Authentication of botanical DNA isolated from dietary supplements
Described herein are methods and kits for authentication of botanical DNA fragments isolated from processed botanical products, including dietary supplements or nutraceutical compositions including botanical extracts.
1. A method of authenticating sterilized chamomile material in a botanical dietary supplement, the method comprising:
isolating genomic DNA from a botanical dietary supplement comprising German chamomile, feverfew, Roman chamomile, and Chinese chamomile, wherein the geomic chamomile DNA isolated from the botanical dietary supplement comprises chamomile DNA fragments, wherein at least some of the chamomile DNA fragments are less than 220 base pairs, wherein the genomic chamomile DNA isolated from the botanical dietary supplement is degraded and undetectable using DNA barcoding, and wherein the chamomile DNA fragments are present in an amount of 1 pg to 1 ng;
ligating adapters to the chamomile DNA fragments to generate adapter-ligated DNA fragments;
amplifying the adapter-ligated DNA fragments using 8-12 cycles of polymerase chain reaction (PCR);
detecting the chamomile DNA fragments;
determining a size range of the chamomile DNA fragments;
hybridizing primers to a target nucleic acid sequence in the chamomile DNA fragments, wherein the primers comprise a species-specific primer set selected from:
a primer comprising a nucleic acid sequence as defined in SEQ ID NO: 1 and a primer comprising a nucleic acid sequence as defined in SEQ ID NO: 2;
a primer comprising a nucleic acid sequence as defined in SEQ ID NO: 3 and a primer comprising a nucleic acid sequence as defined in SEQ ID NO: 4;
a primer comprising a nucleic acid sequence as defined in SEQ ID NO: 5 and a primer comprising a nucleic acid sequence as defined in SEQ ID NO: 6; and
a primer comprising a nucleic acid sequence as defined in SEQ ID NO: 7 and a primer comprising a nucleic acid sequence as defined in SEQ ID NO: 8, and
amplifying the target nucleic acid sequence.
2. The method of claim 1 , further comprising sequencing the target nucleic acid sequence.
3. The method of claim 1 , wherein detecting the chamomile DNA fragments comprises running the amplified adapter-ligated DNA fragments on a gel for detection of the chamomile DNA fragments.
4. The method of claim 1 , wherein the primers are designed on the basis of target species consensus sequence as PCR template and non-target consensus sequences as exclusion sequences.
5. The method of claim 1 , further comprising detecting botanical adulteration in the botanical dietary sample.
6. The method of claim 1 , wherein ligating is performed at 4° C. overnight.
7. The method of claim 1 , further omprising evaluating total DNA after the amplifying the target nucleic acid sequence.
8. The method of claim 1 , further comprising determining the sequence of the chamomile DNA fragments or determining the quantity or quality of the chamomile DNA fragments.
9. The method of claim 1 , wherein the botanical dietary supplement is a processed botanical extract.
10. The method of claim 1 , wherein the chamomile is Matricaria chamomilla, Tanacetum parthenium (feverfew), Chamaemelum nobile ( Anthemis nobilis or Roman chamomile), Chrysanthemum x morifolium or Chrysanthemum indicum (Chinese chamomile).
11. A method of authenicating a processed botanical dietary supplement, the method comprising:
isolating genomic DNA from a processed botanical dietary supplement, wherein the genomic DNA isolated from the processed botanical dietary supplement comprises botanical DNA fragments, wherein at least some of the botanical DNA fragments are less than 220 base pairs, wherein the genomic DNA isolated from the processed botanical dietary supplement is degraded and undetectable using DNA barcoding, and wherein the botanical DNA fragments are present in an amount of 1 pg to 1 ng;
ligating adapters to the botanical DNA fragments to generate adapter-ligated DNA fragments;
amplifying the adapter-ligated DNA fragments using 8-12 cycles of polymerase chain reaction (PCR);
detecting the botanical DNA fragments;
determining a size range of the botanical DNA fragments;
designing primers according to the size range of the botanical DNA fragments;
hybridizing the primers to a target nucleic acid sequence in the botanical DNA fragments; and
amplifying the target nucleic acid sequence,
wherein the processed botanical dietary supplement comprises a botanical material comprising chamomile ( Matricaria chamomilla ), German chamomile ( Matricaria recutita ), feverfew ( Tanacetum parthenium ), Roman chamomile ( Chamaemelum nobile syn anthemis noblis ), Chineses chamomile ( Chrysanthemum x morifolium or Chrysanthemum indicum ), guarna ( Paullinia cupana ), parsley ( Petroselimum crispum ), celery ( Apium graveolens ), fennel ( Foeniculum vulgare ), Siberian ginseng ( Eleutherococcus senticosus ), Dong Quai ( Angelica sinensis ), garden angelica ( Angelica archangelica ), pubescent Angelica ( Angelica pubescens ), dahurain angelica ( Angelica dahurica ), Chinese cinnamon ( Cinnamomum cassia ), true cinnamon ( Cinnanamomum verum syn Cinnamomum zeylanicum ), Indonesian cinnamon ( Cinnamomum burmannii ), ginkgo ( Ginkgo biloba ), Japanese sophora ( Sophora japonica ), buckwheat ( Fagopyrum esculentum ), jujube ( Ziziphus spinosa ), Indian jujube ( Ziziphus mauritiana ), Japanese raisin tree ( Hovenia dulcis ), ginger ( Zingiber officinale ), lesser galangal ( Alpinia officinarum ), greater galangal ( Alpinia galanga ), schisandra ( Schisandra chinensis ), southern schisandra ( Schisandra sphenanthera ), astragalus ( Astragalus membranaceus ), maca ( Lepidium meyenii ), radish ( Raphanus sativus ), turnip ( Brassica rapa ), peppermint ( Mentha piperita ), Chinese mint ( Mentha canadensis ), green tea ( Camellia sinensis ), rosemary ( Rosmarinus officinalis ), bilberry ( Vaccinium myrtillus ), blueberry ( Vaccinium corymbosum ), cranberry ( Vaccinium macrocarpon ), mulberry ( Morus alba ), or guarana ( Paullinia cupana ).
12. The method of claim 11 , further comprising sequencing the target nucleic acid sequence.
13. The method of claim 11 , wherein detecting the botanical DNA fragments comprises running the amplified adapter-ligated DNA fragments on a gel for detection of the chamomile DNA fragments.
14. The method of claim 11 , wherein the primers are designed on the basis of target species consensus sequence as PCR template and non-target consensus sequences as exclusion sequences.
15. The method of claim 11 , further comprising detecting botanical adultaration in the processed botanical dietary supplement.
16. The methof claim 11 , wherein ligating is performed at 4° C. overnight.
17. The method of claim 11 , further comprising evaluating total DNA after the amplifying the target nucleic acid sequence.
18. The method of claim 11 , wherein the processed botanical dietary supplement is a processed botanical extract.