Identification of Echinacea and its imposters using genetic variations
This invention provides methods, compounds, and kits for identifying Echinacea species and for distinguishing between Echinacea and imposter species based upon molecular variations. In particular, amplification primers are provided that amplify specific regions of nucleic acid sequences to generate unique amplification profiles in the different plant species. A plant may be identified by comparing its amplification profile to known amplification profiles of Echinacea and imposter species.
1 . A method for determining whether a plant is an Echinacea species or an imposter species, the method comprising:
a. amplifying a specific region of a nucleic acid sequence from the plant using at least one pair of amplification primers, whereby the primers generate a specific amplification profile in each species; and
b. identifying the plant by comparing its amplification profile against known amplification profiles of Echinacea and imposter species.
2 . The method of claim 1 , wherein the pair of amplification primers comprises a first primer selected from the group consisting of SEQ ID NOs:1, 2, 3, and 4, and a second primer that is a specific universal primer.
3 . The method of claim 2 , wherein the first primer is at least seventy-five percent identical in nucleotide sequence to SEQ ID Nos:1, 2, 3, or 4 and the second primer is at least seventy-five percent identical in nucleotide sequence to the specific universal primer.
4 . The method of claim 1 , wherein the nucleotides comprising the primers are selected from the group consisting of standard, nonstandard, modified, and derivatized nucleotides.
5 . The method of claim 1 , wherein the Echinacea species is selected from the group consisting of E. angustifolia, E. pallida, E. purpurea, E. afrorubens, E. laevigata, E. paradoxa, E. sanguinea, E. simulata , and E. tennesseensis.
6 . The method of claim 1 , wherein the imposter species is selected from the group consisting of Parthenium integrifolium, Eryngium spp., Helianthus spp., Lespedeza capitata, Mahonia aquifolium , and Rudbeckia spp.
7 . The method of claim 1 , wherein the Echinacea species is selected from the group consisting of E. angustifolia, E. pallida , and E. purpurea , and the imposter species is Parthenium integrifolium.
8 . The method of claim 1 , wherein the plant is a material selected from the group consisting of roots, rhizomes, stems, leaves, leaflets, flowers, seed heads, and seeds.
9 . The method of claim 8 , wherein the plant material is selected from the group consisting of whole, shredded, sliced, diced, crushed, pulverized material, and extracted juice.
10 . The method of claim 1 , wherein the plant is a processed plant material selected from the group consisting of capsules, tablets, powders, teas, aqueous extracts, alcohol extracts, glycerine extracts, creams, and gels.
11 . The method of claim 1 , wherein the nucleic acid sequence is deoxyribonucleic acid.
12 . The method of claim 1 , wherein the nucleic acid sequence is amplified using PCR.
13 . The method of claim 1 , wherein the nucleic acid sequence is amplified using real time PCR.
14 . The method of claim 1 , wherein the method further comprises distinguishing among species of Echinacea.
15 . A method for distinguishing among species of Echinacea , the method comprising:
a. amplifying a specific region of DNA from a plant using a first amplification primer selected from the group consisting of SEQ ID NOs:1, 2, and 4 and a second amplification primer that is a specific universal primer, whereby the primers generate a specific amplification profile in each species; and
b. identifying the plant by comparing its amplification profile against known amplification profiles of Echinacea species.
16 . The method of claim 15 , wherein the first primer is at least seventy-five percent identical in nucleotide sequence to SEQ ID Nos:1, 2, 3, or 4 and the second primer is at least seventy-five percent identical in nucleotide sequence to the specific universal primer.
17 . A single stranded oligonucleotide consisting essentially of a nucleotide sequence selected from the group consisting of SEQ ID NOs:1, 2, 3, and 4.
18 . The oligonucleotide of claim 16 , wherein the oligonucleotide is at least seventy-five percent identical in nucleotide sequence to SEQ ID NOs:1, 2, 3, or 4.
19 . An isolated nucleic acid selected from the group consisting of SEQ ID NOs:1, 2, 3, and 4.
20 . The nucleic acid of claim 19 , wherein the nucleic acid is at least seventy-five percent identical in nucleotide sequence to SEQ ID NOs:1, 2, 3, or 4.
21 . A kit for determining whether a plant is an Echinacea species or an imposter species, the kit comprising:
a. amplification primers that, in conjunction with specific universal primers, produce a specific amplification profile in each species;
b. instructions for amplifying a region of deoxyribonucleic from the plant; and
c. amplification profiles from Echinacea and imposter species for identifying the plant by comparing its amplification profile with the standard profiles.
22 . The kit of claim 21 , wherein the amplification primers are selected from the group consisting of SEQ ID NOs:1, 2, 3, and 4.
23 . The kit of claim 22 , wherein the primers are at least seventy-five percent identical in nucleotide sequence to SEQ ID NOs:1, 2, 3, or 4.
24 . The kit of claim 21 , wherein the nucleotides comprising the primers are selected from the group consisting of standard, nonstandard, modified, and derivatized nucleotides.
25 . The kit of claim 21 , wherein the Echinacea species is selected from the group consisting of E. angustifolia, E. pallida, E. purpurea, E. atrorubens, E. laevigata, E. paradoxa, E. sanguinea, E. simulata , and E. tennesseensis.
26 . The kit of claim 21 , wherein the imposter species is selected from the group consisting of Parthenium integrifolium, Eryngium spp., Helianthus spp., Lespedeza capitata, Mahonia aquifolium , and Rudbeckia spp.
27 . The kit of claim 21 , wherein the Echinacea species is selected from the group consisting of E. angustifolia, E. pallida , and E. purpurea , and the imposter species is Parthenium integrifolium.
28 . The kit of claim 21 , wherein the plant is a material selected from the group consisting of roots, rhizomes, stems, leaves, leaflets, flowers, seed heads, and seeds.
29 . The kit of claim 28 , wherein the plant material is selected from the group consisting of whole, shredded, sliced, diced, crushed, pulverized material, and extracted juice.
30 . The kit of claim 21 , wherein the plant is a processed plant material selected from the group consisting of capsules, tablets, powders, teas, aqueous extracts, alcohol extracts, glycerine extracts, creams, and gels.
31 . The kit of claim 21 , wherein the deoxyribonucleic acid is amplified using PCR.
32 . The kit of claim 21 , wherein the deoxyribonucleic acid is amplified using real time PCR.
33 . The kit of claim 21 , wherein the kit further comprises a reaction buffer solution, a dNTP mixture, a divalent cation solution, and Taq DNA polymerase.
34 . The kit of claim 21 , wherein the kit further comprises distinguishing among species of Echinacea.