Genetic Identification And Validation Of Echinacea Species

• US 20090081657A1
• Filed: 03/28/2008
• Published: 03/26/2009
• Est. Priority Date: 03/28/2007
• Status: Active Grant

• Current Assignee: Peter K. Rogan
• Original Assignee: ThinkVillage Corporation
• Inventors: Rogan, Peter K.
• Granted Patent: US 7,811,766 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12Q 1/6895 for plants, fungi or algae

Abstract

A method for identification and validation of Echinacea is disclosed. Primers are designed based on information analysis of sequences from a large number of Echinacea species to amplify certain segments of genomic DNA to identify the species. Primers and methods are also disclosed to amplify other plant species that are frequently found in adulterated herbal samples of Echinacea.

Claims

1. A pair of oligonucleotide molecules for amplification of ribosomal DNA from a plant material, said oligonucleotide molecules being selected from the group consisting of primer set 1 and primer set 2, wherein primer set 1 comprises the oligonucleotides of SEQ ID Nos. 84 and 85, primer set 2 comprises the oligonucleotides of SEQ ID Nos. 86 and 87.

2. A pair of oligonucleotide molecules for amplification of ribosomal DNA from a plant material, said oligonucleotide molecules being selected from the group consisting of primer set 3 and primer set 4, wherein primer set 3 comprises the oligonucleotides of SEQ ID Nos. 88 and 89, primer set 4 comprises the oligonucleotides of SEQ ID Nos. 90 and 91.

3. A method for determining the existence of an organism or its derivatives in a material, said method comprising:

(1) a first DNA amplification of a segment on a ribosomal DNA, wherein a first primer pair is used as PCR primer and DNA from at least one species belonging to the same genus as said organism is used as template;

(2) sequencing the PCR product resulting from the first amplification;

(3) a second DNA amplification using as PCR primer a second primer pair and using as template DNA prepared from said material;

wherein the selection of the first primer pair comprises the steps of;

(a) searching for a divergent segment of the DNA from said at least one species with low average information content determined quantitatively surrounded by two conserved segments of said DNA with high average information content determined quantitatively; and

(b) designing the first primer pair for PCR amplification of said divergent segment by constructing a sequence logo for said DNA such that said primers contain a set of sequences present in said sequence logo that encompass the nucleotide variability of said conserved segments, said primer pair being able to anneal to said conserved segments for amplification of said divergent segment;

and the selection of the second primer pair comprises the steps of;

(a) searching the sequences obtained from step (2) for at least one segment of DNA with significant interspecies variations; and

(b) designing the second primer pair for DNA amplification, wherein said second primer pair comprises at least one interspecies variation.

4. The method of claim 3, wherein the PCR product resulting from the first amplification comprises the ITS region.

5. The method of claim 3, wherein the second primer pair is selected from the group consisting of primer set 1, primer set 2, primer set 3, and primer set 4;

and primer set I comprises the oligonucleotides of SEQ ID Nos. 84 and 85, primer set 2 comprises the oligonucleotides of SEQ ID Nos. 86 and 87, primer set 3 comprises the oligonucleotides of SEQ ID Nos. 88 and 89, and primer set 4 comprises the oligonucleotides of SEQ ID Nos. 90 and 91.

6. A method for determining the existence of a plant material derived from Echinacea based on the primary structure of Echinacea DNA, said method comprising amplification of a segment of the internal transcribed spacer region of the ribosomal DNA of said plant material, said amplification being performed using a pair of oligonucleotides selected from the group consisting of primer set 1, primer set 2, primer set 3, and primer set 4, wherein primer set 1 comprises the oligonucleotides of SEQ ID Nos. 84 and 85, primer set 2 comprises the oligonucleotides of SEQ ID Nos. 86 and 87, primer set 3 comprises the oligonucleotides of SEQ ID Nos. 88 and 89, and primer set 4 comprises the oligonucleotides of SEQ ID Nos. 90 and 91.

7. The method of claim 6 wherein the amplified segment comprises at least one DNA fragment derived from an internal transcribed spacer region selected from the group consisting of ITS1 or ITS2.

8. The method of claim 6 wherein the segment is amplified using primers that specifically amplify DNA from Echinacea species but do not amplify DNA from Parthenium species.

9. The method of claim 6 wherein the segment is amplified using primers that specifically amplify DNA from Parthenium species but do not amplify DNA from Echinacea species.

10. The method of claim 6 further comprising a step of sequencing said amplified segment.

11. The method of claim 6 wherein DNA prepared from a target sample is used as a template for said amplification.

12. The method of claim 11 wherein said target sample is obtained from a previous growing season, said target sample having been stored for at least 6 months.

13. The method of claim 11 wherein the target sample contains specimen from at least two species selected from the group consisting of Echinacea, Parthineum and Rudbeckia.

14. The method of claim 11 wherein the target sample contains specimen from Echinacea and Parthineum.

15. An oligonucleotide primer for sequencing of the ITS region of the Echinacea DNA, said oligonucleotide primer being selected from the group consisting of SEQ ID No. 82 and SEQ ID No. 83.