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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 [
] (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.
The level of DNA degradation was examined on an Agilent 2100 Bioanalyzer (Agilent Technologies, USA), specifically on DNA 100 and high-sensitivity DNA chips.
3. Results
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.
Fig. 1The amplifiability of Bos taurus nuclear DNA (101 bp amplicon) after boiling. NC-negative control, PC-positive control.
Fig. 2The amplifiability of Bos taurus mitochondrial DNA (12S rRNA (111 bp amplicon), CytB (357 bp amplicon), COI (511 bp amplicon), and jgHLCO (710 bp amplicon)) after boiling. NC-negative control, PC-positive control.
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.
References
Karlsson A.O.
Holmlund G.
Identification of mammal species using species-specific DNA pyrosequencing.
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