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Forensic animal DNA analysis using economical two-step direct PCR

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Abstract

Wildlife forensic DNA analysis by amplification of a mitochondrial locus followed by DNA sequencing is routine, yet suffers from being costly and time-consuming. To address these disadvantages we report on a low-cost two-step direct PCR assay to efficiently analyze 12 forensically relevant mammalian sample types without DNA extraction. A cytochrome oxidase I degenerate-universal primer pair was designed and validated for the developed assay. The 12 sample types, which included bone, horn, feces, and urine, were amplified successfully by the assay using a pre-direct PCR dilution protocol. The average amplification success rate was as high as 92.5 % (n = 350), with an average PCR product concentration of 220.71 ± 180.84 ng/μL. Differences in amplification success rate and PCR product quantity between sample types were observed; however, most samples provided high quality sequences, permitting a 100 % nucleotide similarity to their respective species via BLAST database queries. The combination of PBS and Phire® Hot Start II DNA polymerase gave comparable amplification success rate and amplicon quantity with the proprietary commercial kits (P > 0.05, n = 350) but at considerably lower cost. The stability of the assay was tested by successfully amplifying samples that had been stored for up to 12 months. Our data indicate that this low-cost two-step direct amplification assay has the potential to be a valuable tool for the forensic DNA community.

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Acknowledgments

The authors gratefully acknowledge the support of the Prince of Songkla University Research Fund (Grant no. SCI550385S) for TK and WC. We are in debt to Mr. Yingyong Lapwong and Mr. Sukone Pradutkanchana for the voucher specimens. We also appreciate the help of the Princess Maha Chakri Sirindhorn Natural History Museum, Thailand; the Songkhla Zoo, Thailand; the Sawaddee Deer Park, Thailand; and the Chang-Puak Elephant Camp, Thailand, in contributing samples.

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Authors and Affiliations

  1. Forensic Science Program, Department of Applied Science, Faculty of Science, Prince of Songkla University, Songkhla, Thailand

    Thitika Kitpipit & Phuvadol Thanakiatkrai

  2. Department of Molecular Biotechnology and Bioinformatics, Faculty of Science, Prince of Songkla University, Songkhla, Thailand

    Wilaiwan Chotigeat

  3. School of Biological Sciences, Flinders University, Adelaide, 5001, Australia

    Adrian Linacre

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  1. Thitika Kitpipit
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  2. Wilaiwan Chotigeat
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  3. Adrian Linacre
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  4. Phuvadol Thanakiatkrai
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Correspondence to Thitika Kitpipit.

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Kitpipit, T., Chotigeat, W., Linacre, A. et al. Forensic animal DNA analysis using economical two-step direct PCR. Forensic Sci Med Pathol 10, 29–38 (2014). https://doi.org/10.1007/s12024-013-9521-8

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