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<body id="AAVS_Nexus-610" lang="en-GB">
		<div class="Basic-Text-Frame">
			<p class="Type-of-Article">&nbsp;</p>
			<p class="Type-of-Article"><span class="CharOverride-1">Research Article</span></p>
	</div>
		<div class="Basic-Text-Frame">
			<p class="title- ParaOverride-1">&nbsp;</p>
			<p class="title- ParaOverride-1">Identification of some Domestic Animal Species (Camel, Buffalo and Sheep) by PCR-RFLP Analysis of the Mitochondrial Cytochrome b Gene</p>
		</div>
		<div class="Basic-Text-Frame">
			<p class="Authors ParaOverride-1">&nbsp;</p>
			<p class="Authors ParaOverride-1"><span class="CharOverride-2">Mayada Ragab Farag</span><span class="CharOverride-3">1*</span><span class="CharOverride-2">, Tamer Said Imam</span><span class="CharOverride-3">1</span><span class="CharOverride-2">, Kuldeep Dhama</span><span class="CharOverride-3">2</span></p>
		</div>
		<div class="Basic-Text-Frame">
			<p class="Affiliations ParaOverride-1"><span class="CharOverride-4">1</span>Department of Forensic Medicine and Toxicology, Faculty of Veterinary Medicine, Zagazig University, Zagazig 44111, Egypt; <span class="CharOverride-4">2</span>Division of Pathology, Indian Veterinary Research Institute, Izatnagar, Bareilly, 243122, Uttar Pradesh, India.</p>
		</div>
		<div>
			<p class="Abstract ParaOverride-1">&nbsp;</p>
			<p class="Abstract ParaOverride-1"><span class="CharOverride-5">Abstract</span> | The present study describes the use of PCR and PCR-RFLP methods to discriminate between blood samples obtained from domestic camel, buffalo and sheep. The DNA was extracted from 3 blood samples of each species at 3 different ages and subjected to PCR amplification of a mitochondrial cytochrome b (cyt b) gene segment (358 bp) using universal primers. Subsequent cleavage with 4 restriction endonucleases (<span class="CharOverride-6">Alu</span>I, <span class="CharOverride-6">Hae</span>III, <span class="CharOverride-6">Hinf</span>I and <span class="CharOverride-6">Taq</span> I) gaverise to a species-specific pattern on an agarose gel. Results of cleavage by <span class="CharOverride-6">Alu</span>I<span class="CharOverride-6"> and Hae</span>III were 2 fragments of different sizes in all animals; <span class="CharOverride-6">Hinf </span>I produced 2 fragments in camel and sheep and 3 fragments in buffalo; <span class="CharOverride-6">Taq</span> I produced 2 bands in camel and sheep while no fragmentation was generated for buffalo. The results suggest that the method of PCR-RFLP using these restriction enzymes can reliably identify chosen species. Moreover, PCR-RFLP is a rapid and simple method for identification of animal species.</p>
		  <p class="Abstract ParaOverride-1">&nbsp;</p>
			<p class="Abstract ParaOverride-1"><span class="CharOverride-5">Keywords </span>| Species identification, Animals, Cytochrome b gene, PCR-RFLP, Forensic samples</p>
		  <p class="Abstract ParaOverride-1">&nbsp;</p>
		</div>
		<div class="Basic-Text-Frame">
			<p class="Editor----Citation"><span class="CharOverride-8">Editor</span> | Kuldeep Dhama, Indian Veterinary Research Institute, Uttar Pradesh, India.</p>
			<p class="Editor----Citation"><span class="CharOverride-5">Received</span> | January  06, 2015; <span class="CharOverride-5">Revised</span> | January 24, 2015; <span class="CharOverride-5">Accepted</span> | January 25, 2015; <span class="CharOverride-5">Published</span> | January 30, 2015&#9;&#9;</p>
			<p class="Editor----Citation"><span class="CharOverride-5">*Correspondence</span> | Mayada Ragab Farag, Zagazig University, Zagazig, Egypt; <span class="CharOverride-5">Email:</span> dr.mayadarf@gmail.com</p>
			<p class="Editor----Citation"><span class="CharOverride-5">Citation</span> | Farag MR, Imam TS, Dhama K (2015). Identification of some domestic animal species (camel, buffalo and sheep) by PCR-RFLP analysis of the mitochondrial cytochrome b gene. Adv. Anim. Vet. Sci. 3(2): 136-142.  </p>
			<p class="Editor----Citation"><span class="CharOverride-8">DOI</span> | <a href="http://dx.doi.org/10.14737/journal.aavs/2015/3.2.136.142"><span class="Hyperlink">http://dx.doi.org/10.14737/journal.aavs/2015/3.2.136.142</span></a></p>
			<p class="Editor----Citation"><span class="Editor---Citation CharOverride-5" lang="en-US">ISSN </span><span class="Editor---Citation CharOverride-5" lang="en-US">(</span><span class="Editor---Citation CharOverride-5" lang="en-US">Online<span class="Editor---Citation CharOverride-5" lang="en-US">)</span> | 2307-8316; <span class="Editor---Citation CharOverride-5" lang="en-US">ISSN </span><span class="Editor---Citation CharOverride-5" lang="en-US">(Print) </span> | 2309-3331</p>
			<p class="Editor----Citation"><span class="CharOverride-5">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>
		</div>
		<div class="Basic-Text-Frame">
			<p class="Heading-1--Introduction----">&nbsp; </p>
			<p class="Heading-1--Introduction----">&nbsp;</p>
		  <p class="Heading-1--Introduction----">INTRODUCTION </p>
			<p class="Caps-on-First-Para ParaOverride-1">&nbsp;</p>
			<p class="Caps-on-First-Para ParaOverride-1"><span class="_idGenDropcap-1">D</span>etermining the species origin of different kind of samples (blood, meat, skin, etc.) is important in forensic purpose for species differentiation and identification (<a href="#Sherryn-AC--Shanan-ST--Stephen-CD--Julianne-MH--Adrian-MTL--2015-"><span class="Hyperlink">Sherryn et al., 2015</span></a>) and it is also an integral part of food regulatory issue as adulteration and substitution of meat has always been a concern for various reasons such as public health, religious factors, wholesomeness and unhealthy competition in meat market (<a href="#Rashid-PM--Babashekh-MO--Marouf-AS--Amin-KM--2014-.-I"><span class="Hyperlink">Rashid et al., 2014</span></a>; <a href="#Hou-B--Xianrong-M--Liyuan-Z--Jinyue-G--Shaowen-L--Hui-J--2015-"><span class="Hyperlink">Hou et al., 2015</span></a>).Various methods like traditional morphological or protein identification methods, species specific markers or repeats (SSR), mitochondrial DNA restricted fragment length polymorphism (mtDNA-RFLP), and random amplified polymorphic PCR (RAPD-PCR) are employed for detection of the species origin of different forensic samples and meat, and among these DNA based assays are gaining popularity (<a href="#Cheng-X--Weiling-H--Feng-H--Ming-H--Guanghong-Z--2014-."><span class="Hyperlink">Cheng et al., 2014</span></a>; <a href="#Lin-Y--Hsieh-H--James-L--Chung-Ting-H--Der-Yuh-L--Adrian-L--Li-Chin-T--2014-"><span class="Hyperlink">Lin et al., 2014</span></a>). Particularly the mitochondrial DNA (mtDNA) has been the most widely studied region of eukaryotic genomes which has played a critical role in development of population and evolutionary genetics (<a href="#Abo-Hadeed-AH--Mervat-HG--Mayada-RF--2011-"><span class="Hyperlink">Abou-Hadeed et al., 2011</span></a>; <a href="#DeMasi-L--Adelfi-MG--Pignone-D--Laratta-B--2015-."><span class="Hyperlink">DeMasi et al., 2015</span></a>). It has several advantages over nuclear genome for diagnostic studies of animals because of greater abundance in sample extracts and higher copy number making the amplification of mtDNA segment to be a relatively sensitive procedure (<a href="#Sumathi-G-Jeyasekaran-G--JeyaShakila-R--Sivaraman-B--Arunkumar-G--Manimaran-U--Sukumar-D--2015-"><span class="Hyperlink">Sumathi et al., 2015</span></a>).</p>
		  <p class="Caps-on-First-Para ParaOverride-1">&nbsp;</p>
			<p class="Body-Text ParaOverride-1">The cytochrome b gene (cyt b) encodes one of the best known proteins that make up complex III of the mitochondrial phosphorylation system and is the only one encoded by the mitochondrial genome. The cyt b gene is used as an important utility tool in studies of legal medicine and molecular evolution (<a href="#Prusak-B--GrzybowskiG--Zieba-G--2004-."><span class="Hyperlink">Prusak et al., 2004</span></a>; <a href="#Abo-Hadeed-AH--Mervat-HG--Mayada-RF--2011-"><span class="Hyperlink">Abou-Hadeed et al., 2011</span></a>). This  gene has been completely sequenced or partially sequencedfor many species of mammals, birds (<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>), reptiles, amphibian and fishes (<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="#Quinteiro-J--Sotelo-CG--Rehbein-H--Pryde-SE--Perez-Martin-RI--Rey-Mendez-M--Mackie-IM--1998-."><span class="Hyperlink">Quinterio et al., 1998</span></a>; <a href="#Lindstrom-DP--1999-"><span class="Hyperlink">Lidstrom, 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="#Lee-JC--Tasi-LC--Liao-SP--Linacrre-A--Hsieh-HM--2009a-"><span class="Hyperlink">Lee et al., 2009a</span></a>). </p>
		  <p class="Body-Text ParaOverride-1">&nbsp;</p>
			<p class="Body-Text ParaOverride-1">Species identification studies based on the cyt b gene have ranged from both short (&lt; 400 base pair) to long (&gt; 900 base pair) PCR-RFLP DNA sequencing and variable size species specific multiplex PCR (<a href="#Lee-JC--Hsieh-HM--Huang-LH--Kuo-YC--Wu-JH--Chin-SC--Lee-AH--Linacre-A--Tasi-LC--2009b-"><span class="Hyperlink">Lee et al., 2009b</span></a>). An alternative DNA detection system is based on the PCR amplification of a segment of the mitochondrial cyt b gene followed by RFLP analysis based on subsequent cleavage by a restriction enzymes which gives rise to species specific pattern (<a href="#Ahmed-MMM--2007-."><span class="Hyperlink">Ahmed, 2007</span></a>; <a href="#Abdel-Rahman-SM--El-Saadani-MA--Ashry-KM--Haggag-AS--2009-"><span class="Hyperlink">Abd El-Rahman et al., 2009</span></a>). This technique requires less equipment and cost than sequencing.</p>
		  <p class="Body-Text ParaOverride-1">&nbsp;</p>
			<p class="Body-Text ParaOverride-1">One of the most important issues in forensic medicine is to identify the sample’s origin and discriminate whether they descend from human or non-human origins. Therefore, the present study was aimed atexploit the molecular technique of PCR-RFLP analysis of the mitochondrial cytochrome b gene followed by subsequent cleave using 4 restriction enzymes <span class="CharOverride-6">(Alu</span>I, <span class="CharOverride-6">Hae</span>III, <span class="CharOverride-6">Hinf</span>I and <span class="CharOverride-6">Taq</span> I) for differentiation between  camel, buffalo and sheep. These restriction enzymes don’t used before for identification of camel and do not used collectively in these species where <span class="CharOverride-6">Hinf</span>I and <span class="CharOverride-6">Taq </span>I have been used only for buffalo not for camel and sheep identification.</p>
			<p class="Body-Text ParaOverride-1">&nbsp;</p>
		  <p class="Body-Text ParaOverride-1">&nbsp;</p>
			<p class="Heading-1--Introduction----">MATERIALS AND METHODS</p>
		  <p class="Heading-1--Introduction----">&nbsp;</p>
			<p class="Heading-2--History-in-MM-">Samples</p>
			<p class="Body-Text ParaOverride-1">The 5 ml blood samples were collected from domestic camel, buffalo and sheep (3 each from each different age: new born, young and adult), reared in local animal farms in Zagazig, Egypt. The DNA was extracted from the blood samples of camel, buffalo and sheep using QIAamp® tissue kit (QIAGEN GmbH, Hilden, Germany) according to the manufacturer’s instructions. The concentration and purity of the extracted DNA samples were measured by NanoDrop® ND-1000 (Full- Spectrum UV Spectrophotometer at 260/280 nm) as presented in <a href="#Table-1-"><span class="Hyperlink">Table 1</span></a>.</p>
		  <p class="Body-Text ParaOverride-1">&nbsp;</p>
			<p class="Body-Text ParaOverride-1">The blood DNA samples of the three animal species were subjected to PCR amplification of the mitochondrial cytochrome b gene. The primers employed were:  Primer 1 (L 14816) (5<span class="CharOverride-4">´</span>- CCA TCC AAC ATC TCA GCA TGATGA AA – 3’) = P1: conc. 0.25 µM; Primer 2 (H15173) (5’-CCC CTC AGA ATG ATA TTT GTC CTC A-3’)= P2: conc. 0.25 µM (Metabion international AG, Deutschland).The PCR Master mix (RED TaqReadyMix PCR Reaction Mix, with   MgCl<span class="CharOverride-11">2</span>)  used comprised of a total volume of 25 µl containing 20 mMTris- HCl, Ph 8.3, with 100MKCl, 3 mM MgCl<span class="CharOverride-11">2</span>, 0.002% gelatin, 0.4 mMdNTP  mix (dATP, dCTP, dGTP, TTP), stabilizers, and 0.06 unit/µl of Taq DNA polymerase (Sigma) and nuclease free water (NFW, PCR reaction water) (Sigma).The amount of PCR mix, primers, DNA samples and water used in the PCR reaction for amplification of the cyt b gene fragment from the blood samples of the three animal species is presented in <a href="#Table-2-"><span class="Hyperlink">Table 2</span></a>.</p>
			<p class="Body-Text ParaOverride-1">&nbsp;</p>
			<p class="Body-Text ParaOverride-1">&nbsp; </p>
			<p class="Figure--and-Table-Heading ParaOverride-1"><span class="CharOverride-5"><a id="Table-1-"></a>Table 1: </span>The concentration and purity of DNA extracted from blood samples</p>
			<table width="657" height="94" class="Table-Style-1" id="table-1">
				<colgroup>
					<col class="_idGenTableRowColumn-1" />
					<col class="_idGenTableRowColumn-2" />
					<col class="_idGenTableRowColumn-3" />
				</colgroup>
				<tbody>
					<tr class="_idGenTableRowColumn-4">
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-12">The sample</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-12">Concentration (ng/ µl)</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-12">Purity (at 260/280nm)</span></p>
						</td>
					</tr>
					<tr class="_idGenTableRowColumn-5">
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-13">Camel</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-13">40.20</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-13">1.8</span></p>
						</td>
					</tr>
					<tr class="_idGenTableRowColumn-6">
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-13">Buffalo</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-13">90.77</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-13">1.8</span></p>
						</td>
					</tr>
					<tr class="_idGenTableRowColumn-5">
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-13">Sheep</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-13">190.3</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-13">1.4</span></p>
						</td>
					</tr>
				</tbody>
			</table>
			<p class="Figure--and-Table-Heading ParaOverride-1">&nbsp;</p>
			<p class="Figure--and-Table-Heading ParaOverride-1">&nbsp;</p>
		  <p class="Figure--and-Table-Heading ParaOverride-1"><span class="CharOverride-5"><a id="Table-2-"></a>Table 2: </span>PCR reaction - PCR mix, primers, DNA sample and water used in amplification of cyt b gene fragment of three animal species</p>
			<table width="657" height="163" class="Table-Style-1" id="table-2">
				<colgroup>
					<col class="_idGenTableRowColumn-7" />
					<col class="_idGenTableRowColumn-8" />
					<col class="_idGenTableRowColumn-9" />
					<col class="_idGenTableRowColumn-10" />
				</colgroup>
				<tbody>
					<tr class="_idGenTableRowColumn-6">
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-12">PCR requirements </span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-12">Camel</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-12">Buffalo</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-12">Sheep</span></p>
						</td>
					</tr>
					<tr class="_idGenTableRowColumn-5">
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-13">RED Taqreadymix (µl)</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-13">12.5</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-13">12.5</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-13">12.5</span></p>
						</td>
					</tr>
					<tr class="_idGenTableRowColumn-6">
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-13">Forward primer (µl)</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-13">1</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-13">1</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-13">1</span></p>
						</td>
					</tr>
					<tr class="_idGenTableRowColumn-5">
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-13">Reverse primer (µl)</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-13">1</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-13">1</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-13">1</span></p>
						</td>
					</tr>
					<tr class="_idGenTableRowColumn-11">
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-13">DNA template (sample) (µl)</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-13">2.5</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-13">3.5</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-13">4</span></p>
						</td>
					</tr>
					<tr class="_idGenTableRowColumn-12">
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-13">PCR reaction water (µl)</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-13">8.0</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-13">7.0</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-13">6.5</span></p>
						</td>
					</tr>
					<tr class="_idGenTableRowColumn-6">
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-13">Total volume (µl)</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-13">25</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-13">25</span></p>
						</td>
						<td>
							<p class="Basic-Paragraph"><span class="CharOverride-13">25</span></p>
						</td>
					</tr>
				</tbody>
			</table>
			<p class="Figure--and-Table-Heading ParaOverride-1">&nbsp;</p>
			<p class="Figure--and-Table-Heading ParaOverride-1">&nbsp;</p>
		  <p class="Figure--and-Table-Heading ParaOverride-1"><span class="CharOverride-5"><a id="Table-3-"></a>Table 3: </span>RFLP banding pattern of cyt b gene PCR amplified fragment afterdigestion with different restriction endonucleases</p>
			<table width="657" height="121" class="Table-Style-1" id="table-3">
				<colgroup>
					<col class="_idGenTableRowColumn-13" />
					<col class="_idGenTableRowColumn-14" />
					<col class="_idGenTableRowColumn-15" />
					<col class="_idGenTableRowColumn-16" />
					<col class="_idGenTableRowColumn-17" />
				</colgroup>
				<tbody>
					<tr class="_idGenTableRowColumn-6">
						<td rowspan="2">
							<p class="Body-Text ParaOverride-1"><span class="CharOverride-5">Animal species</span></p>
						</td>
						<td colspan="4">
							<p class="Body-Text ParaOverride-1"><span class="CharOverride-5">Restriction endonuclease cleavage pattern</span></p>
						</td>
					</tr>
					<tr class="_idGenTableRowColumn-18">
						<td>
							<p class="Body-Text ParaOverride-1"><span class="CharOverride-15">Alu</span><span class="CharOverride-5"> I</span></p>
						</td>
						<td>
							<p class="Body-Text ParaOverride-1"><span class="CharOverride-15">Hae</span><span class="CharOverride-5">III</span></p>
						</td>
						<td>
							<p class="Body-Text ParaOverride-1"><span class="CharOverride-15">Hinf</span><span class="CharOverride-5">I</span></p>
						</td>
						<td>
							<p class="Body-Text ParaOverride-1"><span class="CharOverride-15">Taq</span><span class="CharOverride-5"> I</span></p>
						</td>
					</tr>
					<tr class="_idGenTableRowColumn-6">
						<td>
							<p class="Body-Text ParaOverride-1">Camel </p>
						</td>
						<td>
							<p class="Body-Text ParaOverride-1">2 (198+160)</p>
						</td>
						<td>
							<p class="Body-Text ParaOverride-1">2 (238+120)</p>
						</td>
						<td>
							<p class="Body-Text ParaOverride-1">2 (300+58)</p>
						</td>
						<td>
							<p class="Body-Text ParaOverride-1">2 (185+173)</p>
						</td>
					</tr>
					<tr class="_idGenTableRowColumn-5">
						<td>
							<p class="Body-Text ParaOverride-1">buffalo </p>
						</td>
						<td>
							<p class="Body-Text ParaOverride-1">2 (290+68)</p>
						</td>
						<td>
							<p class="Body-Text ParaOverride-1">2 (259+99)</p>
						</td>
						<td>
							<p class="Body-Text ParaOverride-1">3(195+145+48)</p>
						</td>
						<td>
							<p class="Body-Text ParaOverride-1">1 (358)</p>
						</td>
					</tr>
					<tr class="_idGenTableRowColumn-6">
						<td>
							<p class="Body-Text ParaOverride-1">Sheep  </p>
						</td>
						<td>
							<p class="Body-Text ParaOverride-1">2  (312+46)</p>
						</td>
						<td>
							<p class="Body-Text ParaOverride-1">2 (278+80)</p>
						</td>
						<td>
							<p class="Body-Text ParaOverride-1">2(200+158)</p>
						</td>
						<td>
							<p class="Body-Text ParaOverride-1">2 (260+98)</p>
						</td>
					</tr>
				</tbody>
			</table><br>
		  <div class="pt" > <a href="http://nexusacademicpublishers.com/uploads/figures/20150131190449.png" target="new"><img class="img_display" src="http://nexusacademicpublishers.com/uploads/figures/20150131190449.png" width="80" height="80"></a>
            
            <p class="Figure--and-Table-Heading ParaOverride-1"><span class="CharOverride-5"><a id="Figure-1-"></a>Figure 1: </span><a href="http://nexusacademicpublishers.com/uploads/figures/20150131190449.png">Agarose gel electrophoresis of PCR amplified products (358 bp) of the cytochrome b gene from blood samples of camel (C), buffalo (B) and sheep (SH)</a></p>
       </div>

			
		  <p class="Body-Text ParaOverride-1">&nbsp;</p>
		  <p class="Body-Text ParaOverride-1">The PCR protocol as described by <a href="#Bartlett-SE--Davidson-WS--1992-."><span class="Hyperlink">Bartlett and Davidson (1992)</span></a> was employed for amplification of the cytochrome b gene with the extracted DNA samples. A 5µl of the extracted DNA sample was used. The PCR amplification reaction conditions comprised of a total volume of 25µl consisting of 1X PCR reaction buffer (20 mMTris- HCl, pH 8.3, with 100 mMKCl, 3 mM MgCl<span class="CharOverride-11">2</span>), 0.4 mMdNTP, 0.25µM each primer and 0.06 unit/µl of Taq DNA polymerase (Perkin Elmer), DNA templates ( 2.5, 3.5 and 4.0µl for camel, buffalo and sheep samples, respectively). The PCR cyclic conditions comprised of 35 cycles at 94<span class="CharOverride-4">°</span>C for 30 seconds, 50<span class="CharOverride-4">°</span>C for 45 seconds and 72<span class="CharOverride-4">°</span>C for 45 seconds including one initial denaturation step at 95<span class="CharOverride-4">°</span>C for 11 minutes in a 9600 GeneAmp (Perkin Elmer) thermocycler. The PCR amplified samples were electrophoresed on 1.6% agarose gel in 0.5 X TBE buffer at 100-150 Ma for 30-45 minutes. The visualization of the DNA fragments was done in a UV cabinet unit and photographed with a Polaroid camera. </p>
		  <p class="Body-Text ParaOverride-1">&nbsp;</p>
			<p class="Heading-2--History-in-MM-">PCR-RFLP Analysis</p>
			<p class="Body-Text ParaOverride-1">For RFLP analysis, the PCR amplified products (10 ul) were directly digested with 5 U of each one of the four restriction endonuclease enzymes <span class="CharOverride-6">viz.,</span> <span class="CharOverride-6">Alu</span>I, <span class="CharOverride-6">Hae</span>III, <span class="CharOverride-6">Hinf </span>I and <span class="CharOverride-6">Taq</span> I separately (Roche Diagnostics, Roche applied science, Mannheim, Germany). The digestion was performed for 2 hours at 37<span class="CharOverride-4">°</span>C and the digested DNA products were separated by electrophoresis on 3% agarose gels, and visualized under UV illumination and photographed with a Polaroid camera. The length of the restriction fragments generated after RE digestion was compared with the standard 100 bp DNA Ladder (100 to 1000 bp scale) (Jena Bioscience, GmbH, Germany). </p>
			<p class="Body-Text ParaOverride-1">&nbsp;</p>
			<p class="Heading-1--Introduction----">RESULTS</p>
		  <p class="Heading-1--Introduction----">&nbsp;</p>
			<p class="Body-Text ParaOverride-1">The universal primers of <span class="CharOverride-6">L14816</span> and <span class="CharOverride-6">H15173</span> were used to amplify part of the cyt b gene (358 bp) from blood samples of camel, buffalo and sheep. The PCR generated a single amplification product for each genomic DNA template from the three animal species. The size of all the PCR products from the different animals tested showed no obvious differences when separated on 1.6 % agarose gel with the expected amplicon size (358 bp) of the cyt b gene as shown in <a href="#Figure-1-"><span class="Hyperlink">Figure 1</span></a>. </p>
		  <p class="Body-Text ParaOverride-1">&nbsp;</p>
			<p class="Body-Text">The different size of bands produced under gel electrophoresis after digestion with the four restriction endonucleases (<span class="CharOverride-6">Alu</span>I, <span class="CharOverride-6">Hae</span>III, <span class="CharOverride-6">Hinf </span>I and <span class="CharOverride-6">Taq</span> I) were identified by comparison of the standard size marker using graphical methods. The cleavage patterns of the four RE enzymes of the cyt b gene PCR amplified products of the three animal species including their band numbers and fragment sizes are presented in <a href="#Table-3-"><span class="Hyperlink">Table 3</span></a>. The RFLP analysis of the cyt b gene region of the three different animal species revealed high specificity and discrimination, even there was no common fragment shared among the studied species. Moreover the resulting fragments were the same for each species in all samples regardless the age (newborn, young and adult) .Restriction endonucleases digestion of the PCR products showed specific patterns as follows: <span class="CharOverride-6">Alu</span> I yielded 2 restriction fragments of different sizes in camel, buffalo and sheep (<a href="#Figure-2-"><span class="Hyperlink">Figure 2A</span></a>); <span class="CharOverride-6">Hae</span>III also revealed 2 restriction fragments of different sizes patterns in all species (<a href="#Figure-2-"><span class="Hyperlink">Figure 2B</span></a>); <span class="CharOverride-6">Hinf </span>I produced 2 fragments of different sizes for both camel and sheep and 3 fragments in buffalo (<a href="#Figure-2-"><span class="Hyperlink">Figure 2C</span></a>); <span class="CharOverride-6">Taq</span> I produced 2 bands differing in their sizes between camel and sheep while no fragmentation was generated for buffalo by this enzyme (<a href="#Figure-2-"><span class="Hyperlink">Figure 2D</span></a>).The resulted fragments by the 4 different restriction enzymes in all species are completely identical to those obtained using hair and meat samples from the same species (data not shown). </p>
			<p class="Body-Text ParaOverride-1">&nbsp;</p>
			<div class="pt" > <a href="http://nexusacademicpublishers.com/uploads/figures/20150131192350.png" target="new"><img class="img_display" src="http://nexusacademicpublishers.com/uploads/figures/20150131192350.png" width="80" height="80"></a>
            
         <p class="Figure--and-Table-Heading ParaOverride-1"><span class="CharOverride-5"><a id="Figure-2-"></a>Figure 2: </span><a href="http://nexusacademicpublishers.com/uploads/figures/20150131192350.png">RFLP banding pattern of the cyt b gene PCR amplified products after digestion with different restriction endonucleases. A: <span class="CharOverride-6">AluI, </span>B: <span class="CharOverride-6">Hae</span>III, C: <span class="CharOverride-6">Hinf </span>I, D: <span class="CharOverride-6">Taq</span> I</a></p>
       </div>

		  
		  <p class="Heading-1--Introduction----">&nbsp;</p>
		  <p class="Heading-1--Introduction----">&nbsp;</p>
		  <p class="Heading-1--Introduction----">DISCUSSION </p>
		  <p class="Heading-1--Introduction----">&nbsp;</p>
			<p class="Body-Text ParaOverride-1">DNA identification methods generally give better resolution and confirmatory identification than the traditional morphological or protein identification methods, and are the most useful tools for determining animal species in commercial foods and animal products (<a href="#Ali-E--Abdur-R--Sharifah-BA--Mahfujur-R--Al-Amin-AJ--Abd-Rashid-NR--Asing--2015-"><span class="Hyperlink">Ali et al., 2015</span></a>; <a href="#Andrea-A--Lisa-G--Lorenzo-C--Priscilla-D--Antonino-M--Renato-M--Daniela-G--Alessandra-G--2015-."><span class="Hyperlink">Andrea et al., 2015</span></a>). When analysis of such animal samples is under taken for species level identification it usually involves sequencing of all or part of the mitochondrial genome which is then compared to known sequences in Gene Bank (NCBI). This technique is accepted, but has many problems including: being costly, time and labour intensive due to the extra step of sequencing products, mixtures cannot be separated, and the generated samples may not produce sufficient sequence data (<a href="#Lenstra-JA--Buntjer-JB--Janssen-FW--2001-"><span class="Hyperlink">Lenstra et al., 2001</span></a>). The more modern techniques now allow the identification of species specific markers or repeats (SSR), mitochondrial DNA restricted fragment length polymorphism (mtDNA-RFLP), and random amplified polymorphic PCR (RAPD-PCR) which have the major advantages over protein analysis and that the samples heated to as high as 120<span class="CharOverride-4">°</span>C can still be analysed and discriminated between species (<a href="#Minarovi--T--Trakovick--A--Rafayov--A--Lieskovsk--Z--2010-"><span class="Hyperlink">Minarovič et al., 2010</span></a>; <a href="#Linacre-A--2012-."><span class="Hyperlink">Linacre, 2012</span></a>; <a href="#Muhammad-S--Yasmeen-J--2015-"><span class="Hyperlink">Muhammad and Yasmeen, 2015</span></a>). </p>
		  <p class="Body-Text ParaOverride-1">&nbsp;</p>
			<p class="Body-Text ParaOverride-1">In the present study, a short DNA fragment (358 bp) of the mitochondrial cytochrome b gene was investigated and exploited for species identification of three domestic animals (camel, buffalo and sheep) because this fragment has by far the widest taxonomic representation in nucleotide databases as reported by <a href="#Guo-X--He-S--Zhang-Y--2005-"><span class="Hyperlink">Guo et al. (2005)</span></a>. The PCR amplification of the cyt b gene, using universal primers (<span class="CharOverride-6">L14816</span> and <span class="CharOverride-6">H15173</span>) generated a specific 358 bp product with DNA samples of all the three animal species tested, which is in accordance to the report of (<a href="#Parson-W--Pegoraro-K--Niederstatter-H--Foger-M--Steinlechner-M--2000-."><span class="Hyperlink">Parson et al., 2000</span></a>).The resulting PCR amplified products when digested with four restriction endonucleases (<span class="CharOverride-6">Alu</span>I, <span class="CharOverride-6">Hae</span>III, <span class="CharOverride-6">Hinf </span>I and <span class="CharOverride-6">Taq</span> I) revealed different RFLP patterns which discriminated the samples with animal species origin. In theoretical case of the same fragments of chosen species we recommend to pick different restriction enzyme for RFLP identification.</p>
		  <p class="Body-Text ParaOverride-1">&nbsp;</p>
			<p class="Body-Text ParaOverride-1">The selection of these enzymes for further experiments was based on their putative ability which discriminate between the samples of different animal species in polyacrylamide gels. In theoretical case of the same fragments of chosen species we recommend to pick different restriction enzyme for RFLP identification.</p>
		  <p class="Body-Text ParaOverride-1">&nbsp;</p>
			<p class="Body-Text ParaOverride-1">The RFLP analysis of the cyt b gene region with the samples of the different animal species (camel, buffalo and sheep) showed high specificity and discriminatory power that even there was no common digestion fragment shared among them. Restriction endonucleases digestion showed specific patterns for each animal species, and these results were confirmatory to that obtained by <a href="#Apostolidis-AP--Alifakiotis-T--Mamuris-Z--Karkavelia-E--2000-"><span class="Hyperlink">Apostolidis et al. (2000)</span></a> who used <span class="CharOverride-6">Alu</span>I, <span class="CharOverride-6">Hae</span>III, <span class="CharOverride-6">Hinf </span>I and <span class="CharOverride-6">Taq</span> I to screen five horse breeds in Greek and found these enzymes to have restriction sites as recognition sites on cyt b gene and were able to identify polymorphism and classify the horse breeds studied. The results of the present study also are in agreement and support of the results obtained by <a href="#Bravi-CM--Liron-JP--Mirol-PM--Ripoli-MV--Garcia-PP--Giovambattista-G--2004-"><span class="Hyperlink">Bravi et al. (2004)</span></a> who reported that the PCR- RFLP method allowed them to identify cattle, horse, donkey, pig, sheep, dog, cat, rabbit, chicken, and human through the different restriction patterns of the enzymes <span class="CharOverride-6">Alu</span>I, <span class="CharOverride-6">Hae</span>III, <span class="CharOverride-6">Hinf </span>I on cyt b gene fragment (358 bp). They concluded that this method may be useful as a first assessment process in forensic evidence and testing for species level identification. </p>
		  <p class="Body-Text ParaOverride-1">&nbsp;</p>
			<p class="Body-Text ParaOverride-1">The results obtained by <span class="CharOverride-6">Alu</span>I enzyme were supported by <a href="#Akasaki-T-Yanagimoro-T--Yamakami-K--Tomonaga-H--Sato-S--2006-."><span class="Hyperlink">Akasaki et al. (2006)</span></a> who found that <span class="CharOverride-6">Alu</span>I enzyme generated different digestion patterns enabling identification of cod fish imported into Japan and consequently they found that the PCR-RFLP methods were sufficient for rapidly screening the cod products. These results were also confirmed by those of <a href="#Minarovi--T--Trakovick--A--Rafayov--A--Lieskovsk--Z--2010-"><span class="Hyperlink">Minarovič et al. (2010)</span></a> who reported that <span class="CharOverride-6">Alu</span> I enzyme generated different digestion patterns enabling identification of <span class="CharOverride-6">Mustelavison</span> (81 bp, 109 bp and 169 bp), <span class="CharOverride-6">Mustelaputoriusfuro</span> (169 bp and 190 bp), <span class="CharOverride-6">Susscrofadomesticus</span> (115 bp and 244 bp), <span class="CharOverride-6">Oryctolaguscunninculus</span> is not cleaved by <span class="CharOverride-6">AluI</span>so it has whole 359 bp fragment on agarose gel, <span class="CharOverride-6">Anseranser</span> (130 bp and 229 bp). <a href="#Abdel-Rahman-SM--El-Saadani-MA--Ashry-KM--Haggag-AS--2009-"><span class="Hyperlink">Abd El-Rahman et al. (2009)</span></a> have also used species specific primers to identify cat’s, dog’s, donkey’s and horse’s meat, they could differentiate between cat’s, dog’s, and horse’s meat but the same PCR amplification size of horse (221 bp) was obtained for donkey and to differentiate between the two species they used restriction enzyme <span class="CharOverride-6">AluI</span> which yielded three fragments in horse’s meat (189, 96 and 74 bp), whereas no fragments were obtained in donkey’s meat. </p>
		  <p class="Body-Text ParaOverride-1">&nbsp;</p>
			<p class="Body-Text ParaOverride-1">Regarding to the results obtained by <span class="CharOverride-6">Hae</span>III enzyme, similar results were reported by <a href="#Viseshakul-N--Sarikaputi-M--2003-.-D"><span class="Hyperlink">Viseshakul and Sarikaputi, (2003)</span></a> in their study to identify <span class="CharOverride-6">Bosgaurus</span> origin from the DNA recovered from other possible ruminant species by PCR-RFLP of cyt b gene using <span class="CharOverride-6">Hae</span>III, and stated that this method could give conclusive results. Similarly, <a href="#Jung-WY--Hsieh-CH--Chen-HM--Hwang-DF--2008-"><span class="Hyperlink">Jung et al. (2008)</span></a> concluded that the PCR-RFLP was proved to be a rapid, reliable and simple method that enabled the identification of six commercial filefish processed species, where they amplified a cyt b gene fragment (465 bp) from processed filefish meats. Then they exposed the PCR amplified fragment to digestion by <span class="CharOverride-6">Hae</span>III enzyme which could differentiate the species of <span class="CharOverride-6">A. monoceros</span> (285+180 bp), <span class="CharOverride-6">A. scriptus </span>(180+159+126 bp), <span class="CharOverride-6">M. chinensis</span> (424+41 bp), <span class="CharOverride-6">T. hypergyrus</span> (180+159+94+32 bp), <span class="CharOverride-6">T. modestus</span> (229+180+56 bp) and <span class="CharOverride-6">C. penicilligerus</span> (285+139+41bp). </p>
		  <p class="Body-Text ParaOverride-1">&nbsp;</p>
			<p class="Body-Text ParaOverride-1">PCR-RFLP of cyt b gene has also been used by <a href="#Nagata-J--Aramilev-VV--BelozorA--Sugimoto-T--McCullough-DR--2005-."><span class="Hyperlink">Nagata et al. (2005)</span></a> to differentiate Tiger (<span class="CharOverride-6">Pantheratigris</span>) and Leopard (<span class="CharOverride-6">Pantherapardus</span>) using <span class="CharOverride-6">Hinf</span>I enzyme which appeared more likely to produce easily distinguishable differences in RFLP banding patterns between the two wild animal species and these results are in agreement with the results of the present study concerning using <span class="CharOverride-6">Hinf</span>I enzyme for identification of animal species.</p>
		  <p class="Body-Text ParaOverride-1">&nbsp;</p>
			<p class="Body-Text ParaOverride-1">Our investigation concerning the use of <span class="CharOverride-6">Taq</span> I enzyme was proved by previous study carried out by <a href="#Ahmed-MMM--Abdel-Rahman-SM--El-Hanafy-AA--2007-."><span class="Hyperlink">Ahmed et al. (2007)</span></a> in which they amplified a segment of cyt b gene (359 bp) and exposed it to digestion by <span class="CharOverride-6">TaqI</span> restriction enzyme to discriminate between buffalo’s and cattle’s meat, which generated two fragments (191 and 168 bp) in buffalo whereas the cyt b gene fragment was not digested in cattle because using of the species specific primers couldn’t differentiate between the two species. The results showed that the cytochrome b gene PCR-RFLP analysis provides rapid and effective method to detect species origin of given samples. The results obtained by the four enzymes used in the present study are totally in agreement with those of recent study of <a href="#Abo-Hadeed-AH--Mervat-HG--Mayada-RF--2011-"><span class="Hyperlink">Abou-Hadeed et al. (2011)</span></a> where these enzymes could successfully differentiate between hair samples of American black bear (<span class="CharOverride-6">Urusamericanus), </span>blue nile monkey<span class="CharOverride-6"> (Ceropithecusmitis), </span>Barbary sheep<span class="CharOverride-6"> (Amotracuslervia), </span>bacterian camel<span class="CharOverride-6"> (Camelusbactrianus)</span>, llama<span class="CharOverride-6"> (Lama glama) </span>and human<span class="CharOverride-6"> (Homo sapiens).</span><span class="CharOverride-17"> </span></p>
		  <p class="Body-Text ParaOverride-1">&nbsp;</p>
			<p class="Heading-1--Introduction----">CONCLUSION</p>
		  <p class="Heading-1--Introduction----">&nbsp;</p>
			<p class="Body-Text ParaOverride-1">This study shows that species identification method based on PCR- amplification of a segment of the mitochondrial cytochrome b gene, processed with RFLP analysis using the four restriction endonucleases (<span class="CharOverride-6">Alu</span>I, <span class="CharOverride-6">Hae</span>III, <span class="CharOverride-6">Hinf</span>I and <span class="CharOverride-6">Taq</span> I) can be very reliable. It can also be considered as a simple, quick and cheap method in forensic identification of the studied animal species (camel, buffalo and sheep). Application of genetic methods is very useful for breeding of livestock and protection of biodiversity as well as differentiation of sample´s origin which could be valuable in detection of adulteration.</p>
		  <p class="Body-Text ParaOverride-1">&nbsp;</p>
			<p class="Heading-1--Introduction----">REFERENCES</p>
		  <p class="Heading-1--Introduction----">&nbsp;</p>
			
			  <li class="References ParaOverride-3" lang="en-US"><a id="Abdel-Rahman-SM--El-Saadani-MA--Ashry-KM--Haggag-AS--2009-"></a>Abdel-Rahman SM, El-Saadani MA, Ashry KM, Haggag AS (2009). Detection of adulteration and identification of cats, dogs, donkeys and horses meat using species specific PCR and PCR-RFLP techniques. Aust. J. Basic Appl. Sci. 3(3): 1716-1719. </li>
				<li class="References ParaOverride-3" lang="en-US"><a id="Abo-Hadeed-AH--Mervat-HG--Mayada-RF--2011-"></a>Abo-Hadeed AH, Mervat HG, Mayada RF (2011). Species identification of some non-domestic animals using PCR-RFLP analysis of cytochrome b gene. Zagazig Vet. J. 39(4): 37-46.</li>
				<li class="References ParaOverride-3" lang="en-US"><a id="Ahmed-MMM--2007-."></a>Ahmed MMM (2007). PCR amplification of species specific repeat for meat DNA identification via genetic markers in cattle and sheep. Biotechnol. Anim. Husb. 21(1-2): 1-11. <a href="http://dx.doi.org/10.2298/BAH0502001A"><span class="Hyperlink">http://dx.doi.org/10.2298/BAH0502001A</span></a></li>
				<li class="References ParaOverride-3" lang="en-US"><a id="Ahmed-MMM--Abdel-Rahman-SM--El-Hanafy-AA--2007-."></a>Ahmed MMM, Abdel-Rahman SM, El-Hanafy AA (2007). Application of species specific polymerase chain reaction and cytochrome b gene for different meat species authentication. Biotechnol. 6(3): 426-430. <a href="http://dx.doi.org/10.3923/biotech.2007.426.430"><span class="Hyperlink">http://dx.doi.org/10.3923/biotech.2007.426.430</span></a></li>
				<li class="References ParaOverride-3" lang="en-US"><a id="Akasaki-T-Yanagimoro-T--Yamakami-K--Tomonaga-H--Sato-S--2006-."></a>Akasaki T,Yanagimoro T, Yamakami K, Tomonaga H, Sato S (2006)<span class="CharOverride-6">. </span>Species identification and PCR-RFLP analysis of cytochrome b gene in cod fish (Order Gadiformes) products. J. Food Sci. 71(3):  190-195. <a href="http://dx.doi.org/10.1111/j.1365-2621.2006.tb15616.x"><span class="Hyperlink">http://dx.doi.org/10.1111/j.1365-2621.2006.tb15616.x</span></a></li>
				<li class="References ParaOverride-3" lang="en-US"><a id="Ali-E--Abdur-R--Sharifah-BA--Mahfujur-R--Al-Amin-AJ--Abd-Rashid-NR--Asing--2015-"></a>Ali E, Abdur R, Sharifah BA, Mahfujur R, Al Amin AJ, Abd Rashid NR, Asing (2015). Multiplex PCR assay for the detection of five meat species forbidden in Islamic foods. Food Chem. 177(15): 214-224. <a href="http://dx.doi.org/10.1016/j.foodchem.2014.12.098"><span class="Hyperlink">http://dx.doi.org/10.1016/j.foodchem.2014.12.098</span></a></li>
				<li class="References ParaOverride-3" lang="en-US"><a id="Andrea-A--Lisa-G--Lorenzo-C--Priscilla-D--Antonino-M--Renato-M--Daniela-G--Alessandra-G--2015-."></a>Andrea A, Lisa G, Lorenzo C, Priscilla D, Antonino M, Renato M, Daniela G, Alessandra G (2015). <span class="articletypelabel CharOverride-13">DNA </span><span class="Heading-2-Char CharOverride-18">and Mini-</span><span class="articletypelabel CharOverride-13">DNA </span><span class="Heading-2-Char CharOverride-18">barcoding for the</span><span class="articletypelabel CharOverride-13"> identification</span><span class="Heading-2-Char CharOverride-18">of Porgies species (family Sparidae) of commercial interest on the internationalmarket</span>. Food Control. 50: 589-596.</li>
				<li class="References ParaOverride-3" lang="en-US"><a id="Andrzej-D--Kamila-K--2005-"></a>Andrzej D, Kamila K (2005). Anew PCR-RFLP with in the domestic pigeon (Columba liviavar, domestica) cytochrome b (MTCYB) gene. J. Appl. Genet. 46(3): 315-317.</li>
				<li class="References ParaOverride-3" lang="en-US"><a id="Apostolidis-AP--Alifakiotis-T--Mamuris-Z--Karkavelia-E--2000-"></a>Apostolidis AP, Alifakiotis T, Mamuris Z, Karkavelia E (2000). PCR-RFLP analysis of mitochondrial cytochrome b gene among Greek horse breeds. Ital. J. Zool. 67(2): 159-162. </li>
				<li class="References ParaOverride-3" lang="en-US"><a id="Bartlett-SE--Davidson-WS--1992-."></a>Bartlett SE, Davidson WS (1992). FINS (forensically informative nucleotide sequencing): A procedure for identifying the animal origin of biological specimens. Bio. Techniques. 12(3): 408-411.</li>
				<li class="References ParaOverride-3" lang="en-US"><a id="Bravi-CM--Liron-JP--Mirol-PM--Ripoli-MV--Garcia-PP--Giovambattista-G--2004-"></a>Bravi CM, Liron JP, Mirol PM, Ripoli MV, Garcia PP, Giovambattista G (2004). A simple method for domestic animal identification in Argentina using PCR-RFLP analysis of cytochrome b gene. J. Legal Med. 6(4): 246-251. <a href="http://dx.doi.org/10.1016/j.legalmed.2004.06.003"><span class="Hyperlink">http://dx.doi.org/10.1016/j.legalmed.2004.06.003</span></a></li>
				<li class="References ParaOverride-3" lang="en-US"><a id="Cheng-X--Weiling-H--Feng-H--Ming-H--Guanghong-Z--2014-."></a>Cheng X, Weiling H, Feng H, Ming H, Guanghong Z (2014). <span class="Heading-2-Char CharOverride-18">Multiplex real-time PCR for the </span><span class="articletypelabel CharOverride-13">identification</span><span class="Heading-2-Char CharOverride-18"> and quantification of </span><span class="articletypelabel CharOverride-13">DNA </span><span class="Heading-2-Char CharOverride-18">from duck, pig and chicken in Chinese blood curds.  </span>Food Res. Int. 60: 30-37.</li>
				<li class="References ParaOverride-3" lang="en-US"><a id="Chow-S--Clarke-ME--Walsh-PJ--1993-."></a>Chow S, Clarke ME, Walsh PJ (1993). PCR-RFLP analysis on thirteen western Atlantic snappers (subfamily Lutjaninae): A simple method for species and stock identification. Fish Bull. 30: 207-224.</li>
				<li class="References ParaOverride-3" lang="en-US"><a id="DeMasi-L--Adelfi-MG--Pignone-D--Laratta-B--2015-."></a>DeMasi L, Adelfi MG, Pignone D, Laratta B (2015). <span class="articletypelabel CharOverride-13">Identification </span><span class="Heading-2-Char CharOverride-18">of Doris verrucosamollusc via mitochondrial 16S rDNA. </span>Biochem. Syst. Ecol. 58: 21-29.</li>
				<li class="References ParaOverride-3" lang="en-US"><a id="Guo-X--He-S--Zhang-Y--2005-"></a>Guo X, He S, Zhang Y (2005). Phylogeny and biogeography of Chinese sisorid catfishes re-examined using mitochondrial cytochrome b and 16S rRNA gene sequences. J. Mole. Phyl. Evol. 35(2): 344-362. <a href="http://dx.doi.org/10.1016/j.ympev.2004.12.015"><span class="Hyperlink">http://dx.doi.org/10.1016/j.ympev.2004.12.015</span></a></li>
				<li class="References ParaOverride-3" lang="en-US"><a id="Hou-B--Xianrong-M--Liyuan-Z--Jinyue-G--Shaowen-L--Hui-J--2015-"></a>Hou B, Xianrong M, Liyuan Z, Jinyue G, Shaowen L, Hui J (2015). <span class="Heading-2-Char CharOverride-18">Development of a sensitive and specific multiplex PCR method for the simultaneous detection of chicken, duck and goose</span><span class="articletypelabel CharOverride-13"> DNA </span><span class="Heading-2-Char CharOverride-18">in meat products</span>. Meat Sci. 101: 90-94.</li>
				<li class="References ParaOverride-3" lang="en-US"><a id="Jung-WY--Hsieh-CH--Chen-HM--Hwang-DF--2008-"></a>Jung WY, Hsieh CH, Chen HM, Hwang DF (2008). Identification of six common species of processed filefish using cytochrome b gene sequence and PCR-RFLP analysis. The Rraffles Bulletin of Zoology. 19: 151-158. </li>
				<li class="References ParaOverride-3" lang="en-US"><a id="Lee-JC--Hsieh-HM--Huang-LH--Kuo-YC--Wu-JH--Chin-SC--Lee-AH--Linacre-A--Tasi-LC--2009b-"></a><a id="Lee-JC--Tasi-LC--Liao-SP--Linacrre-A--Hsieh-HM--2009a-"></a>Lee JC, Tasi LC, Liao SP, Linacrre A, Hsieh HM (2009a). Species identification using the cytochrome b gene of commercial turtle shells. J. Forensic Sci. Int. Gen. 3(2): 67-73. <a href="http://dx.doi.org/10.1016/j.fsigen.2008.10.005"><span class="Hyperlink">http://dx.doi.org/10.1016/j.fsigen.2008.10.005</span></a></li>
				<li class="References ParaOverride-3" lang="en-US">Lee JC, Hsieh HM, Huang LH, Kuo YC, Wu JH, Chin SC, Lee AH, Linacre A, Tasi LC (2009b). Ivory identification by DNA profiling of cytochrome b gene. Int. J. Legal Med. 123(2): 117-121. <a href="http://dx.doi.org/10.1007/s00414-008-0264-0"><span class="Hyperlink">http://dx.doi.org/10.1007/s00414-008-0264-0</span></a></li>
				<li class="References ParaOverride-3" lang="en-US"><a id="Lenstra-JA--Buntjer-JB--Janssen-FW--2001-"></a>Lenstra JA, Buntjer JB, Janssen FW (2001). On the origin of meat-DNA techniques for species identification of meat products. J. Vet. Sci. 2: 1-15. </li>
				<li class="References ParaOverride-3" lang="en-US"><a id="Lin-Y--Hsieh-H--James-L--Chung-Ting-H--Der-Yuh-L--Adrian-L--Li-Chin-T--2014-"></a>Lin Y, Hsieh H, James L, Chung-Ting H, Der-Yuh L, Adrian L, Li-Chin T (2014). <span class="Heading-2-Char CharOverride-18">Establishing a </span><span class="articletypelabel CharOverride-13">DNA identification </span><span class="Heading-2-Char CharOverride-18">system for pigs (Susscrofa) using a multiplex STR amplification</span>. Forensic Sci. Int. Genet. 9: 12-19.</li>
				<li class="References ParaOverride-3" lang="en-US"><a id="Linacre-A--2012-."></a>Linacre A (2012). Capillary electrophoresis of mtDNA cytochrome b gene sequences for animal species identification. Methods Mol. Biol. 830: 321-329. </li>
				<li class="References ParaOverride-3" lang="en-US"><a id="Lindstrom-DP--1999-"></a>Lindstrom DP (1999). Molecular species identification of newly hatched Hawaiian <span class="CharOverride-6">amphidromousgobioid</span> larvae. Mar. Biotech. 1: 167–174.</li>
				<li class="References ParaOverride-3" lang="en-US"><a id="Minarovi--T--Trakovick--A--Rafayov--A--Lieskovsk--Z--2010-"></a>Minarovič T, Trakovická A, Rafayová A, Lieskovská Z (2010). Animal Species Identification by PCR – RFLP of Cytochrome b Scientific Papers: Anim. Sci. Biotechn. 43(1): 296-299. </li>
				<li class="References ParaOverride-3" lang="en-US"><a id="Muhammad-S--Yasmeen-J--2015-"></a>Muhammad S, Yasmeen J (2015). A multiplex-conventional PCR assay for bovine, ovine, caprine and fish species identification in feedstuffs: Highly sensitive and specific. Food Control. 50: 190-194. <a href="http://dx.doi.org/10.1016/j.foodcont.2014.08.048"><span class="Hyperlink">http://dx.doi.org/10.1016/j.foodcont.2014.08.048</span></a></li>
				<li class="References ParaOverride-3" lang="en-US"><a id="Nagata-J--Aramilev-VV--BelozorA--Sugimoto-T--McCullough-DR--2005-."></a>Nagata J, Aramilev VV, BelozorA, Sugimoto T, McCullough DR (2005). Fecal genetic analysis using PCR-RFLP of cytochrome b to identify sympatric carnivores, the tiger <span class="CharOverride-6">pantheratigris</span> and leopard <span class="CharOverride-6">pantherapardus</span>, in far eastern Russia. Conserv. Genent. 6(5): 863-865. </li>
				<li class="References ParaOverride-3" lang="en-US"><a id="Parson-W--Pegoraro-K--Niederstatter-H--Foger-M--Steinlechner-M--2000-."></a>Parson W, Pegoraro K, Niederstatter H, Foger M, Steinlechner M (2000). Species identification by means of the cytochrome <span class="CharOverride-6">b </span>gene. Int. J. Legal Med. 114(1-2): 23-28. <a href="http://dx.doi.org/10.1007/s004140000134"><span class="Hyperlink">http://dx.doi.org/10.1007/s004140000134</span></a></li>
				<li class="References ParaOverride-3" lang="en-US"><a id="Prusak-B--GrzybowskiG--Zieba-G--2004-."></a>Prusak B, GrzybowskiG, Zieba G (2004). Taxonomic position of Bison bison (Linnaeus, 1758) and Bison bonasus (Linnaeus, 1758) based on analysis of cytbgene. Anim. Sci. Pap. Rep. 22(1): 27-35.</li>
				<li class="References ParaOverride-3" lang="en-US"><a id="Quinteiro-J--Sotelo-CG--Rehbein-H--Pryde-SE--Perez-Martin-RI--Rey-Mendez-M--Mackie-IM--1998-."></a>Quinterio J, Sotelo CG, Rehbein H, Pryde SE, Perez-Martin RI, Rey-Mendez M, Mackie IM (1998). Use of mtDNA direct polymerase chain reaction (PCR) sequencing and PCR-restriction fragment length polymorphism methodologies in species identification of canned tuna. J. Agric. Food Chem. 46(4): 1662-1669. <a href="http://dx.doi.org/10.1021/jf970552+"><span class="Hyperlink">http://dx.doi.org/10.1021/jf970552+</span></a></li>
				<li class="References ParaOverride-3" lang="en-US"><a id="Ram-JL--Ram-ML--Baidoun-FF--1996-."></a>Ram JL, Ram ML, Baidoun FF (1996). Authentication of canned tuna and bonito by sequence and restriction site analysis of polymerase chain reaction products of mitochondrial DNA. J. Agric. Food Chem. 44(8): 2460-2467. <a href="http://dx.doi.org/10.1021/jf950822t"><span class="Hyperlink">http://dx.doi.org/10.1021/jf950822t</span></a></li>
				<li class="References ParaOverride-3" lang="en-US"><a id="Rashid-PM--Babashekh-MO--Marouf-AS--Amin-KM--2014-.-I"></a>Rashid PM, Babashekh MO, Marouf AS, Amin KM (2014). Identification of Animal Species in Meat Broth by Simplex and Multiplex PCR. J. Zankoy Sulaimani- Part A (JZS-A). 16(1): 97-102.</li>
				<li class="References ParaOverride-3" lang="en-US"><a id="Sherryn-AC--Shanan-ST--Stephen-CD--Julianne-MH--Adrian-MTL--2015-"></a>Sherryn AC, Shanan ST, Stephen CD, Julianne MH, Adrian MTL (2015). Molecular identification of python species: Development and validation of a novel assay for forensic investigations. Forensic Sci. Int-Gen. 12(16): 64-70.</li>
		  <li class="References ParaOverride-3" lang="en-US"><a id="Sumathi-G-Jeyasekaran-G--JeyaShakila-R--Sivaraman-B--Arunkumar-G--Manimaran-U--Sukumar-D--2015-"></a>Sumathi G,Jeyasekaran G, JeyaShakila R, Sivaraman <span class="hit CharOverride-13">B</span>, Arunkumar G, Manimaran U, Sukumar D (2015). <span class="Hyperlink CharOverride-9">Molecular </span><span class="hit CharOverride-13">identification</span><span class="Hyperlink CharOverride-9"> of grouper</span><span class="hit CharOverride-13"> species</span><span class="Hyperlink CharOverride-9"> using</span><span class="hit CharOverride-13"> PCR-RFLP</span><span class="Hyperlink CharOverride-9"> technique. </span>Food Control. 51: 300-306. </li>
				<li class="References ParaOverride-3" lang="en-US"><a id="Viseshakul-N--Sarikaputi-M--2003-.-D"></a>Viseshakul N, Sarikaputi M (2003). Detection of the cytochrome b gene and its RFLP pattern from Bosgaurus specimen as a forensic application. The 11th International Symposium of the World Association of Veterinary Laboratory Diagnosticians and OIE Seminar on Biotechnology, Sofitel Central Plaza, Bangkok, Thailand, November 9-13.</li>
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