FINAL by: OSAC Program Office; Version 2, January 13, 2020 1

SAC RESEARCH NEEDS ASSESSMENT FORM

Title of research need:

Develop species-specific mitochondrial primers for comingled samples that 

require taxonomic identification of one or more contributors

Describe

the need:

Forensic DNA typing for taxonomic identification (aka barcoding) is used to authenticate 

seized samples by comparison to appropriate databases (1, 2, 3). Taxonomic identification 

of multiple contributors in a DNA mixture allows the makeup of complex samples to be 

elucidated. Identifying different species in a complex mixture can result in identification of 

illegal or protected species when mixed with legal or non-protected species. In addition, 

the ability to use eDNA samples to detect illegal and injurious species in imports of aquatic 

organisms is in demand at US ports of entry to facilitate legal trade. When unknown 

biological material is subjected to DNA sequencing using non-specific, or universal, 

mitochondrial primers, an uninterpretable mixed sequence may be obtained. Developing 

species- or taxon-specific mitochondrial primers would assist in restricting sequencing 

results to a single entity, resulting in the identification of the species/taxon of origin. 

Further, wildlife forensic source materials may be contaminated by human DNA prior to 

submission to the forensic laboratory and developing species/taxon-specific mitochondrial 

primers that do not amplify human DNA would assist in identifying the wildlife species 

present in the sample. Reliable and detailed data on taxon of origin in mixed samples can 

also enhance identification of illegal species in food supplements and traditional medicines 

(4) and that of cryptic species, or species that can be distinguished genetically but not 

necessarily morphologically (5, 6). By targeting species/taxon-specific DNA sequences, the 

sensitivity and accuracy of forensic testing is increased, enabling more specific 

identification of questioned samples that could not be achieved using standard sequencing 

methods (7). 

Keyword(s): Species identification, taxonomic identification, mitochondrial DNA, sequencing, complex 

mixtures, primers, DNA typing, barcoding, eDNA

Submitting subcommittee(s): Wildlife Forensic Biology Date Approved: 10/7/2022

Background Information:

1. Does this research need address a gap(s) in a current or planned standard? (ex.: Field identification system 

for on scene opioid detection and confirmation)

ANSI/ASB Standard 048, Wildlife Forensic DNA Standard Procedures states there must be a protocol to 

interpret “sequence mixtures” (5.1.c) and that for “laboratories that report on the composition of mixture 

samples, a method must be established that specifically addresses analysis and interpretation of such 

mixtures” (6.1.2). 

ANSI/ASB Standard 047, Wildlife Forensics Validation Standard - Validating New Primers for Sequencing sets 

requirements for species specificity of primers (4.3.2), including “species likely to be present in combination 

with the species of interest on evidentiary items (4.3.2.b). 
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2. Are you aware of any ongoing research that may address this research need that has not yet been published 

(e.g., research presented in conference proceedings, studies that you or a colleague have participated in but 

have yet to be published)?

The U.S. Fish and Wildlife Service and US Geological Survey invasive species programs have funded research 

into targeted PCR testing for invasive and injurious aquatic species (e.g., Dreissenid mussels, Burmese 

python, among others). The intent of this research is to deploy rapid testing units at ports of entry to test 

water samples for the target species. 

3. Key bibliographic references relating to this research need: (ex.: Toll, L., Standifer, K. M., Massotte, D., eds. 

(2019). Current Topics in Opioid Research. Lausanne: Frontiers Media SA. doi: 10.3389/978-2-88963-180-3)

1. Chang, C. H., Dai, W. Y., Chen, T. Y., Lee, A. H., Hou, H. Y., Liu, S. H., & Jang-Liaw, N. H. (2018). DNA 

barcoding reveals CITES-listed species among Taiwanese government-seized chelonian 

specimens. Genome, 61(8), 615–624. 

2. Bezeng, B. S., Davies, T. J., Daru, B. H., Kabongo, R. M., Maurin, O., Yessoufou, K., van der Bank, H., & 

van der Bank, M. (2017). Ten years of barcoding at the African Centre for DNA 

Barcoding. Genome, 60(7), 629–638. 

3. Sheth, B. P., & Thaker, V. S. (2017). DNA barcoding and traditional taxonomy: an integrated approach 

for biodiversity conservation. Genome, 60(7), 618–628. 

4. Arulandhu, A. J., 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(10), 

1–18. 

5. Wilson-Wilde, L., Norman, J., Robertson, J., Sarre, S., & Georges, A. (2010). Current issues in species 

identification for forensic science and the validity of using the cytochrome oxidase I (COI) 

gene. Forensic Science, Medicine, and Pathology, 6(3), 233–241. 

6. Vasconcelos, R., Montero-Mendieta, S., Simó-Riudalbas, M., Sindaco, R., Santos, X., Fasola, M., 

Llorente, G., Razzetti, E., & Carranza, S. (2016). Unexpectedly High Levels of Cryptic Diversity 

Uncovered by a Complete DNA Barcoding of Reptiles of the Socotra Archipelago. PloS One, 11(3), 

e0149985. 

7. Tobe, S. S., & Linacre, A. (2010). DNA typing in wildlife crime: recent developments in species 

identification. Forensic Science, Medicine, and Pathology, 6(3), 195–206. 

4. Review the annual operational/research needs published by the National Institute of Justice (NIJ) at 

https://nij.ojp.gov/topics/articles/forensic-science-research-and-development-technology-working-groupoperational#latest? Is your research need identified by NIJ?

Forensic Biology/DNA, Scientific Research, Increased information about the discriminatory power and 

sensitivity of alternate biological analyses (e.g., proteomics, microbiome, plants, animals) to associate 

individuals with crime scene evidence.
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5. In what ways would the research results improve current laboratory capabilities?

Identification of species-specific primer sets would allow labs to increase the number of samples that could 

be tested by not eliminating mixture samples. The success rate of identifying evidentiary samples would be 

increased, and rapid testing would enhance the interception of illegal species. 

6. In what ways would the research results improve understanding of the scientific basis for the 

subcommittee(s)?

Availability of species-specific mitochondrial primers is insufficient to meet the needs of wildlife forensic 

laboratories and law enforcement agencies that submit samples for testing and identification. Many species 

of interest to wildlife law enforcement are difficult to obtain but important to detect in forensic samples, 

however, they are not of general interest to the wider conservation genetics community. The development 

of additional species-specific mitochondrial primers for taxonomic identification would greatly increase the 

ability of species of forensic value to be identified for casework purposes.

7. In what ways would the research results improve services to the criminal justice system?

Wildlife forensic laboratories are relied upon to identify the taxonomic origin of evidentiary materials. When 

the desired laboratory capabilities do not exist because further targeted research is required, investigations 

are delayed or abandoned that could otherwise be easily completed. 

8. Status assessment (I, II, III, or IV): II

Major gap in 

current 

knowledge

Minor gap in 

current 

knowledge

No or limited

current research is 

being conducted I III

Existing current 

research is being 

conducted II IV

This research need has been identified by one or more subcommittees of OSAC and is being provided as an 

informational resource to the community.