1. Development of a simultaneous identification method for 13 animal species using two multiplex real-time PCR assays and melting curve analysis 
Natsumi Ishida, Makoto Sakurada, Hiroshi Kusunoki, Yasuhiro Ueno 
Legal Medicine 
Volume 30, Pages (January 2018)
DOI: /j.legalmed
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2. Fig. 1
Specificity of the primer pairs used for nine domestic animals and four wild animals. (A) PCR amplicons from each species-specific primer pair are as follows. Lanes: M, 100 bp ladder marker (TaKaRa); 1, dog; 2, cat; 3, rabbit; 4, pig; 5, goat; 6, horse (primer pairs, current study); lanes 7, cattle; 8, chicken; 9, sheep (commercial primer pairs from BEX Co. see Materials and methods). (B) PCR amplicons from each species-specific primer pair are as follows. Lanes: M, 100 bp ladder marker (TaKaRa); 1, deer; 2, raccoon-dog; 3, monkey; 4, bear (black); 5, bear (brown) (original primer pairs).
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3. Fig. 2
Melting curve analysis for singleplex real-time PCR assays. (A) DNA (1 ng) from nine domestic animals (dog, cat, rabbit, cattle, pig, chicken, goat, sheep, and horse) was amplified using species-specific primer pairs. (B) DNA (1 ng) from four wild animals (deer, raccoon-dog, monkey, black or brown bear) was amplified using species-specific primer pairs.
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4. Fig. 3
Melting curve analysis of the negative control used for multiplex real-time PCR (nine domestic animals). Distilled water was used as the negative control for the multiplex real-time PCRs.
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5. Fig. 4
Sensitivities of the primer pairs used for singleplex real-time PCR. The assay was evaluated using DNA from each animal serially diluted from 1 ng to 0.1 pg for each species-specific primer pair. (A) dog; (B) cat; (C) rabbit; (D) cattle; (E) pig; (F) chicken; (G) goat; (H) sheep; (I) horse. NC, negative control.
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6. Fig. 5
Sensitivities of the primer pairs used for singleplex PCR with AmpliTaq Gold® 360 Master Mix. The assay was evaluated using DNA from each animal serially diluted from 1 ng to 0.1 pg (the panels represent 1 ng to  ng) for each species-specific primer pair.
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7. Fig. 6
Amplification using vertebrate universal primers. PCR amplicons from the vertebrate universal primer pair are as follows: lanes: M, 100 bp ladder marker (TaKaRa); 1, dog; 2, cat; 3, rabbit; 4, cattle; 5, pig; 6, chicken; 7, goat; 8, sheep; 9, horse; 10, negative control; 11, deer; 12, raccoon-dog; 13, monkey; 14, black bear; 15, brown bear; 16, axis deer; 17, capuchin monkey; 18, negative control.
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8. Fig. 7
Species identification of experimental forensic samples using multiplex real-time PCR for nine animal species. (A) hair samples (dog, cat); (B) bone samples (cattle, pig). Numbers adjacent to the melting peaks indicate their melting temperatures.
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9. Fig. 8
Melting curve analysis of the practical forensic samples. (A)–(C) Samples 01–03 were analyzed for the presence of DNA from nine animal species using multiplex real-time PCR. (D) Sample 03 was tested for the presence of DNA from four wild animal species using multiplex real-time PCR. Numbers adjacent to melting peaks indicate their melting temperatures.
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10. Fig. 9
Comparison of the melting temperatures of PCR amplicons from pig and wild boar. DNA (1 ng) from pig and wild boar was amplified using pig-specific primer pairs.
Legal Medicine  , 64-71DOI: ( /j.legalmed )
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