Progress of DNA-based Methods for Species Identification

HU Zhen,ZHANG Su-hua,WANG Zheng,BIAN Ying-nan,LI Cheng-tao

（1.Department of Forensic Medicine,Medical College of Soochow University,Suzhou 215123,China; 2.Shanghai Key Laboratory of Forensic Medicine,Institute of Forensic Science,Ministry of Justice,P.R.China,Shanghai 200063,China）

·綜述·

Progress of DNA-based Methods for Species Identification

HU Zhen1,2,ZHANG Su-hua2,WANG Zheng2,BIAN Ying-nan2,LI Cheng-tao2

（1.Department of Forensic Medicine,Medical College of Soochow University,Suzhou 215123,China; 2.Shanghai Key Laboratory of Forensic Medicine,Institute of Forensic Science,Ministry of Justice,P.R.China,Shanghai 200063,China）

Species identification of biological samples is widely used in such fields as forensic science and food industry.A variety of accurate and reliable methods have been developed in recent years.The current review shows common target genes and screening criteria suitable for species identification,and described various DNA-based molecular biology methods about species identification.Additionally,it discusses the future development of species identification combined with real-time PCR and sequencing technologies.

forensic genetics;species identification;review[publication type]

Article IC:1004-5619（2015）02-0129-03

Introduction

Identifying unknown species is of concern in many areas,such as in forensic science.Determination of species can be used to assist investigating the source of stains or remains littered in a crime scene[1],solving the issues about poaching[2-3],and distinguishing the meat adulteration[4-5].Especially the meat authenticity and traceability are issues of primary importance in the recent cases regarding lamb adulteration.

Theserological,cytologicalandbiochemical methods applied to species identification.Such approaches,however,required higher quality samples, which were not suitable for those poor samples destructed by external contamination or degradation[6]. As a source of powerful information,DNA-based methodshavebeengivenrisetoattention,and nowadays a numerous protocols of DNA analysis for species identification have been developed.

The current review presents an overview on DNA marker selection for species identification and thedifferentanalyticalmethodsbasedonDNA analysis.

Genes for species identification

Selection of DNA markers

In practice,it is complex to analyze multi-component mixings and degraded DNA samples;therefore,the choice of DNA marker is extremely important.Screening target gene is required to meetsome conditions[7].There are sufficient variable mutated genes among species especially in relatively close relationship;low intra-specific variations exist across geographical distribution area;and reference sequence information should be extensively examined for a large number of species in database, which can be compared with the nucleotidesequence of an unknown sample.

Genes in nuclear DNA and mitochondrial DNA （mtDNA）for species identification have been mainly reported to be satisfied above criteria,including cytochrome b[8],12S rRNA[9],16S rRNA,D-loop, COⅠ,the 3′UTR of the SON gene[10],STR,and 28S rRNA.

The main target genes

STR loci

STR loci have good length polymorphisms and sequence polymorphisms.High specific primers are designed according to the polymorphism of humanspecific information,and such human-specific primers only amplify the products from human and primate species.Crouse et al.[11]analyzed 23 different species using 9 human-specific STR primers system.PCR products were detected in human,rhesus,gorilla, chimpanzee and orangutan,and however,were not observed in the other 18 species.

mtDNA gene

The mtDNA sequences are highly species-specific and characterized by multicopy;thus the polymorphic loci in mtDNA are the best selection as target regions.Since mtDNA is maternally inherited as haplotype,recombination does not occur.In addition,mitochondrial genes contain more sequence diversity information than nuclear gene;they can facilitate the identification of phylogenetically related species.

For now,almost half of molecular identifica-tion studies target the cytochrome b（44%）,and followed is the 12S rRNA（11%）,16S rRNA（8%）and D-loop（8%）[7].Cytochrome b gene is studied most in biology science.There were more than 50 000 sequences for cytochrome b available in GenBank in 2004.Hsieh et al.[8]designed two universal primers of cytochrome b to amplify a 402 bp fragment,and to be aligned by Pileup program of GCG computer package;consequently,such a sequence could be capable of identifying animal species.

DNA-based biomolecular methods

PCR-random amplified polymorphic DNA（PCRRAPD）

PCR-RAPD is a technique which uses random primers to amplify DNA fragments,and then separate obtained fingerprints by gel electrophoresis based on sizes,according to compare the DNA bands of the fingerprints to identify different species.Wu et al.[12]used this technique to identify different species belonging to the family cervidae（sika deer, sambar deer,rusa deer,tufted deer,black muntjac and reeve's muntjac）.However,PCR-RAPD is limited in practice,because it needs DNA of high quality and quantity and obtains a poor reproducibility of results.

PCR-restrictionfragmentlengthpolymorphism （PCR-RFLP）

PCR-RFLP is a technique to analyze length polymorphism.It amplifies DNA using universal primers and digests products with restriction enzymes such as Alu I,Hha I,Apo I and Bsp TI.As a result,it can identify species by analyzing fragment size via electrophoresis.PCR amplification of a 456 bp fragment in themitochondrial12S rRNA genewas used to identify beef,buffalo,mutton and chevon[13]. Species-specific primers for PCR

Species-specific PCR is based on the characteristics of the target gene to design highly specific primers,including single or multiple primers,and corresponding amplicons show a single or multiple fragments.Amplicons can be identified by comparing the different sizes on the agarose gel.Montiel-Sosa et al.[14]managed to identify pork meat fromothermeatproductsusingspecies-specific primers targeting the mitochondrial D-loop genes.

In short,multiplex PCR,which uses various primers together for more than one target region,is an efficient and reliable analysis for the simultaneous identification of various meat species[15].

PCR-sequencing

Sequencing is the most direct method of obtainingspeciesinformationfromPCRproducts. PCR-sequencing has been used to identify the various origins of meats[16-17].Such technique has a tendency to focus on the amplification of mtDNA sequences.Owing tothehighmutationandgreat availability of the sequences in the databases,the cytochrome b gene,12S and 16S rRNA are the most commonly used genes for species identification[18].

Balitzki-Korte et al.[19]succeeded in identifying the species of an unknown skin by pyrosequencing 149 bp amplicons generated from the mitochondrial 12S rRNA gene.La Neve et al.[20]targeted a 232bp amplicon from cytochrome b gene and sequenced the amplicon using a minisequencing reaction,thus identifying meats from Capreolus capreolus,Cervus elaphus,Capra ibex,and Rupicapra rupicapra.

Real-time PCR

Real-time PCR is such that the amounts of amplified DNA could be detected as the reaction progresses in“real time”through the fluorescence signal intensity[21],so it allows the detection of micro samples from different animals,which is considered as one of the most promising molecular tools for meat authentication[22].

Bowers et al.[23]identified pheasant and quail DNA in commercial food products using species-specific primers and TaqMan probes designed on the mitochondrial cytochrome b gene.Rojas et al.[24]developed a real-time PCR assay based on specific primers and probes designed on the mitochondrial 12S rRNA gene for each target species.The assay canidentify thecommercialmeatproductsfrom game birds including quail,pheasant,partridge,guinea fowl,pigeon,eurasian woodcock and song thrush.

Compared with the conventional PCR,real-time PCR technique is characterized by high sensitivity and specificity as well as by quickness and convenience in designing specific primers and universal probes;therefore such a technique can a useful tool for species identification.

Prospects

A number of DNA-based methods are potentially available for species identification.Although they vary in their complexity and cost and have different strengths and weaknesses,the use of these techniques is likely to increase in forensic samples and food products.Two suggestions can be made on the development of species identification:the mtDNA polymorphism markers should be selected as more targets as possible;and real-time PCR can be a preferable technique for a wide range of species identified application thanks to its high specificity and sensitivity,good reproducibility,less pollution and other advantages.

Acknowledgements

This review was supported by the grants from the 12th Five-year Plan National Key Technology R&D Program of the Ministry of Science and Tech-nology of P.R.China（2012BAK16B01）and the NationalNaturalScienceFoundationofP.R.China （81330073,81222041）.

Reference:

[1]Brenig B,Beck J,Schütz E.Shotgun metagenomics of biological stains using ultra-deep DNA sequencing[J]. Forensic Sci Int Genet,2010,4（4）:228-231.

[2]An J,Lee MY,Min MS,et al.A molecular genetic approach for species identification of mammals and sex determination of birds in aforensiccaseof poaching from South Korea[J].Forensic Sci Int,2007, 167（1）:59-61.

[3]Gupta SK,Thangaraj K,Singh L.A simple and inexpensive molecular method for sexing and identification of the forensic samples of elephant origin[J]. J Forensic Sci,2006,51（4）:805-807.

[4]Eurlings MC,Lens F,Pakusza C,et al.Forensic identification of Indian snakeroot（Rauvolfia serpentina Benth.ex Kurz）using DNA barcoding[J].J Forensic Sci,2013,58（3）:822-830.

[5]Wasser SK,Shedlock AM,Comstock K,et al.Assigning African elephant DNA to geographic region of origin:applications to the ivory trade[J].Proc Natl Acad Sci U S A,2004,101（41）:14847-14852.

[6]Bottero MT,Dalmasso A.Animal species identificationinfoodproducts:evolutionofbiomolecular methods[J].Vet J,2011,190（1）:34-38.

[7]Teletchea F,Maudet C,H?nni C.Food and forensic molecularidentification:updateandchallenges[J]. Trends Biotechnol,2005,23（7）:359-366.

[8]Hsieh HM,Chiang HL,Tsai LC,et al.Cytochrome b gene for species identification of the conservation animals[J].Forensic Sci Int,2001,122（1）:7-18.

[9]Cawthorn DM,Steinman HA,Witthuhn RC.Evaluation of the 16S and 12S rRNA genes as universal markersfortheidentificationofcommercialfish species in South Africa[J].Gene,2012,491（1）:40-48.

[10]Soteriou B,Fisher RA,Khan IM,et al.Conserved gene sequences for species identification:PCR analysis of the 3'UTR of the SON gene distinguishes human and other mammalian DNAs[J].Forensic Sci Int,1995,73（3）:171-181.

[11]Crouse CA,Schumm J.Investigation of species specificity using nine PCR-based human STR systems[J]. J Forensic Sci,1995,40（6）:952-956.

[12]Wu X,Liu H,Jiang Z.Identification primers for sika deer（Cervus nippon）from a sequence-characterised amplified region（SCAR）[J].New Zealand Journal of Zoology,2006,33（1）:65-71.

[13]Girish PS,Anjaneyulu AS,Viswas KN,et al.Meat species identification by polymerase chain reaction-restriction fragment length polymorphism（PCR-RFLP） of mitochondrial 12S rRNA gene[J].Meat Sci,2005, 70（1）:107-112.

[14]Montiel-Sosa JF,Ruiz-Pesini E,Montoya J,et al. Direct and highly species-specific detection of pork meat and fat in meat products by PCR amplification of mitochondrial DNA[J].J Agric Food Chem,2000, 48（7）:2829-2832.

[15]Tobe SS,Linacre AM.A multiplex assay to identify 18 European mammal species from mixtures using the mitochondrial cytochrome b gene[J].Electrophoresis,2008,29（2）:340-347.

[16]Quinteiro J,Vidal R,Izquierdo M,et al.Identification of Hake species（Merluccius Genus）using sequencing and PCR-RFLP analysis of mitochondrial DNA control region sequences[J].J Agric Food Chem,2001, 49（11）:5108-5114.

[17]Jér?me M,Lemaire C,Verrez-Bagnis V,et al.Direct sequencing method for species identification of canned sardine and sardine-type products[J].J Agric Food Chem,2003,51（25）:7326-7332.

[18]Karlsson AO,Holmlund G.Identification of mammal species using species-specific DNA pyrosequencing[J]. Forensic Sci Int,2007,173（1）:16-20.

[19]Balitzki-Korte B,Anslinger K,Bartsch C,et al.Species identification by means of pyrosequencing the mitochondrial 12S rRNA gene[J].Int J Legal Med,2005, 119（5）:291-294.

[20]La Neve F,Civera T,Mucci N,et al.Authentication of meat from game and domestic species by SNaPshot minisequencing analysis[J].Meat Sci,2008,80（2）: 216-224.

[21]López-Andreo M,Lugo L,Garrido-Pertierra A,et al. Identification and quantitation of species in complex DNA mixtures by real-time polymerase chain reaction[J].Anal Biochem,2005,339（1）:73-82.

[22]K?ppel R,Zimmerli F,Breitenmoser A.Heptaplex real-time PCR for the identification and quantification of DNA from beef,pork,chicken,turkey,horse meat,sheep（mutton）and goat[J].European Food Research and Technology,2009,230（1）:125-133.

[23]Bowers HA,Tengs T,Glasgow HB Jr,et al.Development of real-time PCR assays for rapid detection of Pfiesteria piscicida and related dinoflagellates[J]. Appl Environ Microbiol,2000,66（11）:4641-4648.

[24]Rojas M,González I,Pavón MA,et al.Novel Taq-Man real-time polymerase chain reaction assay for verifying the authenticity of meat and commercial meat products from game birds[J].Food Addit Contam Part A Chem Anal Control Expo Risk Assess, 2010,27（6）:749-763.

（Received date:2015-01-23）

（Editor:LIU Yan）

DF795.2 Document code:A

10.3969/j.issn.1004-5619.2015.02.013

Author:HU Zhen（1989—）,master,major in forensic genetics;E-mail:fly_huzhen@163.com

LI Cheng-tao,Ph.D,research fellow, postgraduate tutor in forensic genetics;E-mail:lichengtaohla@ 163.com