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- Nachgewiesen in: USPTO Patent Grants
- Sprachen: English
- Patent Number: 6,140,046
- Publication Date: October 31, 2000
- Appl. No: 08/904,290
- Application Filed: July 31, 1997
- Assignees: University of Florida (Gainesville, FL)
- Claim: What is claimed is
- Claim: 1. A probe for detecting a strain of Citrus Tristeza Virus, the probe comprising an isolated nucleic acid molecule that hybridizes at 42 degrees Celsius in the presence of 6.times.SSC and 1% SDS to a complement of an oligonucleotide 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, and SEQ ID NO:9, wherein the nucleic acid molecule is 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, and SEQ ID NO:9.
- Claim: 2. The probe of claim 1 further comprising a detectable label.
- Claim: 3. The probe of claim 2 wherein the detectable label is biotin.
- Claim: 4. A probe for detecting a strain of Citrus Tristeza Virus, the probe comprising an isolated nucleic acid molecule that hybridizes at 42 degrees Celsius in the presence of 6.times.SSC and 1% SDS to an oligonucleotide 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, and SEQ ID NO:9, wherein the nucleic acid molecule is selected from the group consisting of the complements 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, and SEQ ID NO:9.
- Claim: 5. The probe of claim 4 further comprising a detectable label.
- Claim: 6. The probe of claim 5 wherein the detectable label is biotin.
- Claim: 7. A method for differentiating among strains of Citrus Tristeza Virus comprising the steps of
- Claim: providing a test sample comprising at least one strain of Citrus Tristeza Virus to be identified;
- Claim: providing at least a first oligonucleotide probe and a second oligonucleotide probe, said first oligonucleotide probe hybridizing at 42 degrees Celsius in the presence of 6.times.SSC and 1% SDS to a nucleic acid molecule selected from the group consisting of SEQ ID NOS 1-8 and the complements of SEQ ID NOS 1-8, and said second oligonucleotide probe hybridizing at 42 degrees Celsius in the presence of 6.times.SSC and 1% SDS to a nucleic acid molecule selected from the group consisting of SEQ ID NOS 1-8 and the complements of SEQ ID NOS 1-8 but not to a complement of the nucleotide sequence of said first oligonucleotide probe; and wherein said first oligonucleotide probe and said second oligonucleotide probes comprise nucleic acid molecules selected from the group consisting of SEQ ID NOS 1-8 and the complements of SEQ ID NOS 1-8
- Claim: contacting said first and said second oligonucleotide probes to the test sample; and
- Claim: analyzing binding of said first and said second oligonucleotide probes to the test sample.
- Claim: 8. The method of claim 7 further comprising the step of
- Claim: isolating nucleic acid from the test sample.
- Claim: 9. The method of claim 8 further comprising the step of: amplifying the nucleic acid isolated from the test sample using polymerase chain reaction.
- Claim: 10. The method of claim 9 wherein the amplifying step comprises the step of adding a first oligonucleotide primer and a second oligonucleotide primer to the nucleic acid isolated from the test sample, the first and second oligonucleotide primers together capable of selectively mediating amplification of a polynucleotide derived from a Citrus Tristeza Virus to which the oligonucleotide probe can hybridize.
- Claim: 11. The method of claim 10 wherein the first oligonucleotide primer consists essentially of SEQ ID NO:10, and the second oligonucleotide primer consists essentially of SEQ ID NO:11.
- Claim: 12. The method of claim 7, wherein said first oligonucleotide probe and said second oligonucleotide probe comprise nucleic acid molecules 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, and SEQ ID NO:8.
- Claim: 13. A kit for identifying a strain of Citrus Tristeza Virus, the kit comprising
- Claim: a first oligonucleotide primer and a second oligonucleotide primer, the first and second oligonucleotide primers together capable of selectively mediating amplification of a polynucleotide derived from Citrus Tristeza Virus; and
- Claim: at least a first oligonucleotide probe and a second oligonucleotide probe, said first oligonucleotide probe hybridizing at 42 degrees Celsius in the presence of 6.times.SSC and 1% SDS to a nucleic acid molecule selected from the group consisting of SEQ ID NOS 1-8 and the complements of SEQ ID NOS 1-8, and said second oligonucleotide probe hybridizing at 42 degrees Celsius in the presence of 6.times.SSC and 1% SDS to a nucleic acid molecule selected from the group consisting of SEQ ID NOS 1-8 and the complements of SEQ ID NOS 1-8 but not to a complement of the nucleotide sequence of said first oligonucleotide probe; and wherein said first oligonucleotide probe and said second oligonucleotide probes comprise nucleic acid molecules selected from the group consisting of SEQ ID NOS 1-8 and the complements of SEQ ID NOS 1-8.
- Claim: 14. The kit of claim 13 further comprising a control oligonucleotide probe wherein the control oligonucleotide probe is shared among substantially all strains of Citrus Tristeza Virus and is set forth in SEQ ID NO 9.
- Claim: 15. An isolated nucleic acid molecule that hybridizes at 42 degrees Celsius in the presence of 6.times.SSC and 1% SDS to an oligonucleotide selected from the group consisting of: SEQ ID NOS 1-9 and the complements of SEQ ID NOS 1-9, wherein the isolated nucleic acid molecule is selected from the group consisting of: SEQ ID NOS 1-9 and the complements of SEQ ID NOS 1-9.
- Claim: 16. The isolated nucleic acid molecule of claim 15, wherein the nucleic acid molecule is selected from the group consisting of: SEQ ID NOS:1-9.
- Claim: 17. The isolated nucleic acid molecule of claim 16, wherein the nucleic acid molecule is SEQ ID NO:1.
- Claim: 18. The isolated nucleic acid molecule of claim 16, wherein the nucleic acid molecule is SEQ ID NO:2.
- Claim: 19. The isolated nucleic acid molecule of claim 16, wherein the nucleic acid molecule is SEQ ID NO:3.
- Claim: 20. The isolated nucleic acid molecule of claim 16, wherein the nucleic acid molecule is SEQ ID NO:4.
- Claim: 21. The isolated nucleic acid molecule of claim 16, wherein the nucleic acid molecule is SEQ ID NO:5.
- Claim: 22. The isolated nucleic acid molecule of claim 16, wherein the nucleic acid molecule is SEQ ID NO:6.
- Claim: 23. The isolated nucleic acid molecule of claim 16, wherein the nucleic acid molecule is SEQ ID NO:7.
- Claim: 24. The isolated nucleic acid molecule of claim 16, wherein the nucleic acid molecule is SEQ ID NO:8.
- Claim: 25. The isolated nucleic acid molecule of claim 16, wherein the nucleic acid molecule is SEQ ID NO:8.
- Claim: 26. The isolated nucleic acid molecule of claim 16, wherein the nucleic acid molecule is SEQ ID NO:9.
- Current U.S. Class: 435/6; 435/911; 536/234
- Current International Class: C12Q 168; C12P 1934; C07H 2104
- Patent References Cited: 5104789 April 1992 Permar et al. ; 5593836 January 1997 Niemiec et al.
- Other References: Chassin et al., accession No. L34839, 1996. ; Pappu et al., accession No. L12175, 1993. ; Cepeda-Nieto et al., acession No. U32116, 1995. ; Gyapay et al., accession No. Z23729, 1996. ; Karasev et al. Virology, vol. 208, pp. 511-520, 1995. ; Mawassi et al. Virus Genes, vol. 7, pp. 265-275, 1993. ; Pappu et al. PNAS vol. 90, pp. 3641-3644, 1993. ; Pappu, H.R., et al., "Molecular characterization of a structural epitope that is largely conserved among severe isolates of a plant virus," Proc. Natl. Acad. Sci. USA, 90:3641-3644 (Apr. 1993). ; Pappu, H.R., et al., "Mutagenic Analysis and Localization of a Highly Conserved Epitope Near the Amino Terminal End of the Citrus Tristeza Closterovirus Capsid Protein," Phytopathology, 85(10):1311-1315 (1995). ; Permar, T.A., et al., "A Monoclonal Antibody That Discriminates Strains of Citrus Tristeza Cirus," Phytopathology, 80(9):224-228 (1990).
- Primary Examiner: Jones, W. Gary
- Assistant Examiner: Souaya, Jehanne
- Attorney, Agent or Firm: Quarles & Brady LLP
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