Magalhães, A., Nunes, M., & Oliveira, M. (2013). High resolution melting of trnL analysis. Genome Research, 16(4), 451–465.
amplicons in fruit juices authentication. Food Control, 33(1), 136–141. Moore, J. C., Spink, J., & Lipp, M. (2012). Development and application of a database of
Galimberti, A., De Mattia, F., Losa, A., Bruni, I., Federici, S., Casiraghi, M., et al. (2013). DNA food ingredient fraud and economically motivated adulteration from 1980 to 2010.
barcoding as a new tool for food traceability. Food Research International, 50(1), Journal of Food Science, 77(4), R118–R126.
55–63. Mural, R. J., Adams, M. D., Myers, E. W., Smith, H. O., Miklos, G. L. G., Wides, R., et al.
Ganopoulos, I., Bazakos, C., Madesis, P., Kalaitzis, P., & Tsaftaris, A. (2013). Barcode DNA (2002). A comparison of whole-genome shotgun-derived mouse chromosome 16
high-resolution melting (Bar-HRM) analysis as a novel close-tubed and accurate and the human genome. Science, 296(5573), 1661–1671.
tool for olive oil forensic use. Journal of the Science of Food and Agriculture, 93(9), Ortola-Vidal, A., Schnerr, H., Rojmyr, M., Lysholm, F., & Knight, A. (2007). Quantitative
2281–2286. identification of plant genera in food products using PCR and Pyrosequencing® tech-
Hebert, P. D., Ratnasingham, S., & de Waard, J. R. (2003). Barcoding animal life: Cyto- nology. Food Control, 18(8), 921–927.
chrome c oxidase subunit 1 divergences among closely related species. Proceedings Venkatesh, B., Kirkness, E. F., Loh, Y. -H., Halpern, A. L., Lee, A. P., Johnson, J., et al. (2006).
of the Royal Society of London, Series B: Biological Sciences, 270(Suppl. 1), S96–S99. Ancient noncoding elements conserved in the human genome. Science, 314(5807),
Hollingsworth, P. M., Forrest, L. L., Spouge, J. L., Hajibabaei, M., Ratnasingham, S., van der 1892-1892.
Bank, M., et al. (2009). A DNA barcode for land plants. Proceedings of the National Wallace, L. J., Boilard, S. M., Eagle, S. H., Spall, J. L., Shokralla, S., & Hajibabaei, M. (2012).
Academy of Sciences, 106(31), 12794–12797. DNA barcodes for everyday life: Routine authentication of Natural Health Products.
Kumar, S., Kahlon, T., & Chaudhary, S. (2011). A rapid screening for adulterants in olive oil Food Research International, 49(1), 446–452.
using DNA barcodes. Food Chemistry, 127(3), 1335–1341. Warriss, P. D. (2010). Meat science: An introductory text. Cabi.
Kusaba, S., Honda, C., & Kano‐Murakami, Y. (2001). Isolation and expression analysis of Wong, E. H. -K., & Hanner, R. H. (2008). DNA barcoding detects market substitution in
gibberellin 20-oxidase homologous gene in apple. Journal of Experimental Botany, North American seafood. Food Research International, 41(8), 828–837.
52(355), 375–376. Yeh, R. -F., Lim, L. P., & Burge, C. B. (2001). Computational inference of homologous gene
Liu, P., Keller, J. R., Ortiz, M., Tessarollo, L., Rachel, R. A., Nakamura, T., et al. (2003). Bcl11a structures in the human genome. Genome Research, 11(5), 803–816.
is essential for normal lymphoid development. Nature Immunology, 4(6), 525–532.

Please cite this article as: Mi, X., et al., Potential DNA markers as a rapid tracing tool for animal adulterants in vegetarian food, Food Research In-
ternational (2015), http://dx.doi.org/10.1016/j.foodres.2015.04.007

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