MICROCHIP FOR IDENTIFYING PHELLINUS SPECIES AND THE METHOD THEREOF

• US 20110111970A1
• Filed: 11/06/2009
• Published: 05/12/2011
• Est. Priority Date: 11/06/2009
• Status: Active Grant

• Current Assignee: National Taiwan University (Government of The Republic of China)
• Original Assignee: National Taiwan University (Government of The Republic of China)
• Inventors: Shu, Po-Yao, Tzean, Yuh, Tzean, Shean-Shong
• Granted Patent: US 8,580,514 B2
• Time in Patent Office: Days
• US Class Current: 506/9
• CPC Class Codes: C12Q 1/6895 for plants, fungi or algae

Abstract

The developed oligonucleotide microchip for simultaneous, rapid identification of multiple crucial forest Phellinus pathogens was based on the DIG or biotin-labeled specific probes derived form ribosomal DNA genes (ITS1-5.8S-ITS2), by using reverse-dot hybridization. The chip can precisely and accurately identify and diagnose seventeen Phellinus species, including notorious hardwood and conifer tree killer, P. noxius and P. weirii, with a sensitivity of 1 pg DNA/μl on Nylon membrane, and 100 fg DNA/μl on plastic chip, respectively. Verification and identification of forest Phellinus pathogens infested authentic samples or voucher specimens can be accomplished within 7 hr.

Claims

1. A microchip for identifying Phellinus species, comprising:

a substrate; and

a multiple of DNA probes immobilized on the substrate,

wherein the DNA probes are selected from the group consisting of the sequences of SEQ ID NO;

1, SEQ ID NO;

2, SEQ ID NO;

3, SEQ ID NO;

4, SEQ ID NO;

5, SEQ ID NO;

6, SEQ ID NO;

7, SEQ ID NO;

8, SEQ ID NO;

9, SEQ ID NO;

10, SEQ ID NO;

11, SEQ ID NO;

12, SEQ ID NO;

13, SEQ ID NO;

14, SEQ ID NO;

15, SEQ ID NO;

16 and SEQ ID NO;

17, a complement sequence of any one of SEQ ID NO;

1˜

17, a degeneration sequence of any one of SEQ ID NO;

1˜

17, and the combination thereof.

2. The microchip for identifying Phellinus species as claimed in claim 1, wherein the substrate is a nylon membrane.

3. The microchip for identifying Phellinus species as claimed in claim 1, wherein the substrate is a plastic material.

4. The microchip for identifying Phellinus species as claimed in claim 1 further comprising a position marker.

5. The microchip for identifying Phellinus species as claimed in claim 1, wherein the position marker is oligo-(dT)₁₀ labeled with digoxigenin or biotin at the 5′

end.

6. A method of identifying a Phellinus species, comprising:

(a) extracting DNA from a sample;

(b) amplifying an internal transcribed spacer (ITS) of a 18S-28S rDNA gene of the extracted DNA to obtain an amplification product;

(c) hybridizing the amplification product with the microchip as claimed in claim 1; and

(d) determining whether the amplification product detectably hybridize to the DNA probes of the microchip.

7. The method of identifying a Phellinus species as claimed claim 6, wherein the substrate of the microchip is a nylon membrane.

8. The method of identifying a Phellinus species as claimed in claim 6, wherein the substrate of the microchip is a plastic material.

9. The method of identifying a Phellinus species as claimed in claim 6, wherein the microchip further comprises a position marker.

10. The method of identifying a Phellinus species as claimed in claim 9, wherein the position marker is oligo-(dT)₁₀ labeled with digoxigenin or biotin at the 5′

end. being oligo-(dT)₁₀ labeled with digoxigenin or biotin at the 5′

end.

11. The method of identifying a Phellinus species as claimed in claim 6, wherein the step (b) further comprised a step of performing a electrophoresis to confirm the amplification product.

13. The method of identifying a Phellinus species as claimed in claim 6, wherein seven thymine residues were further added to the 3′

end of the probes of the microchip to elevate the sensitivity of the probes.

14. The method of identifying a Phellinus species as claimed in claim 6, wherein a forward primer having the sequence of SEQ ID NO:

18 and a reverse primer ITS4e having the sequence of SEQ ID NO;

19 are used for amplification in step (b).

15. The method of identifying a Phellinus species as claimed in claim 6, wherein the forward primer and reverse primer used in step (b) are labeled with digoxin (DIG).

16. The method of identifying a Phellinus species as claimed in claim 5, wherein the forward primer and reverse primer used in step (b) are labeled with biotin.

17. A probe composition for identifying Phellinus species, consisting of one or more DNA probes selected from the group consisting of SEQ ID NO:

1, SEQ ID NO;

2, SEQ ID NO;

3, SEQ ID NO;

4, SEQ ID NO;

5, SEQ ID NO;

6, SEQ ID NO;

7, SEQ ID NO;

8, SEQ ID NO;

9, SEQ ID NO;

10, SEQ ID NO;

11, SEQ ID NO;

12, SEQ ID NO;

13, SEQ ID NO;

14, SEQ ID NO;

15, SEQ ID NO;

16 and SEQ ID NO;

17, a complement sequence of any one of SEQ ID NO;

1˜

17, a degeneration sequence of any one of SEQ ID NO;

1˜

17, and the combination thereof.