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			<p class="Type-of-Article" xml:lang="en-GB">&nbsp;</p>
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			<p class="Type-of-Article" xml:lang="en-GB">Review Article</p>
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			<p class="title- ParaOverride-1" xml:lang="en-GB">&nbsp;</p>
			<p class="title- ParaOverride-1" xml:lang="en-GB">Identification of Different Animal Species in Meat and Meat Products: Trends and Advances</p>
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			<p class="Authors ParaOverride-1" xml:lang="en-GB">&nbsp;</p>
			<p class="Authors ParaOverride-1" xml:lang="en-GB"><span class="CharOverride-1">Mayada Ragab Farag</span><span class="CharOverride-2">1*</span><span class="CharOverride-1">, Mahmoud Alagawany</span><span class="CharOverride-2">2</span><span class="CharOverride-1">, Mohamed Ezzat Abd El-Hack</span><span class="CharOverride-2">2</span><span class="CharOverride-1">, Ruchi Tiwari</span><span class="CharOverride-2">3</span><span class="CharOverride-1">, Kuldeep Dhama</span><span class="CharOverride-2">4</span></p>
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			<p class="Affiliations ParaOverride-1" xml:lang="en-GB"><span class="CharOverride-3">1</span>Forensic Medicine and Toxicology Department, Veterinary Medicine Faculty; <span class="CharOverride-3">2</span>Poultry Department, Faculty of Agriculture, Zagazig University, Zagazig 44111, Egypt; <span class="CharOverride-3">3</span>Department of Veterinary Microbiology and Immunology, College of Veterinary Sciences, Uttar Pradesh Pandit Deen Dayal Upadhayay Pashu Chikitsa Vigyan Vishwa&#160;Vidyalaya Evum Go-Anusandhan Sansthan (DUVASU), Mathura (U.P.) – 281001, India; <span class="CharOverride-3">4</span>Division of Pathology, Indian Veterinary Research Institute, Izatnagar, Bareilly (U.P.) –243122, Uttar Pradesh, India.</p>
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			<p class="Abstract" xml:lang="en-GB">&nbsp;</p>
			<p class="Abstract" xml:lang="en-GB"><span class="CharOverride-4">Abstract</span> | Identification of animal species of origin in meat and meat products is a matter of great concerns such as religious, economical, legal as well as medical aspects. Thus, several analytical techniques have been suggested for the identiﬁcation of meat species either in individual or in mixed samples to protect consumers from the fraudulent and bad habits of marketing. DNA-based techniques especially the techniques based on polymerase chain reaction (PCR) are recognized as the most appropriate methods employed for species identification in raw and processed meat. PCR techniques including randomly amplified polymorphic DNA (PCR-RAPD), restriction fragment length polymorphism (PCR-RFLP), PCR with species-specific primers, real-time PCR and PCR-nucleotide sequencing allow identification of meat species under different processing conditions. But the variability of DNA content on the level of species as well as target tissue make the DNA-based methods somewhat unsuitable for the quantiﬁcation of exact percentages of different species in meat and meat products. For these reasons the proteomic approaches depending on identiﬁcation of different peptide biomarkers has been developed and employed to give information on the different composition of food. To broad the knowledge about these technologies, this review is compiled in an attempt to provide an overview of the possible PCR-based analytical techniques that could help in identifying the meat species of origin in meat and meat products and threw the light on the identiﬁcation of species specific peptide biomarkers by proteomic technologies as a new and attractive alternative that could overcome some of the limitations that faced DNA- based methods especially when used for meat exposed to intensive heating of processing as well as for meat mixtures.</p>
		  <p class="Abstract" xml:lang="en-GB">&nbsp;</p>
			<p class="Abstract ParaOverride-1" xml:lang="en-GB"><span class="CharOverride-4">Keywords</span> | Species identification, animal, meat, meat products, DNA, PCR, proteomics</p>
		  <p class="Abstract ParaOverride-1" xml:lang="en-GB">&nbsp;</p>
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			<p class="Editor----Citation" xml:lang="en-GB"><span class="CharOverride-4">Editor</span> | Muhammad Zubair Shabbir (DVM, M. Phil, Ph D), Assistant Professor, Quality Operations Laboratory, University of Veterinary and Animal Sciences, Lahore, Pakistan.</p>
			<p class="Editor----Citation" xml:lang="en-GB"><span class="CharOverride-4">Received</span> | April 17, 2015; <span class="CharOverride-4">Revised</span> | April 28, 2015; <span class="CharOverride-4">Accepted</span> | April 29, 2015; <span class="CharOverride-4">Published</span> | May 07, 2015&#9;&#9;</p>
			<p class="Editor----Citation" xml:lang="en-GB"><span class="CharOverride-4">*Correspondence</span> | Mayada R. Farag, Zagazig University, Zagazig, Egypt; <span class="CharOverride-4">Email:</span> dr.mayadarf@gmail.com</p>
			<p class="Editor----Citation" xml:lang="en-GB"><span class="CharOverride-4">Citation</span> | Farag MR, Alagawany M, Abd El-Hack ME, Tiwari R, Dhama K (2015). Identification of different animal species in meat and meat products: trends and advances. Adv. Anim. Vet. Sci. 3(6): 334-346.  </p>
			<p class="Editor----Citation" xml:lang="en-GB"><span class="CharOverride-4">DOI</span> | <a href="http://dx.doi.org/10.14737/journal.aavs/2015/3.6.334.346"><span class="Hyperlink">http://dx.doi.org/10.14737/journal.aavs/2015/3.6.334.346</span></a></p>
			<p class="Editor----Citation" xml:lang="en-GB"><span class="Editor---Citation CharOverride-4">ISSN (Online)</span> | 2307-8316; <span class="Editor---Citation CharOverride-4">ISSN (Print)</span> | 2309-3331</p>
			<p class="Editor----Citation" xml:lang="en-GB"><span class="CharOverride-4">Copyright</span> © 2015 Farag et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.</p>
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			<p class="Heading-1--Introduction----" xml:lang="en-GB">&nbsp;</p>
		  <p class="Heading-1--Introduction----" xml:lang="en-GB">INTRODUCTION</p>
			<p class="Caps-on-First-Para ParaOverride-1" xml:lang="en-GB">&nbsp;</p>
			<p class="Caps-on-First-Para ParaOverride-1" xml:lang="en-GB"><span class="_idGenDropcap-1">M</span><span>eat </span>identiﬁcation in various feedstuts and foods including processed meat products deserves an increasing interest owing to many considerations. Rapid examination of adulteration are very critical issues for healthical requirements, speciﬁc food allergies, religious affairs, fraud and malicious marketing practices in addition to economic and legal concerns (<a href="#Koh-MC--Lim-CH--Chua-SB--Chew-ST--Phang-STW--1998-"><span class="Hyperlink">Koh et al., 1998</span></a>; <a href="#Arslan-A--Ilhak-2006"><span class="Hyperlink">Arslan et al., 2006</span></a>; <a href="#Mane-BG--Mendiratta-SK--Tiwari-AK--2009"><span class="Hyperlink">Mane et al., 200</span></a><span class="Hyperlink">9</span>). Furthermore, identifying the meat authenticity in meat products is an important issue in food regulatory control for determination of fraudulent replacement of higher commercial valued meat species by inferior, cheaper or undesirable alternatives, the presence of undeclared species, and replacement of animal meat by plant proteins, accurate food labelling (<a href="#Ballin-NZ--Vogensen-FK--Karlsson-AH--2009"><span class="Hyperlink">Ballin et al., 2009</span></a>) and for the evaluation of food composition and providing consumer needed information to achieve food safety (<a href="#Stamoulis-O--Stamatis-C--Sara-dou-T--Mamuris-Z--2010"><span class="Hyperlink">Stamoulis et al., 2010</span></a>).</p>
		  <p class="Caps-on-First-Para ParaOverride-1" xml:lang="en-GB">&nbsp; </p>
			<p class="Body-Text ParaOverride-1" xml:lang="en-GB">Protection of consumers and producers from mislabelled meat products, fraudulent actions, and bad practices of meat adulterations through processing and marketing and the prevention of illegal sale of protected species were always critical concerns that enforce legal authorities as well as many researchers to develop different techniques and analytical methods for species identiﬁcation present in meat or their products including a wide range of degraded and processed materials that were broadly based on measuring either DNA or protein (<a href="#Matsunaga-T--Chikuni-K-"><span class="Hyperlink">Matsunaga et al., 1999</span></a>; <a href="#Calvo-JH--Zaragoza-P--Osta-R--2001-"><span class="Hyperlink">Calvo et al., 2001</span></a>; <a href="#Herman-BL--2001"><span class="Hyperlink">Herman, 2001</span></a>; <a href="#Myers-MJ--Yancy-HF--Farrell-DE--2003"><span class="Hyperlink">Myers et al., 2003</span></a>; <a href="#Peter-C--Brunen-Nieweler-C--Cammann-K--Borchers-T--2004-."><span class="Hyperlink">Peter et al., 2004</span></a>; <a href="#Aida-AA--Che-Man-YB--Wong-CMVL--Raha-AR--Son-R--2005-."><span class="Hyperlink">Aida et al., 2005</span></a>). </p>
		  <p class="Body-Text ParaOverride-1" xml:lang="en-GB">&nbsp;</p>
			<p class="Body-Text ParaOverride-1" xml:lang="en-GB">The species-specific protein biomarkers have been identified using electrophoretic and chromatographic techniques (<a href="#Vallejo-Cordoba-B--Gonz-lez-C-rdova-AF--Mazorra-Manzano-MA--Rodr-guez-Ram-rez-R--2005"><span class="Hyperlink">Vallejo-Cordoba et al., 2005</span></a>; <a href="#Chou-CC--Lin-SP--Lee-KM--Hsu-CT--Vickroy-TW--Zen-JM--2007"><span class="Hyperlink">Chou et al., 2007</span></a>), or enzyme-linked immunosorbent assay (ELISA) (<a href="#Berger-RG--Margeau--RP--Schwab-B--Johnston-RW--1988"><span class="Hyperlink">Berger et al., 1988</span></a>; <a href="#Andrews-CD--Berger-RG--Mageau-RP--Schwab-B--Jhonston-RW--1992"><span class="Hyperlink">Andrews et al., 1992</span></a>; <a href="#Chen-FC--Hsieh-YH--2000-."><span class="Hyperlink">Chen and Hsieh, 2000</span></a>) and isoelectric focusing (IEF) (<a href="#King-NL--1984"><span class="Hyperlink">King, 1984</span></a>; <a href="#Kim-H--Shelef-LA--1986"><span class="Hyperlink">Kim and Shelef, 1986</span></a>; <a href="#Scarpeid-HJ--Kvaal-K--Hildrum-KI--1998"><span class="Hyperlink">Scarpeid et al., 1998</span></a>). These methods have been suggested to resolve proteins of skeletal muscle based on the differences in their isoelectric point or molecular weight (<a href="#Bauer-F--Hofmann-K--1989"><span class="Hyperlink">Bauer and Hofmann, 1989</span></a>; <a href="#K-uffer-K--Beneke-B--Bentler-W--1990-."><span class="Hyperlink">Käuffer et al., 1990</span></a>; <a href="#Di-Luccia-A--Santoro-A--Anastasio-A--Sarli-T-"><span class="Hyperlink">Di Lucciaet al., 1992</span></a>; <a href="#Hsieh-YHP--2006"><span class="Hyperlink">Hsieh, 2006</span></a>) and could be used for mapping of the skeletal muscle proteins of different animal species such as cattle (<a href="#Bouley-J--Chambon-C--Piccard-B--2004"><span class="Hyperlink">Bouley et al., 2004</span></a>; <a href="#Chaze-T--Bouley-J--Chambon-C--Barboiron-C--Picard-B--2006-."><span class="Hyperlink">Chaze et al., 2006</span></a>), swine (<a href="#Kim-SH--Huang-TS--Seymour-TA-Wei-C--Kempf-SC--Bridgman-CR--Clemens-RA--An-H--2004"><span class="Hyperlink">Kim et al., 2004</span></a>; <a href="#Hollung-K--Grove--H--Faergestad--EM--Sidhu--MS--Berg-P--2009"><span class="Hyperlink">Hollung et al., 2009</span></a>; <a href="#Xu-YJ--Jin-ML--Wang-LJ--Z"><span class="Hyperlink">Xu et al., 2009</span></a>), poultry (<a href="#Doherty-MK--McLean-L--Hayter-JR--Pratt-JM--Robertson-DHL--El-Shafei-A--Gaskell-SJ--Beynon-RJ--2004-."><span class="Hyperlink">Doherty et al., 2004</span></a>) and sheep (<a href="#Herman-BL--2001"><span class="Hyperlink">Hamelin, 200</span></a><span class="Hyperlink">1</span>). The protein based methods has been reported to be non-suitable for species identification in heated meat products due to denaturation of protein by intensive heating during food processing which in turn lead to modifications in the antigenic activity of molecules and their mobility after electrophoresis (<a href="#Jemmi-T--Schlosser-H--1991"><span class="Hyperlink">Jemmi and Schlosser, 1991</span></a>; <a href="#Guoli-Z--Mingguang-Z--Zhijiang-Z--Hongsheng-O--Qiang-L--1999"><span class="Hyperlink">Guoli et al., 1999</span></a>; <a href="#Giovannacci-I--Guizard-C--Carlier-M--Duval-V--Martin-JL--Demeulemester-C--2004"><span class="Hyperlink">Giovannacci et al., 2004</span></a>) consequently, change the ability of antibody to identify its target protein (<a href="#Owusu-Apenten-RK--2002-"><span class="Hyperlink">Owusu-Apenten, 2002</span></a>), moreover, the possible cross-reaction between closely related species (<a href="#Hsieh-YHP--Sheu--SC--Bridgman-RC--1998"><span class="Hyperlink">Hsieh et al., 1998</span></a>). For these reasons protein-based methods have been replaced by DNA-based ones. DNA characterized by more stability under intensive heating, pressures, and chemical processing, has conserved structure in whole body cells, has a great identification power since they are rely on the recognition of speciﬁc DNA segments sequence of a particular tissue or animal (<a href="#Calvo-JH--Zaragoza-P--Osta-R--2001-"><span class="Hyperlink">Calvo et al., 2001</span></a>; <a href="#Frezza-D--Favaro-M--Vaccari-G--Von-Holst-C--Giambra-V--Anklam-E--2003"><span class="Hyperlink">Frezza et al., 2003</span></a>; <a href="#Girish-PS--Anjaneyulu-2004"><span class="Hyperlink">Girish et al., 2004</span></a>; <a href="#Lanzilao-I--Burgalassi-F--Fancell-S--Settimelli-M--Fani-R--2005-."><span class="Hyperlink">Lanzilao et al., 2005</span></a>; <a href="#Akasaki-T--Yanagimoro-T--Yamakami-K--Tomonaga-H--Sato-S--2006"><span class="Hyperlink">Akasaki et al., 2006</span></a>; <a href="#Arslan-A--Ilhak-2006"><span class="Hyperlink">Arslan et al., 2006</span></a>; <a href="#Rashid-PMA--Babashekh-MO--Marouf-AS--Am"><span class="Hyperlink">Rashid et al., 2014</span></a>). From DNA-based techniques, polymerase chain reaction (PCR) is the most employed, simple, time saving, sensitive and specific method that could identify the species of origin exposed to different processing conditions (<a href="#Mafra-I--Ferreira-I--Oliveira-M.--2008"><span class="Hyperlink">Mafra et al., 2008</span></a>; <a href="#Bottero-MT--Dalmasso-A.--2011-"><span class="Hyperlink">Bottero and Dalmasso, 2011</span></a>; <a href="#Floren-C--Wiedemann-I--Brenig-B--Sch-tz-E--Beck-J--2015-"><span class="Hyperlink">Floren et al., 2015</span></a>). In addition, the use of PCR in food analysis has provided various analytical methods for rapid detection and identiﬁcation at species and intra-species level; however DNA-based methods still face some important limitations especially for quantitative measurements of food composition (<a href="#Woolfe-M--Primrose-S--2004"><span class="Hyperlink">Woolfe and Primrose, 2004</span></a>). To overcome these limitations attention has been paid to the development of new technologies that could be successfully used when quantitation assessments are required. Among the attractive newly developed analytical techniques that used for quantitative determination for different composition present in meat processed under high temperature or complex mixes is the proteomic technology that depends on analysis of protein and peptide biomarkers as described by many researchers (<a href="#Jorfi-R--Shuhaimi-M--Yaakob-BCM--Dzul"><span class="Hyperlink">Jorfi et al., 2012</span></a>; <a href="#Giaretta--N--Di-Giuseppe-AM--Lippert-M--Parente-A--Di-Maro-A--2013"><span class="Hyperlink">Giaretta et al., 2013</span></a>; <a href="#Montowska-M--Pospiech-E--2011a-."><span class="Hyperlink">Montowska and Pospiech, 2013</span></a>; <a href="#Boyac---H-Temiz-HT--Uysal-RS--Velio-lu-HM--Yadegari-RJ--Rishkan-MM.--2014-"><span class="Hyperlink">Boyaci et al.; 2014</span></a>; <a href="#Zhao-M--Downey-G--O-Donnell-CP--2014"><span class="Hyperlink">Zhao et al., 2014</span></a>).</p>
		  <p class="Body-Text ParaOverride-1" xml:lang="en-GB">&nbsp;</p>
			<p class="Body-Text ParaOverride-1" xml:lang="en-GB">Within this context, the aim of this review is to provide an overview of the main PCR-based techniques that are published concerning the species identification of meat and meat products with special reference to the advantages and disadvantages of each method and the mitochondrial genes that have been reported to be used for species identification in meat and meat products. PCR-based techniques most frequently used for meat species identiﬁcation include randomly amplified polymorphic DNA (PCR-RAPD), restriction fragment length polymorphism (PCR-RFLP), PCR with species-specific primers, real-time PCR and PCR-nucleotide sequencing. Besides, the advances in proteomic technology for species identification have also been covered.</p>
		  <p class="Body-Text ParaOverride-1" xml:lang="en-GB">&nbsp;</p>
			<p class="Heading-1--Introduction---- ParaOverride-1" xml:lang="en-GB">POLYMERASE CHAIN REACTION (PCR)-BASED TECHNIQUES</p>
		  <p class="Heading-1--Introduction---- ParaOverride-1" xml:lang="en-GB">&nbsp; </p>
			<p class="Heading-2--History-in-MM- ParaOverride-1" xml:lang="en-GB">Randomly Amplified Polymorphic DNA (PCR-RAPD) </p>
			<p class="Body-Text ParaOverride-1" xml:lang="en-GB">The PCR-RADP depends on the use of a single arbitrary primer to initiate and activate the reaction of elongation of strands of the amplified fragment and give a species-specific “fingerprints” followed by isolation of ampliﬁed fragments based on size of fragments by gel electrophoresis. So, there is no need for DNA sequencing, restriction enzymes or hybridization (<a href="#Wu-XB--Liu-H--Jiang-ZG--2006"><span class="Hyperlink">Wu et al., 2006</span></a>) it is simple, cheap, makes it possible to reveal genetic variability without previous knowledge of the sequence of the tested DNA. But, it requires a known standard for species identification and could not be used to identify composition of meat mixtures or severely (autoclaved) heat treated meat (<a href="#Koh-MC--Lim-CH--Chua-SB--Chew-ST--Phang-STW--1998-"><span class="Hyperlink">Koh et al., 1998</span></a>) and the obtained results were non-reproducible (<a href="#Wolko----Witucka-Wall-H--Siemieniako-B--Wolko"><span class="Hyperlink">Wolko et al., 2004</span></a>).</p>
		  <p class="Body-Text ParaOverride-1" xml:lang="en-GB">&nbsp;</p>
			<p class="Body-Text ParaOverride-1" xml:lang="en-GB"><a href="#Koh-MC--Lim-CH--Chua-SB--Chew-ST--Phang-STW--1998-"><span class="Hyperlink">Koh et al. (1998)</span></a> could identify buffalo, wild boar, kangaroo and red deer meats by RAPD technique. Meats from buffalo, Elk, reindeer, kangaroo, ostrich and some domestic species could be identified by RAPD under different conditions including fresh, freezing and canning (<a href="#Mart--nez-I--Yman-IM--1998-"><span class="Hyperlink">Martı´nez and Yman, 1998</span></a>). <a href="#Martinez-I--Dan-elsd-ttir-AK--2000"><span class="Hyperlink">Martı´nez and Danielsdottir (2000)</span></a> designed a primer based on cyt b gene that help in identifying different types of meat products of seal and whale under different processing situations by RAPD and PCR-SSCP. RAPD technology was also employed by <a href="#Huang-MC--Horng-YM--Huang-HL--Sin-YL--Chen-MJ--2003"><span class="Hyperlink">Huang et al. (2003)</span></a> for authentication of quail, ostrich, pheasant, emu and dove meats. <a href="#Saez-R---Sanz-Y--Toldr--F---2004"><span class="Hyperlink">Saez et al. (2004)</span></a> generated species-speciﬁc ﬁnger printings by RAPD- and AP-PCR methods to identify meat species. These methods help in identification of beef, pork, lamb and poultry. <a href="#Arslan-A--Ilhak-2005"><span class="Hyperlink">Arslan et al. (2005)</span></a> discriminated meats from certain domestic animals as goat, cattle, camel, sheep, pork and rabbit as well as meat from wild swine, donkey, dog and cat by PCR-RAPD using 10 base primer on both individual and mixed meat samples. Also, this method was used to identify meats from different fish species (<a href="#Jin-LG--Cho-JY--Seong-KB--Park-JY--Kong-IS--Hong-YK--2006"><span class="Hyperlink">Jin et al., 2006</span></a>).</p>
		  <p class="Body-Text ParaOverride-1" xml:lang="en-GB">&nbsp;</p>
			<p class="Heading-2--History-in-MM- ParaOverride-1" xml:lang="en-GB">Restriction Fragment Length Polymorphism (PCR-RFLP)</p>
			<p class="Body-Text" xml:lang="en-GB">PCR-RFLP is one of the main genetic techniques conducted by many researchers for species identiﬁcation in meat and meat products obtained from mammals, poultry or fish. This technique based on amplification of a DNA fragment of various sequences followed by its digestion with an appropriately selected restriction enzyme allowing species differentiation of even closely related species (<a href="#Pascoal-A--Prado-M--Castro-J--Cepeda-A--Barros-Vel-zquez-J--2004"><span class="Hyperlink">Pascoal et al., 2004</span></a>). The PCR-RFLP method characterized by its simplicity and non-expensive costs and easy application in the inspection purposes (<a href="#Pfeiffer-I--Burger-J--Brenig-B--2004-."><span class="Hyperlink">Pfeiffer et al., 2004</span></a>). But unfortunately it could not be employed for identify composition of meat mixtures or severely heat treated meat due to degradation of DNA and the data recorded after digestion of the PCR products might show a combination of diverse restriction types representing all the possible kinds included in the adulterated sample (<a href="#Girish-PS--Anjaneyulu-ASR"><span class="Hyperlink">Girish et al., 2005</span></a>; <a href="#Girish-PS--Anjaneyulu-2007"><span class="Hyperlink">Girish et al., 2007</span></a>). Another defect of the PCR-RFLP method is the possibility of developing erroneous results due to the possible incomplete digestion of the restriction site or occurrence of intra-specific differences which may result in removal or development of restriction locations (<a href="#Gil-LA--2007"><span class="Hyperlink">Gil, 2007</span></a>), where, relatively large amplicons are commonly need to perform enzymatic restriction of DNAs (<a href="#Fajardo-V--Gonza-2006"><span class="Hyperlink">Fajardo et al., 2006</span></a>). RFLP were developed and applied on the PCR products of certain mitochondrial DNA like mitochondrial displacement (D-loop) region and cytochrome b (cyt b) as well as 12S and 16S rRNA genes as reviewed in our work. </p>
		  <p class="Body-Text" xml:lang="en-GB">&nbsp;</p>
			<p class="Body-Text ParaOverride-1" xml:lang="en-GB"><a href="#Meyer-W--Seger-H--Hulmann-G--1995"><span class="Hyperlink">Meyer et al. (1995)</span></a> ampliﬁed the 359-bp fragment of the cyt b gene followed by digestion with <span class="CharOverride-9">RsaI</span>, TaqI, <span class="CharOverride-9">AluI</span> and <span class="CharOverride-9">HinfI</span> to identify cattle, swine, buﬀalo, wild boar, goat, sheep, horse, turkey and chicken meat. On this context, the PCR-RFLP technique allowed identify of 25 animal species in frozen meat or freeze-dried protein samples using tRNAGlu/cyt b and 11 various restriction enzymes (<a href="#Wolf-C--Rentsch-J--Hubner-P--1999"><span class="Hyperlink">Wolf et al., 1999</span></a>). In another report, <a href="#Partis-L--Croan-D--Guo-Z--Clark-R--Coldham-T--Murby-J--2000"><span class="Hyperlink">Partis et al. (2000)</span></a> stated that PCR-RFLP on the basis of CYT b1 and CYT b2 using C1 and C2 primers which amplify the gene coding cytochrome b that yield products of 359 bp and 464 bp after digestion with HaeIII and <span class="CharOverride-9">HinfI</span> can be applied to analyse both raw and cooked meat species as it could differentiate all the tested species except buffalo and kangaroo, but they do not recommend this method to determine species composition of mixed meats. </p>
		  <p class="Body-Text ParaOverride-1" xml:lang="en-GB">&nbsp;</p>
			<p class="Body-Text ParaOverride-1" xml:lang="en-GB">The discrimination power of cyt b gene has been proved by various studies for example, the study of <a href="#Bellagamba-F--Moretti-VM--Comincini-S--Valfre-F--2001-."><span class="Hyperlink">Bellagamba et al. (2001)</span></a> in which PCR- RFLP products of cyt b gene conducted to identify species in meat meal and animal feed stuffs. <a href="#Bravi-CM--Liron-JP--Mirol-PM--Ripoli-MV--Garcia-PP--Giovambattista-G--2004-"><span class="Hyperlink">Bravi et al. (2004)</span></a> amplified a fragment of cyt b to identify meat of cattle, horse, donkey, pig, sheep, dog, cat, rabbit, chicken, and human using universal primers and 3 restriction enzymes (<span class="CharOverride-9">AluI</span>, <span class="CharOverride-9">HaeIII</span>, and <span class="CharOverride-9">HinfI</span>). <a href="#Ahmed-MMM--Abdel-Rahman-SM--El-Hanafy-AA--2007-"><span class="Hyperlink">Ahmed et al. (2007)</span></a> amplified a segment of cyt b gene (359 bp) followed by digestion with <span class="CharOverride-9">TaqI</span> to differentiate between cattle’s and buffalo’s meat. The PCR products were 2 fragments (191 and 168 bp) in buffalo with no digestion for cattle. <a href="#Ahmed-MMM--Abdel-Rahman-SM--El-Hanafy-AA--2007-"><span class="Hyperlink">Ahmed et al. (2007)</span></a> used PCR-RFLP to differentiate between horse and donkey meat by restriction enzyme <span class="CharOverride-9">AluI </span>that digest the PCR product of cyt b gene amplification to give three fragments in horse’s meat (189, 96 and 74 bp), while no digestion in donkey.  </p>
		  <p class="Body-Text ParaOverride-1" xml:lang="en-GB">&nbsp;</p>
			<p class="Body-Text ParaOverride-1" xml:lang="en-GB"><a href="#Doosti-A--Ghasemi-Dehkordi-P--Rahimi-E--2014-"><span class="Hyperlink">Doosti et al. (2014)</span></a> investigated the PCR-RFLP analysis of the mitochondrial cyt b gene to differentiate between beef, sheep, pork, chicken, donkey, and horse meats in meat products (sausages, frankfurters, hamburgers, hams and cold cut meats) and suggested that this method provide a potential technique to rely on for authentication of halal (lawful or permitted) meat origin. <a href="#Rahman-MM---Ali-ME---Hamid-SB---Mustafa-S--Hashim-U--Hanapi-UK--2014"><span class="Hyperlink">Rahman et al. (2014)</span></a> assessed the presence of dog meat in meatball by PCR assay for amplification of 100-bp region of canine mitochondrial cyt b gene in different circumstances (pure, raw, processed and mixed conditions). This assay tested with many other animal and plant species used in the formation of meatball and is found to be simple, stable, sensitive and specific to detect dog meat in processed food which is very important for halal authentication purposes. </p>
		  <p class="Body-Text ParaOverride-1" xml:lang="en-GB">&nbsp;</p>
			<p class="Body-Text ParaOverride-1" xml:lang="en-GB"><a href="#Fei-S--Okayama-T--Yamanoue-M--Nishikawa-I--Mannen-H--Tsuji-S--1996"><span class="Hyperlink">Fei et al. (1996)</span></a>; <a href="#Montiel-Sosa-JF--Ruiz-Pesini-E-"><span class="Hyperlink">Monteil-Sosa et al. (2000)</span></a> and <a href="#Mane-BG--Mendiratta-SK--Tiwari-AK--2009-296"><span class="Hyperlink">Mane et al. (2009)</span></a> tried to differentiate the chicken from other meat species by designing a primer pair on the basis of mitochondrial D-loop gene to amplify 442bp of DNA fragments followed by subjecting the resulted fragments to digestion by HaeIII and Sau3AI enzymes where ampliﬁcation of 442 bp DNA fragment was observed only in chicken even after cross testing with red meat species investigated (cattle, buffalo, sheep, goat, pig, duck, guinea fowl, turkey and quail) indicating the high specificity of this PCR assay for chicken meat that provide a useful tool for detecting of meat species even in ad-mixed meat and meat products under different processing conditions. </p>
		  <p class="Body-Text ParaOverride-1" xml:lang="en-GB">&nbsp;</p>
			<p class="Body-Text" xml:lang="en-GB">The PCR-RFLP of 12S rRNA were used by several researchers to discriminate between various animal species (<a href="#Prakash-S--Patole-MS--Ghumatkar-SV--Nandode-SK--Yogesh-S--Shouche--2000-"><span class="Hyperlink">Prakash et al., 2000</span></a>; <a href="#Girish-PS--Anjaneyulu-2004"><span class="Hyperlink">Girish et al., 2004</span></a>; <a href="#Rodriguez-MA--Garc---a--T2004"><span class="Hyperlink">Rodriguez et al., 2004</span></a> and <a href="#Rodriguez-MA--Garc-a-T-2005"><span class="Hyperlink">2005</span></a>). More recent study by <a href="#Girish-PS--Anjaneyulu-ASR"><span class="Hyperlink">Girish et al. (2005)</span></a> stated that the method of PCR amplification of 456-bp from the 12SrRNA gene followed by digestion with <span class="CharOverride-9">AluI</span>, HhaI, ApoI and BspTI could differentiate between beef, buﬀalo meat, mutton and chevonin fresh and processed meat but not in meat mixtures. <a href="#Girish-PS--Anjaneyulu-2007"><span class="Hyperlink">Girish et al. (2007)</span></a> also amplified a DNA fragment of the same length (456 bp) from the 12SrRNA gene using universal primers followed by digestion with <span class="CharOverride-9">HinfI</span>, Mph1103I, MvaI, Eco47I, that help in identification of duck, chicken, turkey, guinea fowl where the PCR-RFLP method identified all the poultry species in fresh meats, chicken meat detection was also possible in heated products. Similarly, <a href="#Rajput-N--Shrivastav-AB--Parmar-SNS--Ranjan-R--Singh-S--Joseph-E--2013"><span class="Hyperlink">Rajput et al. (2013)</span></a> amplified a 440 bp length fragment from the 12SrRNA gene using universal primers to differentiate the meat of sambar and chital (wild animals) from meat of sheep and goat, where they used PCR-RFLP and sequencing to differentiate between these species. <span class="CharOverride-9">AluI</span> and <span class="CharOverride-9">RsaI</span> succeeded to differentiated between the meat samples from sambar and chital and the meat samples from sheep and goat. BsrI could differentiate chital from the all other species. DdeI helped in differentiation of chital and sambar from each other. Also, 16S rRNA was used for species identification by PCR-RFLP method (<a href="#Borgo-R--Souty-Grosset-C--Bouchon-Gomot--DL--1996"><span class="Hyperlink">Borgo et al, 1996</span></a>; <a href="#Sawyer-J--Wood-C--Shanahan-D--Gout-S--McD"><span class="Hyperlink">Sawyer et al., 2003</span></a>). <a href="#Chikuni-K--Tabata-T--Kosugiyama-M---Monma-M--1994-"><span class="Hyperlink">Chikuni et al. (1994)</span></a> discriminated goat and sheep meat by PCR-RFLP of satellite I DNA sequence using restriction enzyme of ApaI. Restriction proﬁle of melanocortin gene was used as DNA marker for discrimination of Hanwoo meat from meats of Angus and Holstein (<a href="#Chung-ER--Kim--WT--Kim--YS--Han--SK--2000"><span class="Hyperlink">Chung et al., 2000</span></a>). </p>
		  <p class="Body-Text" xml:lang="en-GB">&nbsp;</p>
			<p class="Heading-2--History-in-MM-" xml:lang="en-GB">PCR with the Use of Species-Specific Primers</p>
			<p class="Body-Text ParaOverride-1" xml:lang="en-GB">The identification of species origin of meat by PCR using species-speciﬁc primers is precise, sensitive, cheap and not a time consuming method that help the novel identification of many mammalian and bird species in meat and meat products as compared to other PCR based assay (<a href="#Mane-BG--Mendiratta-SK--Tiwari-AK--2009-296"><span class="Hyperlink">Mane et al., 2007</span></a>). The PCR methods targets genomic and mitochondrial DNA for the purpose of the identiﬁcation of meat species in large number of samples, even in cooked meat under different processing conditions without the need for further sequencing or digestion of the PCR products with restriction endonucleases (<a href="#Di-Pinto-A--Forte-2005"><span class="Hyperlink">Di Pinto et al., 2005</span></a>; <a href="#Arslan-A--Ilhak-2006"><span class="Hyperlink">Arslan et al., 2006</span></a>; <a href="#Mafra-I--Ferreira-I--Oliveira-M.--2008"><span class="Hyperlink">Mafra et al., 2008</span></a>; <a href="#Rojas-M--Gonza--lez-2009b"><span class="Hyperlink">Rojas et al., 2009b</span></a>), but the most important requirement is that the nucleotide sequence of the gene used for species identification should be known for the purposes of primer designing ( <a href="#Spychaj-A--Paul-EM--Edward-P--2009"><span class="Hyperlink">Spychaj et al., 2009</span></a>).</p>
		  <p class="Body-Text ParaOverride-1" xml:lang="en-GB">&nbsp;</p>
			<p class="Body-Text" xml:lang="en-GB">The species specific PCR assay were also used by <a href="#Hopwood-AJ--Fairbrother-KS--Lockley-AK--Bardsley-RG--1999-"><span class="Hyperlink">Hopwood et al. (1999)</span></a> to identify chicken meat in fresh or cooked meat admixtures including meat of other species as beef, lamb, pork, horse, duck and pheasant. <a href="#Calvo-JH--Zaragoza-P--Osta-R--2001-"><span class="Hyperlink">Calvo et al. (2002)</span></a> designed speciﬁc primers for detection of pork meat in different meat products. Similarly, <a href="#Kitpipit-T----Kuangtiwa-S--Phuvadol-T--2013"><span class="Hyperlink">Kitpipit et al (2013)</span></a> used this method to differentiate between pork, mutton and chicken meat.</p>
		  <p class="Body-Text" xml:lang="en-GB">&nbsp;</p>
			<p class="Body-Text ParaOverride-1" xml:lang="en-GB">Species-speciﬁc primers designing based on the mitochondrial cytochrome b gene has been reported by many researchers like <a href="#Matsunaga-T--Chikuni-K-"><span class="Hyperlink">Matsunaga et al. (1999)</span></a> who could qualitatively identify fresh and thermally processed meats of pigs, cattle, sheep, goat, horse and chicken using seven primers, a forward primer designed for the conservative sequence of the cyt b gene in mitochondria and six reverse primers specific for each of tested kinds except the meat of horse exposed to intensive heating.</p>
		  <p class="Body-Text ParaOverride-1" xml:lang="en-GB">&nbsp;</p>
			<p class="Body-Text" xml:lang="en-GB"><a href="#Colombo-F--Marchisio-E--Pizzini-A--Cantoni-C--2002-"><span class="Hyperlink">Colombo et al. (2002)</span></a> designed species-specific primers based on the cyt b mitochondrial gene that help in the identification of goose (Anseranser) meat in salami meat product in presence or absence of pork or duck meat. <a href="#Hird-H--Goodier-R--Hill-M--2003"><span class="Hyperlink">Hird et al. (2003)</span></a> designed species-specific primers depending on the cyt b gene for speciation of chicken and turkey meat under different manufacturing conditions where the products of amplification were of 120 bp for the chicken and 101 bp for the turkey. The species identification of chicken, turkey, duck, goose, pheasant, quail and guinea fowl in meat and meat products was also conducted by <a href="#Schw-gele-F--Stirtzel-S--Andree-S--2007"><span class="Hyperlink">Schwägele et al. (2007)</span></a> who used cyt b gene to design species specific primers where there was no cross-reactivity with any other species.</p>
		  <p class="Body-Text" xml:lang="en-GB">&nbsp;</p>
			<p class="Body-Text ParaOverride-1" xml:lang="en-GB"><a href="#Haunshi-S--Basumatary-R--Girish--PS--Doley-S--Bardoloi-RK--Kumar-A--2009-"><span class="Hyperlink">Haunshi et al. (2009)</span></a> designed primers specific for pigeon identification based on cyt b and species-speciﬁc markers for chicken duck and pig D-loop mitochondrial genes that could strictly identify the mentioned species in fresh and processed meats. <a href="#Barakat-H--El-Garhy-HA--Moustafa-MM--2014"><span class="Hyperlink">Barakat et al. (2014)</span></a> amplified the mitochondrial cyt b and D-loop genes using porcine-specific primers followed by QIAxcel capillary electrophoresis system to detect and quantify the pork meat in “halal” meat products using raw and cooked sausages as models. This method proved rapidity and sensitivity as it gives specific DNA fragments for pork meat only. </p>
		  <p class="Body-Text ParaOverride-1" xml:lang="en-GB">&nbsp;</p>
			<p class="Body-Text ParaOverride-1" xml:lang="en-GB">The mitochondrial D loop genehas been employed for designing a pair of primers specific to the buffalo meat that used by <a href="#Girish-PS--Haunshi-2013"><span class="Hyperlink">Girish et al. (2013)</span></a> to examine a different method for authentication of buffalo tissues based on DNA extraction by alkaline lysis from meat, liver, heart and kidney samples of buffalo and other related species like cattle, sheep and goat. This species specific PCR resulted in an amplicon of size 482 pb for buffalo and no amplification in the other species. <a href="#Karabasanavar-NS--Singh-SP--Kumar-D--Shebannavar-SN--2014-"><span class="Hyperlink">Karabasanavar et al. (2014)</span></a> designed a new species specific primers specific for the mitochondrial D-loop region of pigs that give a unique amplicon containing 712 pb providing a very sensitive and specific PCR assay for detecting pork meat from many other species including meat of mammals, birds, rodents as well as fish. The developed assay also could detect the authenticity of pig tissues in different processing conditions (raw, cooked, autoclaved, micro-oven) helping in the purposes of forensic identification of pig species as well as adulteration of pig meat with other species meat. </p>
		  <p class="Body-Text ParaOverride-1" xml:lang="en-GB">&nbsp;</p>
			<p class="Body-Text ParaOverride-1" xml:lang="en-GB"><a href="#Fajardo-V--Gonz-lez-2007"><span class="Hyperlink">Fajardo et al. (2007)</span></a> used a PCR assay based on the ampliﬁcation of DNA fragments of D-loop and 12SrRNA gene using species-specific primers to identify various cervid and wild ruminant meats including the meats from different deer species as red deer (Cervuselaphus), fallow deer (Damadama), and roe deer (Capreoluscapreolus). Species-speciﬁc primers depending on D-loop and 12S rRNA genes were similarly used by <a href="#Rojas-M--Gonza--lez-2009b"><span class="Hyperlink">Rojas et al. (2009b)</span></a>; <a href="#Rojas-M--Gonz-lez-I-"><span class="Hyperlink">Rojas et al. (2010)</span></a> for the identiﬁcation of some species of game bird species. Similarly, <a href="#Mart-n-I--Garc--T---Fajardo-V--Ine--sLo--pe"><span class="Hyperlink">Martı´n et al. (2007)</span></a> used the specific primers based on 12S rRNA for identification of four duck species in meat mixtures and speciﬁc identiﬁcation of Muscovy duck even if used on highly damaged DNA. Species-speciﬁc primers targeting 12S and 16 rRNA were applied for detection of some animal species like deer and some ruminant animals in meat products by <a href="#Ha-JC--Jung-WT--Nam-YS--Moon-TW--2006-"><span class="Hyperlink">Ha et al. (2006)</span></a>. Mule duck was identified by the primer sets of 12S and 5S ribosomal RNA, and a-actin genes (<a href="#Rodriguez-MA--Garc--2001"><span class="Hyperlink">Rodriguez et al. 2001</span></a>, <a href="#Rodriguez-MA--Garc---a-2003a"><span class="Hyperlink">2003a</span></a>, <a href="#Rodriguez-MA--Garc--2003b"><span class="Hyperlink">2003b</span></a> and <a href="#Rodriguez-MA--Garc---a--T2004"><span class="Hyperlink">2004</span></a>).</p>
		  <p class="Body-Text ParaOverride-1" xml:lang="en-GB">&nbsp;</p>
			<p class="Body-Text" xml:lang="en-GB">Simplex and multiplex PCR using species specific primers based on the different mitochondrial genes were employed for identification of seven different animal species in meat broth samples instead of using meat directly where they used primers based on cyt b gene for bovine, goat and sheep, 12S RNA for poultry and pig , 16SRNA for ruminants, ND4 for cat and ND2 for donkey (<a href="#Rashid-PMA--Babashekh-MO--Marouf-AS--Am"><span class="Hyperlink">Rashid et al., 2014</span></a>). </p>
		  <p class="Body-Text" xml:lang="en-GB">&nbsp;</p>
			<p class="Heading-2--History-in-MM-" xml:lang="en-GB">Real-Time PCR</p>
			<p class="Body-Text ParaOverride-1" xml:lang="en-GB">The real-time PCR is promising method used for detection of meat authenticity in very complicated mixtures even if the target species present in very small amounts (<a href="#Koppel-R--Zimmerli-F--Breitenmoser-A--2009-"><span class="Hyperlink">Koppel et al., 2009</span></a>). Real-time PCR techniques help to achieve the quantitative determination of gene expression by detecting the received signals resulted from application of fluorescent pigments that help monitoring of PCR products generated in each PCR reaction cycle depending on the fluorescence intensity of these products so skipping of electrophoresis and gel staining that usually must carried out after completing the PCR reaction i.e. do not require additional detection steps. Furthermore, the possibility of contamination is rare (<a href="#Rodriguez-MA--Garc-a-T-2005"><span class="Hyperlink">Rodriguez et al., 2005</span></a>).</p>
		  <p class="Body-Text ParaOverride-1" xml:lang="en-GB">&nbsp;</p>
			<p class="Body-Text ParaOverride-1" xml:lang="en-GB">One of the chemical methods based on ﬂuorescence detection of  real-time PCR products is TaqMan probe technique. Species-speciﬁc primers and TaqMan ﬂuorogenic probes reported to be useful in inspection procedures to ensure the prober labelling of raw and heat-processed meat and meat products. TaqMan probe method based on cyt b gene was employed to identify some closely related species by <a href="#Chisholm-J--Conyers-C--Booth-C--Lawley-W--Hird-H--2005-."><span class="Hyperlink">Chisholm et al. (2005)</span></a> who developed a real time PCR to design species-specific primers to amplify cytochrome b gene, this assay could detect the meat of horse and donkey species in commercial products on the levels of 1 pg and 25 pg, respectively. Cytochrome b gene by this technique could identify beef, pork, lamb, chicken and turkey meats occurring in mixtures from raw (<a href="#Dooley-JJ--Paine-KE--Garrett-SD--Brown-HM--2004"><span class="Hyperlink">Dooley et al., 2004</span></a>) and duck meat (<a href="#Hird-H--Chisholm-2005"><span class="Hyperlink">Hird et al., 2005</span></a>). Real-time TaqMan technology based on cyt b was used for identification of deer and some domestic species (<a href="#Hird-H--Goodier-R-2004"><span class="Hyperlink">Hird et al., 2004</span></a>), and for quantiﬁcation of DNA from ostrich and other meat species (<a href="#Lopez-Andreo-M--Garrido-Pertierra-A--P"><span class="Hyperlink">Lopez-Andreo et al., 2006</span></a>). <a href="#Chisholm-J--Conyers-C--Booth-C--Lawley-W--Hird-H--2005-."><span class="Hyperlink">Chisholm et al. (2008)</span></a> used species-speciﬁc primers and TaqMan probes based on the mitochondrial cyt b gene to identify DNA from quail and pheasant in commercial food products. While, pork meat was identified by developed RT-PCR and TaqMan probe that based on amplification of the mitochondrial fragment of the 12S rRNA gene (<a href="#Rodriguez-MA--Garc-a-T-2005"><span class="Hyperlink">Rodriguez et al., 2005</span></a>). Similarly, <a href="#Rojas-M--Gonza--lez-2009b"><span class="Hyperlink">Rojas et al. (2010b)</span></a> used the same gene to identify pheasant, quail, pigeon, guinea fowl, partridge, Eurasian woodcock and song thrush. The real-time PCR technique was also succeeded to detect the different component of meat mixture congaing red deer, fallow deer, roe deer, chamois and pyrenean ibex as reported by <a href="#Fajardo-V--Gonza-2008b"><span class="Hyperlink">Fajardo et al. (2008b</span></a> and <a href="#Fajardo-V--Gonza--2008c"><span class="Hyperlink">2008c</span></a>) using species-speciﬁc primers designed on D-loop genes and 12S rRNA. TaqMan probe method was also employed for identiﬁcation of meat and meat products from different pigeon species common pigeon, woodpigeon, and stock pigeon (Columba oenas) by <a href="#Rojas-M--Gonza---lez-I--Pavo--n-MA-"><span class="Hyperlink">Rojas et al. (2012)</span></a> depending mitochondrial 12S rRNA and the nuclear 18S rRNA gene from eukaryotic DNA.</p>
		  <p class="Body-Text ParaOverride-1" xml:lang="en-GB">&nbsp;</p>
			<p class="Body-Text ParaOverride-1" xml:lang="en-GB"><a href="#Kesmen-Z--Gulluce-A--Sahin-F--Yetima-H--2009"><span class="Hyperlink">Kesmen et al. (2009)</span></a> in their study to identify meat species, designed sensitive and speciﬁc real-time PCR to design speciﬁc primers and TaqMan probes based on mitochondrial ND2 gene for donkey, ND5 gene for pork and ATP 6-8 gene for horse for differentiation and quantification of their meats in raw and cooked products. The used assay succeeded to detect very minute amounts of DNA (0.0001ng) of different tested species and meat mixtures and showed no cross-reaction was detected among the tested species and could differentiate them from chicken, turkey, ovine and bovine meats.</p>
		  <p class="Body-Text ParaOverride-1" xml:lang="en-GB">&nbsp;</p>
			<p class="Body-Text ParaOverride-1" xml:lang="en-GB"><a href="#Druml-B--Mayer-W--Cichna-Markl-M--Hochegger-R--2015"><span class="Hyperlink">Druml et al. (2015)</span></a> developed a TaqMan real-time PCR assay to quantify the roe deer content in different meat products. The percentage of roe deer content was detected depending on the myostin gene. The diluted DNA extracted from roe deer was analyzed serially and the efficiency of the amplification obtained was 93.9%, indicating the high specificity of this assay for roe deer and importance of it in detecting meat adulteration.</p>
		  <p class="Body-Text ParaOverride-1" xml:lang="en-GB">&nbsp;</p>
			<p class="Heading-2--History-in-MM-" xml:lang="en-GB">PCR-Sequencing</p>
			<p class="Body-Text ParaOverride-1" xml:lang="en-GB">PCR nucleotide sequence might be of great value for identification on the species level. Sequencing usually involves part or all of the mitochondrial genome followed by its comparison with known sequences in Gene Bank (NCBI). PCR technique is suitable and accepted but it is expensive, and needs to more time and labour consuming due to the further step of sequencing products, mixtures cannot be separated, and the generated samples may not produce enough sequence results (<a href="#Lenestra-JA--Bunjet-JB--Janssen-FW--2001-"><span class="Hyperlink">Lenstra et al., 2001</span></a>).</p>
		  <p class="Body-Text ParaOverride-1" xml:lang="en-GB">&nbsp;</p>
			<p class="Body-Text ParaOverride-1" xml:lang="en-GB">DNA Sequencing gives much information with no need for more steps like digestion with enzymes or analysis of the given data. With the help of a universal primer bands can be obtained for different animal species after PCR amplification that could help their differentiation (<a href="#Kocher-TD--Thomas-WK--Meyer-A--Edwards-SV--Paabo-S--Villablanca-FX--1989"><span class="Hyperlink">Kocher et al., 1989</span></a>). The most appropriate mitochondrial genes used for species identification using sequencing technology are cyt b, 12S and 16S rRNA genes could give a considerable amount of mutations and there are also many information found on data bases concerning their sequences (<a href="#Karlsson-AO--Holmlund-G--2007"><span class="Hyperlink">Karlsson and Holmlund, 2007</span></a>). </p>
		  <p class="Body-Text ParaOverride-1" xml:lang="en-GB">&nbsp;</p>
			<p class="Body-Text ParaOverride-1" xml:lang="en-GB">The cyt b gene has been completely partially or sequenced for identification of numerous different species of birds, mammals (<a href="#Bravi-CM--Liron-JP--Mirol-PM--Ripoli-MV--Garcia-PP--Giovambattista-G--2004-"><span class="Hyperlink">Bravi et al., 2004</span></a>; <a href="#Andrzej-D--Kamila-K--2005-"><span class="Hyperlink">Andrzej and Kamila, 2005</span></a>), fishes, amphibian and reptiles (<a href="#Chow-S--Clarke-ME--Walsh-PJ--1993"><span class="Hyperlink">Chow et al., 1993</span></a>; <a href="#Ram-JL--Ram-ML--Baidoun-FF--1996-."><span class="Hyperlink">Ram et al., 1996</span></a>; <a href="#Quinterio-J--Sotelo-CG--Rehbein-H--Pryd"><span class="Hyperlink">Quinterio et al., 1998</span></a>; <a href="#Lindstrom-DP--1999"><span class="Hyperlink">Lindstrom, 1999</span></a>; <a href="#Parson-W--Pegoraro-K--Niederstatter-H--Foger-M--Steinlechner-M--2000"><span class="Hyperlink">Parson et al., 2000</span></a>) and also some invertebrates (<a href="#Lenestra-JA--Bunjet-JB--Janssen-FW--2001-"><span class="Hyperlink">Lee et al., 2009</span></a>). In addition, <a href="#Chikuni-K--Tabata-T--Kosugiyama-M---Monma-M--1994-"><span class="Hyperlink">Chikuni et al. (1994)</span></a>; <a href="#Matsunaga-T--Chikuni-K-"><span class="Hyperlink">Matsunaga et al. (1998)</span></a>; <a href="#La-Neve-F--Civera--T--Mucci--N--Bottero-MT--2008-"><span class="Hyperlink">La Neve et al. (2008)</span></a> used cyt b gene sequence to identify of meats and meat products of red deer, roe deer, song thrush, pyrenean ibex, chamois, quail and sparrow. </p>
		  <p class="Body-Text ParaOverride-1" xml:lang="en-GB">&nbsp;</p>
			<p class="Body-Text" xml:lang="en-GB">The variations in the sequences of mitochondrial 12S and 16S rRNA gene are suitable and suﬃcient for identiﬁcation between different species from high number of vertebrates such as birds, fish, reptiles, mammals and amphibians (<a href="#Kocher-TD--Thomas-WK--Meyer-A--Edwards-SV--Paabo-S--Villablanca-FX--1989"><span class="Hyperlink">Kocher et al., 1989</span></a>; <a href="#Prakash-S--Patole-MS--Ghumatkar-SV--Nandode-SK--Yogesh-S--Shouche--2000-"><span class="Hyperlink">Prakash et al., 2000</span></a>; <a href="#Kitano-T--Umetsu-K--Tian-W--Osawa-M--2007"><span class="Hyperlink">Kitano et al., 2007</span></a>; <a href="#Karlsson-AO--Holmlund-G--2007"><span class="Hyperlink">Karlsson and Holmlund, 2007</span></a>). Mitochondrial 12S rRNA gene sequenced to identify ostrich, emu, guinea fowl, and quail meats (<a href="#Girish-PS--Anjaney2009"><span class="Hyperlink">Girish et al., 2009</span></a>). <a href="#Rastogi-G--Dharne-M--Bhar"><span class="Hyperlink">Rostogi et al. (2004)</span></a> assessed the use of  the amplification and sequencing of 450 pb fragment of mitochondrial 12S rRNA gene universal primers to identify the species of origin of raw and cooked meat samples, viscera, blood and semen. This technique was found to identify all studied samples on species level, even if samples exposed to preservation at ambient temperature for long times. They then detected the meat adulteration using conformation-sensitive gel electrophoresis (CSGE) that was found to be of importance in authentication of meat in the field of forensic food analysis. </p>
		  <p class="Body-Text" xml:lang="en-GB">&nbsp;</p>
			<p class="Body-Text ParaOverride-1" xml:lang="en-GB"><a href="#Abuzinadah-OH--Yacoub-HA--El-Ashmaoui-HM--Ramadan-HA--2013"><span class="Hyperlink">Abuzinadah et al. (2013)</span></a> used sequencing of DNA fragment of the mitochondrial 12SrRNA gene followed by confirmation using species specific primer to detect the adulteration of chicken products including (Luncheon, burger, sausage and minced meat). They could detect the substitution of all samples used by inedible parts of turkey. <a href="#Li-B--Bai-SY--Xu-YC--Zhang-W--Ma-JZ--2006"><span class="Hyperlink">Li et al. (2006)</span></a> identify cervid species by sequence analysis of 12S rRNA and cyt b genes. <a href="#Cawthorn--DM---Harris-A-Steinman--Louwrens-C-Hoffman--2013-"><span class="Hyperlink">Cawthorn et al. (2013)</span></a> used a DNA-based LCD array followed by confirmation Species specific PCR and DNA and sequencing of Cyt b, 12SrRNA and ND2 genes to detect the fraud and mislabeling of meat products. 68% of samples used found to contain species that are not indicated on the product label. The diglycerideacyltransferase 1 (DGAT1) or 18S rRNA have been sequenced for the differentiation of meats from these species buffalo, crocodile and kangaroo (<a href="#Matsunaga-T--Chikuni-K--Tanabe-R--Muroya--S--Shibata--Yamada-J--Shinmura-Y--1999-."><span class="Hyperlink">Matsunaga et al., 1998</span></a>; <a href="#Venkatachalapathy-RT--Sharma-A--Sukla-S--Hattacharya-TK--2008"><span class="Hyperlink">Venkatachalapathy et al., 2008</span></a>).</p>
		  <p class="Body-Text ParaOverride-1" xml:lang="en-GB">&nbsp;</p>
			<p class="Heading-1--Introduction----" xml:lang="en-GB">THE ROLE OF PROTEOMIC TECHNOLOGY AND PEPTIDE BIOMARKERS IN IDENTIFICATION OF MEAT ORIGIN</p>
			<p class="Heading-1--Introduction----" xml:lang="en-GB">&nbsp;</p>
			<p class="Body-Text ParaOverride-1" xml:lang="en-GB">The great advances in the application of mass spectrometry in the peptides and proteins analysis make the proteomic technology gaining attention as alternative to the other methods used for species identification and meat authentication issues (<a href="#Montowska-M--Pospiech-E--2012a"><span class="Hyperlink">Montowska and Pospiech 2011a</span></a> and <a href="#Montowska-M--Pospiech-E--2012a"><span class="Hyperlink">2012a</span></a>). Mass spectrometry has also been successfully employed to study the protein maps of muscles that differ in their composition of fiber (<a href="#Hamelin-M--Sayd-T--Chambon-C--Bouix-J--Bibe-B--Milenkovic-D--2007"><span class="Hyperlink">Hamelin et al., 2007</span></a>), or in the identification of muscles derived from different genetic origins (<a href="#McDonagh-MB--Ferguson-KL--Bacic-A--Gardner-GE--Hegarty-RS--2006-"><span class="Hyperlink">McDonagh et al., 2006</span></a>; <a href="#Hollung-K--Grove--H--Faergestad--EM--Sidhu--MS--Berg-P--2009"><span class="Hyperlink">Hollung et al., 2009</span></a>) with a discriminating activity near to DNA- based analytical methods. However, the proteomic technology can overcome some of the limitations that face methods based on the analysis of DNA, in particularly the quantitative analysis of thermally processed food and meats that exposed to high degrees of temperature during processing as the peptide´s amino acid sequences are highly resistant to different processing conditions than DNA additionally the proteins and peptides are easily extracted compared to DNA (<a href="#Ortea-I--Ca-as-B--Calo-Mata-P--Barros-Vel-zquez-J--Gallardo-JM--2009"><span class="Hyperlink">Ortea et al., 2009</span></a>; <a href="#Montowska-M--Pospiech-E--2012a"><span class="Hyperlink">Montowska and Pospiech 2011b</span></a>; <a href="#Montowska-M--Pospiech-E--2013"><span class="Hyperlink">Montowska and Pospiech, 2013</span></a>). Mass spectrometry method could help in identification of different meats depending on the mass differences between hemoglobins and myoglobins of the different meat species. This method was helpful in detection of horse hemoglobin that was present in a mixture with that of beef (<a href="#Taylor-AJ--Linforth-R--Weir-O--Hutto"><span class="Hyperlink">Taylor et al., 1993</span></a>).</p>
		  <p class="Body-Text ParaOverride-1" xml:lang="en-GB">&nbsp;</p>
			<p class="Body-Text ParaOverride-1" xml:lang="en-GB">Proteomic technology could also successfully identify specific proteins of the different ﬁsh species in both fresh and processed ﬁsh products (<a href="#Carrera-M--Ca-as-B--Pi-eiro-C--V-zquez-J--Gallardo-JM--2007-."><span class="Hyperlink">Carrera et al., 2007</span></a>; <a href="#Mazzeo-MF--De-Giulio-B--Gue"><span class="Hyperlink">Mazzeo et al., 2008</span></a>; <a href="#Ortea-I--Ca-as-B--Calo-Mata-P--Barros-Vel-zquez-J--Gallardo-JM--2009"><span class="Hyperlink">Ortea et al., 2009</span></a>) based on the use of MALDI–TOF MS and MALDI–TOF and LC–ESI–MS/MS as a rapid screening method. By the same spectrometry, <a href="#Sentandreu-MA--Fraser-PD--Halket-J--Patel-R--Bramley-PM--2010-."><span class="Hyperlink">Sentandreu et al. (2010)</span></a> reported that the proteomic technology could differentiate between turkey and chicken meats by the use of species-speciﬁc peptides derived from digestion of myosin light chain 3 (MLC-3) using in-solution trypsin digestion. On the same context, <a href="#Montowska-M--Pospiech-E--2012b"><span class="Hyperlink">Montowska and Pospiech (2011b</span></a> and <a href="#Montowska-M--Pospiech-E--2012b"><span class="Hyperlink">2012b</span></a>) observed the inter-species differences in myosin light chain isoforms (MLC) in the raw and processed meat and meat products of some animal species including some poultry species (chicken, turkey, duck and goose) in addition to pig and cattle depending on the species-speciﬁc electrophoretic mobility. <a href="#Zhao-M--Downey-G--O-Donnell-CP--2014"><span class="Hyperlink">Zhao et al. (2014)</span></a> reported that mid-infrared ATR spectroscopy could help in identifying the authentic higher and lower quality beef burger samples from other samples adultrated by beef offal under fresh and freezing conditions. Raman spectroscopy enables rapid determination of beef adulteration with horsemeat with high accuracy, few seconds for analysis and no sample preparation (<a href="#Boyac---H-Temiz-HT--Uysal-RS--Velio-lu-HM--Yadegari-RJ--Rishkan-MM.--2014-"><span class="Hyperlink">Boyaci et al., 2014</span></a>).</p>
		  <p class="Body-Text ParaOverride-1" xml:lang="en-GB">&nbsp;</p>
			<p class="Body-Text ParaOverride-1" xml:lang="en-GB">Proteomic approaches depending on specific peptide biomarkers were also investigated by <a href="#Montowska-M--Pospiech-E--2013"><span class="Hyperlink">Montowska and Pospiech (2013)</span></a> who used some regulatory proteins, nmetabolic enzymes and myoﬁbrillar proteins (troponin T and tropomodulin) to identify the different meat species. They observed inter-species differences in protein expression in raw meat, thermally processed meat and ready-made products. Especially in albumin and apolipoprotein B; the regulatory proteins (HSP27 and H-FABP) and the metabolic enzymes ATP synthase, cytochromebc-1 subunit 1 and alpha-ETF that were greatly differs in their species-speciﬁc electrophoretic mobility. The differences in the sequences of obtained fragments were species-spesific and very valuable in identification of poultry meat (chicken and turkey) as well as cattle and pig. The use of specific peptide sequences was highly valuable  in differentiation between gelatin from bovine and porcine as described by <a href="#Zhang-G-F--Liu-T--Wang-Q--Chen-L"><span class="Hyperlink">Zhang et al. (2009)</span></a> although the sequence of collagen in mammals are highly homogenous. <a href="#Shibata-M--Matsumoto-K--Oe-M"><span class="Hyperlink">Shibata et al. (2009)</span></a> observed many species differences in the proteomes of Japanese Black Cattle that fed on grain than those fed on grass. </p>
		  <p class="Body-Text ParaOverride-1" xml:lang="en-GB">&nbsp;</p>
			<p class="Body-Text" xml:lang="en-GB">Moreover, the proteomic approaches depending on identiﬁcation of different peptide biomarkers could also employed to give information on the different composition of food. The quantity of individual type of sarcoplasmic and myoﬁbrillar proteins found to be different from one type of muscle to another of the same animal as described for white and red skeletal muscles of pig (<a href="#Kim-SH--Huang-TS--Seymour-TA-Wei-C--Kempf-SC--Bridgman-CR--Clemens-RA--An-H--2004"><span class="Hyperlink">Kim et al., 2004</span></a>), sheep (<a href="#Hamelin-M--Sayd-T--Chambon-C--Bouix-J--Bibe-B--Milenkovic-D--2007"><span class="Hyperlink">Hamelin et al., 2007</span></a>) and Bayonne ham (<a href="#Th-ron-L--Sayd-T--Pinguet-J--Chambon-C--Robert-N--Sant--Lhoutellier-V--2"><span class="Hyperlink">Théron et al., 2011</span></a>). </p>
		  <p class="Body-Text" xml:lang="en-GB">&nbsp;</p>
			<p class="Body-Text ParaOverride-1" xml:lang="en-GB"><a href="#Jorfi-R--Shuhaimi-M--Yaakob-BCM--Dzul"><span class="Hyperlink">Jorfi et al. (2012)</span></a> used the amino acid content of meat as markers for halal meat authentication, their method succeeded to identify pork meat from other meat species including mutton , beef, chevon and chicken by the use of reverse phase-high performance liquid chromatography (RP-HPLC) followed by o-phthalaldehyde (OPA) derivatization and ultraviolet (UV) detection where histidine, alanine, serine, valine and arginine found to be the highly discriminative ones between porcine and other species meat. <a href="#Giaretta--N--Di-Giuseppe-AM--Lippert-M--Parente-A--Di-Maro-A--2013"><span class="Hyperlink">Giaretta et al. (2013)</span></a> stated that myoglobin could be used as a marker in identifying the pork meat in the raw beef burger using ultra-performance liquid chromatography (UPLC). Where, the percentages of pork and beef meat can be quantified in premixed minced meat samples from different animal origin like beef, chicken, horse, ostrich, pig and water buffalo.</p>
		  <p class="Body-Text ParaOverride-1" xml:lang="en-GB">&nbsp;</p>
			<p class="Heading-1--Introduction----" xml:lang="en-GB">CONCLUSION</p>
		  <p class="Heading-1--Introduction----" xml:lang="en-GB">&nbsp;</p>
			<p class="Body-Text ParaOverride-1" xml:lang="en-GB">The problem of protein denaturation after exposure to high temperature during food processing makes protein based techniques not adequate for meat species identification because this denaturation of protein could lead to many problems such as changing the antigenicity of the molecules and the possible cross reaction between the closely related species. So the attention was directed towards applying of the DNA-based methods. Because DNA is more stable under different processing conditions and its conserved structure gives it a high discriminating properties. Both mitochondrial and nuclear genes have been broadly targeted for the identiﬁcation of species of meat and meat products. The mitochondrial markers were proved to be more valuable than nuclear markers in species identiﬁcation and authentication since the mitochondrial DNA is maternally inherited, mitochondrial genes have variable regions that are found in thousands per individual cell that facilitates PCR ampliﬁcation improve the assay sensitivity allowing the achievement of positive result even if the DNA was severely fragmented or damaged under intense conditions of food processing. These properties give the probability to mitochondrial DNA to identify origin of meat in processed meat products and make mitochondrial markers highly efficient than nuclear ones in identiﬁcation and authentication of meat species in fresh, cooked and autoclaved meat and meat products. Among the mitochondrial genes, the cyt b geneD-loop12S rRNA16S rRNA have been used for species identiﬁcation. PCR is the most well developed DNA based methods until now that provides a wide range of analytical method which could be used for rapid detection and identiﬁcation at species and intra-species level. PCR-based methods most frequently used for meat species identiﬁcation include PCR-RFLP, PCR-RAPD Species specific primers, RT-PCR and PCR-nucleotide sequencing. Nevertheless, the PCR-RAPD is not suitable for identification of meat species in meat mixtures and intensively heated products. Also the PCR-RLFP technique could not give accurate information on the composition of meat present in mixtures. Sequencing of mitochondrial genes is costly, time and labour consuming and interpretation of the results is difficult. Additionally, DNA still facing certain limitations in both quantitative analysis and degradation due to temperature and pressure used for food processing that may alter the results of PCR ampliﬁcation especially with long DNA fragments. So many researchers directed toward developing new technologies that could overcome these limitations. One of the new developing technologies that could be successfully used to assess the meat authenticity is the proteomic technology that depends on analysis of protein and peptide biomarkers. This technique has been proved by many researchers to have a discriminating power comparable to that of DNA-based analysis and could be used for quantitative determination of the composition of thermally processed meats as the sequences of peptide’s amino acids are easily extracted and highly resistant at high temperature than DNA. </p>
		  <p class="Body-Text ParaOverride-1" xml:lang="en-GB">&nbsp;</p>
			<p class="Heading-1--Introduction----" xml:lang="en-GB">ACKNOWLEDGEMENTS</p>
		  <p class="Heading-1--Introduction----" xml:lang="en-GB">&nbsp;</p>
			<p class="Body-Text ParaOverride-1" xml:lang="en-GB">All the authors of the manuscript thank and acknowledge their respective Universities and Institutes.</p>
			<p class="Heading-1--Introduction----" xml:lang="en-GB">&nbsp;</p>
			<p class="Heading-1--Introduction----" xml:lang="en-GB">REFERENCES</p>
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