![handle is hein.journals/injlegame136 and id is 1 raw text is: International Journal of Legal Medicine (2022) 136:1-12
https://doi.org/l 0.1007/s00414-021-02711-y
ORIGINAL ARTICLE
Development and validation of simultaneous identification of 26
mammalian and poultry species by a multiplex assay
Chikahiro Mori - Shuichi Matsumura3
Received: 9 July 2021 / Accepted: 24 September 2021 / Published online: 9 October 2021
© The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature 2021
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.
Keywords Species identification - Forensic science - DNA mixtures - Multiplex PCR - DNA degradation
Introduction
Non-human biological samples are frequently encountered
in routine forensic investigations. Forensic scientists are
required to identify mammalian species in many forensic
cases; for example, traffic accidents involving animals [1],
animal cruelty [2-5], livestock robbery [6, 7], animal attacks
[5, 7, 8], murder cases [9], and postmortem investigations
[10]. In addition, species identification has become impor-
tant in wildlife forensics [11-15], conservation of endan-
gered animals [16-19], and the detection of food fraud
[20-22].
Chikahiro Mori
cmori2010gp@gmail.com
The United Graduate School of Agricultural Science, Gifu
University, 1-1 Yanagido, Gifu 501-1193, Japan
2 Forensic Science Laboratory, Gifu Prefectural Police
Headquarters, 2-1-1 Yabutaminami, Gifu 500-8501, Japan
3 Faculty of Applied Biological Sciences, Gifu University, 1-1
Yanagido, Gifu 501-1193, Japan
Currently, mitochondrial DNA (mtDNA) markers are
commonly used for species identification because the high
copy number of mtDNA per cell [23-25] allows for success-
ful identification, even in samples that contain insufficient
amounts of nuclear DNA (e.g., partial bone fragments and
hair shafts). The cytochrome b (cyt b) gene, the cytochrome
c oxidase I (COI) gene, and the D-loop region are typical
mtDNA markers in forensic, phylogenetic, and biodiversity
studies [7, 13, 15, 26-32]. Other genes, such as the 12S and
16S ribosomal RNA genes, have also been studied for spe-
cies identification [33-35].
In forensic investigations, non-human biological sam-
ples recovered from crime scenes are often contaminated
with human DNA [25]. In other cases, samples may con-
tain DNA from more than one non-human species. Typical
DNA mixture samples include feces containing materi-
als from ingested species, mixed meat or meat products,
and saliva collected from a bite mark on a victim's body.
Species identification from these mixtures is a challenge
for forensic scientists. This is mainly because conven-
tional Sanger sequencing using universal primers, which
1 Springer](ViewImageLocal?handle=hein.journals/injlegame136&div=1&collection=&method=preview&ext=.png&size=3)
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