The aim of our study was to monitor the quality and quantity of DNA in bone samples that were boiled for 48 h. Bos taurus bone disks were sampled every hour for 48 h. The subsequent DNA analysis used multiple mitochondrial DNA (mtDNA) targets (100–700 bp) to evaluate the quality and quantity of the DNA extracted. The DNA extracted from bone disks remained typeable after boiling for 48 h. We have proven that DNA typing results can be obtained even after long-term boiling.
Traditional Chinese medicine (TCM) has been practiced for thousands of years, but only within the last few decades has its use become more widespread outside of Asia. One of the most trafficked products of TCM is the broth from Panthera tigris (PT). The traditional recipe for the preparation of Panthera tigris broth requires boiling the PT bones with removed bone marrow for 7 days and 7 nights. The final PT broths analyzed by our laboratory in the past did not yield any typeable DNA for use in DNA-based species identification.
2. Materials and methods
2.1 Sample preparation
One-centimeter cuts of Bos taurus femur bones were boiled for 48 h. Samples were taken every hour for subsequent DNA analysis. Heat-processed bones were cut into 1,5 × 1,5 cm samples, bleached and washed in water and ethanol. Cleaned samples were ground in a cryogenic mill 6770 Freezer/Mill® (SPEX SamplePrep, USA). DNA was extracted by a PrepFiler BTA Forensic DNA extraction kit (ThermoFisher, USA) according to the protocol provided by the manufacturer.
The presence of Bos taurus DNA was verified by specific Bos taurus STR primers targeting BLAD (101 bp) (designed using GenBank: KX666090.1 sequence). DNA degradation was tested by mtDNA primers commonly used for species identification (12S rRNA [
] (111 bp), CytB [
- Karlsson A.O.
- Holmlund G.
Identification of mammal species using species-specific DNA pyrosequencing.
Forensic Sci. Int. 2007; 173: 16-20
- Parson W.
- Pegoraro K.
- Niederstätter H.
- Föger M.
- Steinlechner M.
Species identification by means of the cytochrome b gene.
Int. J. Legal Med. 2000; 114: 23-28
] (357 bp), COI [
- Branicki W.
- Kupiec T.
- Pawlowski R.
Validation of cytochrome b sequence analysis as a method of species identification.
J. Forensic Sci. 2003; 48: 1-5
] (511 bp), jgHLCO [
- Kitpipit T.
- Chotigeat W.
- Linacre A.
- Thanakiatkrai P.
Forensic animal DNA analysis using economical two-step direct PCR.
Forensic Sci. Med. Pathol. 2014; 10: 29-38
] (710 bp)). PCRs were carried out in 25 μl reaction volumes containing 0,4 μM of each primer, 1x Gold buffer, 2 mM MgCl2, 400 μM of each deoxy-nucleotide triphosphates, 0,625 U of AmpliTaq Gold DNA polymerase (ThermoFisher, USA) and 10 μl of DNA extract.
- Geller J.
- Meyer C.
- Parker M.
- Hawk H.
Redesign of PCR primers for mitochondrial cytochrome c oxidase subunit I for marine invertebrates and application in all‐taxa biotic surveys.
Mol. Ecol. Resour. 2013; 13: 851-861
The level of DNA degradation was examined on an Agilent 2100 Bioanalyzer (Agilent Technologies, USA), specifically on DNA 100 and high-sensitivity DNA chips.
We found that a 101 bp amplicon of nuclear DNA is amplifiable even after 48 h of boiling (Fig. 1). Mitochondrial targets 12S rRNA (111 bp), CytB (357 bp), COI (511 bp), and jgHLCO were amplifiable after 48, 16, 8, and 8 h of boiling, respectively (Fig. 2). The level of DNA degradation (Agilent 2100 Bioanalyzer) is shown on Fig. 3.
Nuclear DNA extracted from boiled Bos taurus bones is amplifiable (101 bp target) even after 48 h of boiling. The longer the mitochondrial DNA amplicon is, the shorter the DNA “survival” time. After 8 h of boiling, 711 bp mtDNA fragments can be amplified; 357 bp fragments can be amplified after 16 h of boiling, and 111 bp fragments are amplifiable after 48 h of boiling. The analysis of mtDNA is commonly used for species identification. The mtDNA fragment length (111 bp) is sufficient for species identification in the majority of cases. This experiment has shown that DNA is typeable even after long-term boiling. Another possible approach to species identification in TCM products would be the analysis of proteins or MPS (massive parallel sequencing).
Declaration of Competing Interest
The authors of this manuscript declare no conflicts of interest. This project was supported by research project VH20182021028 of the Ministry of Interior Czech Republic.
- Identification of mammal species using species-specific DNA pyrosequencing.Forensic Sci. Int. 2007; 173: 16-20
- Species identification by means of the cytochrome b gene.Int. J. Legal Med. 2000; 114: 23-28
- Validation of cytochrome b sequence analysis as a method of species identification.J. Forensic Sci. 2003; 48: 1-5
- Forensic animal DNA analysis using economical two-step direct PCR.Forensic Sci. Med. Pathol. 2014; 10: 29-38
- Redesign of PCR primers for mitochondrial cytochrome c oxidase subunit I for marine invertebrates and application in all‐taxa biotic surveys.Mol. Ecol. Resour. 2013; 13: 851-861
Published online: September 23, 2019
Accepted: September 21, 2019
Received: September 12, 2019
© 2019 Elsevier B.V. All rights reserved.