Skip to main content
SpringerLink
Log in

Development and validation of simultaneous identification of 26 mammalian and poultry species by a multiplex assay

  • Original Article
  • Published:
International Journal of Legal Medicine Aims and scope Submit manuscript

Abstract

A multiplex PCR assay was developed to simultaneously identify 22 mammalian species (alpaca, Asiatic black bear, Bactrian camel, brown rat, cat, cattle, common raccoon, dog, European rabbit, goat, horse, house mouse, human, Japanese badger, Japanese wild boar, masked palm civet, pig, raccoon dog, red fox, sheep, Siberian weasel, and sika deer) and four poultry species (chicken, domestic turkey, Japanese quail, and mallard), even from a biological sample containing a DNA mixture of multiple species. The assay was designed to identify species through multiplex PCR and capillary electrophoresis, with a combination of amplification of a partial region of the mitochondrial D-loop by universal primer sets and a partial region of the cytochrome b (cyt b) gene by species-specific primer sets. The assay was highly sensitive, with a detection limit of 100 copies of mitochondrial DNA. The assay’s ability to identify species from complex DNA mixtures was demonstrated using an experimental sample consisting of 10 species. Efficacy, accuracy, and reliability of the assay were validated for use in forensic analysis with the guidelines of Scientific Working Group on DNA Analysis Methods (SWGDAM). The multiplex PCR assay developed in this study enables cost-effective, highly sensitive, and simultaneous species identification without massively parallel sequencing (MPS) platforms. Thus, the technique described is straightforward and suitable for routine forensic investigations.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5

Similar content being viewed by others

Availability of data and material

Not applicable.

Code availability

Not applicable.

References

  1. Schneider PM, Seo Y, Rittner C (1999) Forensic mtDNA hair analysis excludes a dog from having caused a traffic accident. Int J Legal Med 112:315–316. https://doi.org/10.1007/s004140050257

    Article  CAS  PubMed  Google Scholar 

  2. Lorenzini R (2005) DNA forensics and the poaching of wildlife in Italy: A case study. Forensic Sci Int 153:218–221. https://doi.org/10.1016/j.forsciint.2005.04.032

    Article  CAS  PubMed  Google Scholar 

  3. Sato I, Nakaki S, Murata K et al (2010) Forensic hair analysis to identify animal species on a case of pet animal abuse. Int J Legal Med 124:249–256. https://doi.org/10.1007/s00414-009-0383-2

    Article  PubMed  Google Scholar 

  4. Stern AW, Lamm CG (2011) Utilization of paw prints for species identification in the Canidae family. J Forensic Sci 56:1041–1043. https://doi.org/10.1111/j.1556-4029.2011.01768.x

    Article  PubMed  Google Scholar 

  5. Tillmar AO, Dell’Amico B, Welander J, Holmlund G (2013) A universal method for species identification of mammals utilizing next generation sequencing for the analysis of DNA mixtures. PLoS ONE 8:e83761. https://doi.org/10.1371/journal.pone.0083761

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Bravi CM, Lirón JP, Mirol PM et al (2004) A simple method for domestic animal identification in Argentina using PCR-RFLP analysis of cytochrome b gene. Leg Med 6:246–251

    Article  CAS  Google Scholar 

  7. Nakamura H, Muro T, Imamura S, Yuasa I (2009) Forensic species identification based on size variation of mitochondrial DNA hypervariable regions. Int J Legal Med 123:177–184. https://doi.org/10.1007/s00414-008-0306-7

    Article  PubMed  Google Scholar 

  8. Naue J, Lutz-Bonengel S, Pietsch K et al (2012) Bite through the tent. Int J Legal Med 126:483–488. https://doi.org/10.1007/s00414-012-0674-x

    Article  PubMed  Google Scholar 

  9. Savolainen P, Lundeberg J (1999) Forensic evidence based on mtDNA from dog and wolf hairs. J Forensic Sci 44:77–81. https://doi.org/10.1016/S1353-1131(99)90078-0

    Article  CAS  PubMed  Google Scholar 

  10. Schulz I, Schneider PM, Olek K et al (2006) Examination of postmortem animal interference to human remains using cross-species multiplex PCR. Forensic Sci Med Pathol 2:95–101. https://doi.org/10.1385/FSMP:2:2:95

    Article  CAS  PubMed  Google Scholar 

  11. Alacs EA, Georges A, FitzSimmons NN, Robertson J (2010) DNA detective: A review of molecular approaches to wildlife forensics. Forensic Sci Med Pathol 6:180–194. https://doi.org/10.1007/s12024-009-9131-7

    Article  CAS  PubMed  Google Scholar 

  12. Johnson RN, Wilson-Wilde L, Linacre A (2014) Current and future directions of DNA in wildlife forensic science. Forensic Sci Int Genet 10:1–11. https://doi.org/10.1016/j.fsigen.2013.12.007

    Article  CAS  PubMed  Google Scholar 

  13. Ewart KM, Frankham GJ, McEwing R et al (2018) An internationally standardized species identification test for use on suspected seized rhinoceros horn in the illegal wildlife trade. Forensic Sci Int Genet 32:33–39. https://doi.org/10.1016/j.fsigen.2017.10.003

    Article  CAS  PubMed  Google Scholar 

  14. Summerell AE, Frankham GJ, Gunn P, Johnson RN (2019) DNA based method for determining source country of the short beaked echidna (Tachyglossus aculeatus) in the illegal wildlife trade. Forensic Sci Int 295:46–53. https://doi.org/10.1016/j.forsciint.2018.11.019

    Article  CAS  PubMed  Google Scholar 

  15. Ouso DO, Otiende MY, Jeneby MM et al (2020) Three-gene PCR and high-resolution melting analysis for differentiating vertebrate species mitochondrial DNA for biodiversity research and complementing forensic surveillance. Sci Rep 10:1–13. https://doi.org/10.1038/s41598-020-61600-3

    Article  CAS  Google Scholar 

  16. Hsieh HM, Chiang HL, Tsai LC et al (2001) Cytochrome b gene for species identification of the conservation animals. Forensic Sci Int 122:7–18. https://doi.org/10.1016/S0379-0738(01)00403-0

    Article  CAS  PubMed  Google Scholar 

  17. Iyengar A (2014) Forensic DNA analysis for animal protection and biodiversity conservation: A review. J Nat Conserv 22:195–205. https://doi.org/10.1016/j.jnc.2013.12.001

    Article  Google Scholar 

  18. Arulandhu AJ, Staats M, Hagelaar R, et al (2017) Development and validation of a multi-locus DNA metabarcoding method to identify endangered species in complex samples. Gigascience 6. https://doi.org/10.1093/gigascience/gix080

  19. Conte J, Potoczniak MJ, Mower C, Tobe SS (2019) ELEquant: a developmental framework and validation of forensic and conservation real-time PCR assays. Mol Biol Rep 46:2093–2100. https://doi.org/10.1007/s11033-019-04660-7

    Article  CAS  PubMed  Google Scholar 

  20. Mafra I, Ferreira IMPLVO, Oliveira MBPP (2008) Food authentication by PCR-based methods. Eur Food Res Technol 227:649–665. https://doi.org/10.1007/s00217-007-0782-x

    Article  CAS  Google Scholar 

  21. Galimberti A, De Mattia F, Losa A et al (2013) DNA barcoding as a new tool for food traceability. Food Res Int 50:55–63. https://doi.org/10.1016/j.foodres.2012.09.036

    Article  CAS  Google Scholar 

  22. Dobrovolny S, Blaschitz M, Weinmaier T et al (2019) Development of a DNA metabarcoding method for the identification of fifteen mammalian and six poultry species in food. Food Chem 272:354–361. https://doi.org/10.1016/j.foodchem.2018.08.032

    Article  CAS  PubMed  Google Scholar 

  23. Robin ED, Wong R (1988) Mitochondrial DNA molecules and virtual number of mitochondria per cell in mammalian cells. J Cell Physiol 136:507–513. https://doi.org/10.1002/jcp.1041360316

    Article  CAS  PubMed  Google Scholar 

  24. Holland MM, Parsons TJ (1999) Mitochondrial DNA Sequence Analysis - Validation and Use for Forensic Casework. Forensic Sci Rev 11:21–50

    CAS  PubMed  Google Scholar 

  25. Tobe SS, Linacre AMT (2008) A technique for the quantification of human and non-human mammalian mitochondrial DNA copy number in forensic and other mixtures. Forensic Sci Int Genet 2:249–256. https://doi.org/10.1016/J.FSIGEN.2008.03.002

    Article  PubMed  Google Scholar 

  26. Kocher TD, Thomas WK, Meyer A et al (1989) Dynamics of mitochondrial DNA evolution in animals: amplification and sequencing with conserved primers. Proc Natl Acad Sci 86:6196–6200. https://doi.org/10.1073/pnas.86.16.6196

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Johns GC, Avise JC (1998) A comparative summary of genetic distances in the vertebrates from the mitochondrial cytochrome b gene. Mol Biol Evol 15:1481–1490. https://doi.org/10.1093/oxfordjournals.molbev.a025875

    Article  CAS  PubMed  Google Scholar 

  28. Parson W, Pegoraro K, Niederstätter H et al (2000) Species identification by means of the cytochrome b gene. Int J Legal Med 114:23–28. https://doi.org/10.1007/s004140000134

    Article  CAS  PubMed  Google Scholar 

  29. Ratnasingham S, Hebert PDN (2007) BOLD: The Barcode of Life Data System: Barcoding. Mol Ecol Notes 7:355–364. https://doi.org/10.1111/j.1471-8286.2007.01678.x

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Dawnay N, Ogden R, McEwing R et al (2007) Validation of the barcoding gene COI for use in forensic genetic species identification. Forensic Sci Int 173:1–6. https://doi.org/10.1016/j.forsciint.2006.09.013

    Article  CAS  PubMed  Google Scholar 

  31. Tobe SS, Linacre AMT (2008) A multiplex assay to identify 18 European mammal species from mixtures using the mitochondrial cytochrome b gene. Electrophoresis 29:340–347. https://doi.org/10.1002/elps.200700706

    Article  CAS  PubMed  Google Scholar 

  32. Ramón-Laca A, Linacre AMT, Gleeson DM, Tobe SS (2013) Identification multiplex assay of 19 terrestrial mammal species present in New Zealand. Electrophoresis 34:3370–3376. https://doi.org/10.1002/elps.201300324

    Article  CAS  PubMed  Google Scholar 

  33. Kitano T, Umetsu K, Tian W, Osawa M (2007) Two universal primer sets for species identification among vertebrates. Int J Legal Med 121:423–427. https://doi.org/10.1007/s00414-006-0113-y

    Article  PubMed  Google Scholar 

  34. Pereira F, Carneiro J, Matthiesen R et al (2010) Identification of species by multiplex analysis of variable-length sequences. Nucleic Acids Res 38:e203–e203. https://doi.org/10.1093/nar/gkq865

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Ishida N, Sakurada M, Kusunoki H, Ueno Y (2018) Development of a simultaneous identification method for 13 animal species using two multiplex real-time PCR assays and melting curve analysis. Leg Med 30:64–71. https://doi.org/10.1016/J.LEGALMED.2017.11.007

    Article  CAS  Google Scholar 

  36. Mori C, Matsumura S (2021) Current issues for mammalian species identification in forensic science: a review. Int J Legal Med 135:3–12. https://doi.org/10.1007/s00414-020-02341-w

    Article  PubMed  Google Scholar 

  37. Coghlan ML, Haile J, Houston J et al (2012) Deep sequencing of plant and animal DNA contained within traditional Chinese medicines reveals legality issues and health safety concerns. PLoS Genet 8:e1002657. https://doi.org/10.1371/journal.pgen.1002657

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Köppel R, van Velsen F, Ganeshan A et al (2020) Multiplex real-time PCR for the detection and quantification of DNA from chamois, roe, deer, pork and beef. Eur Food Res Technol 246:1007–1015. https://doi.org/10.1007/s00217-020-03468-1

    Article  CAS  Google Scholar 

  39. Scientific Working Group on DNA Analysis Methods (SWGDAM) (2016) Validation Guidelines for DNA Analysis Methods. https://1ecb9588-ea6f-4feb-971a-73265dbf079c.filesusr.com/ugd/4344b0_813b241e8944497e99b9c45b163b76bd.pdf. Accessed 1 Jul 2020

  40. Kumar S, Stecher G, Tamura K (2016) MEGA7: Molecular Evolutionary Genetics Analysis Version 7.0 for Bigger Datasets. Mol Biol Evol 33:1870–1874. https://doi.org/10.1093/molbev/msw054

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Pääbo S, Gifford JA, Wilson AC (1988) Mitochondrial DNA sequences from a 7000-year old brain. Nucleic Acids Res 16:9775–9787. https://doi.org/10.1093/nar/16.20.9775

    Article  PubMed  PubMed Central  Google Scholar 

  42. Matsunaga T, Chikuni K, Tanabe R et al (1999) A quick and simple method for the identification of meat species and meat products by PCR assay. Meat Sci 51:143–148. https://doi.org/10.1016/S0309-1740(98)00112-0

    Article  CAS  PubMed  Google Scholar 

  43. Andrews RM, Kubacka I, Chinnery PF et al (1999) Reanalysis and revision of the Cambridge reference sequence for human mitochondrial DNA. Nat Genet 23:147–147. https://doi.org/10.1038/13779

    Article  CAS  PubMed  Google Scholar 

  44. Katoh K, Rozewicki J, Yamada KD (2019) MAFFT online service: Multiple sequence alignment, interactive sequence choice and visualization. Brief Bioinform 20:1160–1166. https://doi.org/10.1093/bib/bbx108

    Article  CAS  PubMed  Google Scholar 

  45. Letunic I, Bork P (2021) Interactive Tree Of Life (iTOL) v5: an online tool for phylogenetic tree display and annotation. Nucleic Acids Res gkab301. https://doi.org/10.1093/nar/gkab301

  46. ThermoFisher Scientific (2018) User Guide: 3500/3500xL Genetic Analyzer with 3500 Series Data Collection Software 3.1 Revision C. https://assets.thermofisher.com/TFS-Assets/LSG/manuals/100031809_3500_3500xL_Software_v3_1_UG.pdf. Accessed 30 Apr 2021

  47. Smith RN (1995) Accurate size comparison of short tandem repeat alleles amplified by PCR. Biotechniques 18:122–128

    CAS  PubMed  Google Scholar 

  48. Bendall KE, Sykes BC (1995) Length heteroplasmy in the first hypervariable segment of the human mtDNA control region. Am J Hum Genet 57:248–256

    CAS  PubMed  PubMed Central  Google Scholar 

  49. Sekiguchi K, Imaizumi K, Fujii K et al (2008) Mitochondrial DNA population data of HV1 and HV2 sequences from Japanese individuals. Leg Med 10:284–286. https://doi.org/10.1016/j.legalmed.2008.02.002

    Article  CAS  Google Scholar 

  50. Chen F, Dang YH, Yan CX et al (2009) Sequence-length variation of mtDNA HVS-I C-stretch in Chinese ethnic groups. J Zhejiang Univ Sci B 10:711–720. https://doi.org/10.1631/jzus.B0920140

    Article  PubMed  PubMed Central  Google Scholar 

  51. Li M, Tian S, Yeung CKL et al (2014) Whole-genome sequencing of Berkshire (European native pig) provides insights into its origin and domestication. Sci Rep 4:1–7. https://doi.org/10.1038/srep04678

    Article  CAS  Google Scholar 

  52. Takahashi R (2018) Detection of inobuta from wild boar population in Japan by genetic analysis. Rev Agric Sci 6:61–71. https://doi.org/10.7831/ras.6.61

    Article  Google Scholar 

  53. Anderson D, Toma R, Negishi Y et al (2019) Mating of escaped domestic pigs with wild boar and possibility of their offspring migration after the Fukushima Daiichi Nuclear Power Plant accident. Sci Rep 9:1–6. https://doi.org/10.1038/s41598-019-47982-z

    Article  CAS  Google Scholar 

  54. Amorim A, Pereira F, Alves C, García O (2020) Species assignment in forensics and the challenge of hybrids. Forensic Sci Int Genet 48:102333. https://doi.org/10.1016/j.fsigen.2020.102333

    Article  CAS  PubMed  Google Scholar 

  55. Lopez-Oceja A, Nuñez C, Baeta M et al (2017) Species identification in meat products: A new screening method based on high resolution melting analysis of cyt b gene. Food Chem 237:701–706. https://doi.org/10.1016/j.foodchem.2017.06.004

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

The authors gratefully acknowledge the following individuals or organizations for their help in obtaining samples: Dr. Makoto Asano, Dr. Takashi Hayakawa, Dr. Satoshi Nagaoka, and Dr. Ryo Tadano (Gifu University); Dr. Hiroo Imai (Kyoto University); Kyoto City Zoo (Japan); Shunan Tokuyama Zoo (Japan); Takaoka Kojyo Zoo (Japan); KE’KEN Textile Testing & Certification Center (Japan). The authors would also like to thank Dr. Kohei Nakamura (Gifu University) for the use of the Nanodrop 1000 Spectrophotometer.

This work was supported in part by the Cooperative Research Program of the Primate Research Institute, Kyoto University (No. 2016-D-19).

Funding

The authors did not receive support from any organization for the submitted work.

Author information

Authors and Affiliations

  1. The United Graduate School of Agricultural Science, Gifu University, 1-1 Yanagido, Gifu, 501-1193, Japan

    Chikahiro Mori

  2. Forensic Science Laboratory, Gifu Prefectural Police Headquarters, 2-1-1 Yabutaminami, Gifu, 500-8501, Japan

    Chikahiro Mori

  3. Faculty of Applied Biological Sciences, Gifu University, 1-1 Yanagido, Gifu, 501-1193, Japan

    Shuichi Matsumura

Authors
  1. Chikahiro Mori
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Shuichi Matsumura
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

Chikahiro Mori: Conceptualization, methodology, validation, formal analysis, investigation, resources, data curation, writing (original draft), visualization, project administration. Shuichi Matsumura: conceptualization, methodology, resources, writing (review and editing), supervision.

Corresponding author

Correspondence to Chikahiro Mori.

Ethics declarations

Ethics approval

Not applicable.

Consent to participate

Not applicable.

Consent for publication

Not applicable.

Conflict of interest

The authors declare they have no competing interests.

Additional information

Publisher's note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary Information

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Mori, C., Matsumura, S. Development and validation of simultaneous identification of 26 mammalian and poultry species by a multiplex assay. Int J Legal Med 136, 1–12 (2022). https://doi.org/10.1007/s00414-021-02711-y

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00414-021-02711-y

Keywords

Search

Navigation