Claims
- 1. An isolated oligonucleotide not more than 60 nucleotides in length comprising:
(a) a nucleotide sequence of at least 10 contiguous nucleotides from a sequence selected from the group consisting of SEQ ID NOS:1, 2 and 3; (b) a nucleotide sequence having 90% sequence identity to a nucleotide sequence of (a); or (c) complements of (a) and (b).
- 2. The oligonucleotide of claim 1, wherein the oligonucleotide is a nucleotide sequence of at least 10 contiguous nucleotides from a sequence selected from the group consisting of SEQ ID NOS: 1, 2 and 3.
- 3. The oligonucleotide of claim 1, wherein the nucleotide sequence comprises SEQ ID NO:1.
- 4. The oligonucleotide of claim 1, wherein the nucleotide sequence comprises SEQ ID NO:2.
- 5. The oligonucleotide of claim 1, wherein the nucleotide sequence comprises SEQ ID NO:3.
- 6. The oligonucleotide of claim 5, further comprising a detectable label at the 5′-end and/or the 3′-end.
- 7. The oligonucleotide of claim 6, wherein the detectable label is a fluorescent label selected from the group consisting of 6-carboxyfluorescein (6-FAM), tetramethyl rhodamine (TAMRA), and 2′, 4′, 5′, 7′,-tetrachloro-4-7-dichlorofluorescein (TET).
- 8. A set of two primers for amplifying Hepatitis A virus nucleic acid, said set of primers consisting of the primers consisting of SEQ ID NO: 1 and SEQ ID NO:2.
- 9. A method of detecting Hepatitis A virus (HAV) in a biological sample, the method comprising:
isolating nucleic acids from a biological sample suspected of containing HAV; amplifying the isolated nucleic acids using at least two primers derived from the 5′ UTR of the HAV genome, wherein each of the primers is from 10 to 60 nucleotides in length and is sufficiently complementary to a portion of the sense and antisense strands, respectively, of the isolated nucleic acid to hybridize therewith; and detecting the presence of the amplified nucleic acids as an indication of the presence or absence of HAV in the sample.
- 10. The method of claim 9, wherein (a) one of the primers comprises a nucleotide sequence of at least 10 contiguous nucleotides from SEQ ID NO: 1 and the other primer comprises a nucleotide sequence of at least 10 contiguous nucleotides from SEQ ID NO:2, or (b) primers having 90% sequence identity to a nucleotide sequence of (a), and
detecting the presence of the amplified nucleic acids as an indication of the presence or absence of HAV in the sample.
- 11. The method of claim 9, wherein the nucleic acids are isolated from the biological sample by a method comprising:
(a) contacting a solid support comprising capture nucleic acids associated therewith with a biological sample under hybridizing conditions wherein target nucleic acid strands hybridize with the capture nucleic acids; and (b) separating the solid support from the sample.
- 12. The method of claim 11, wherein the solid support comprises beads.
- 13. The method of claim 12, wherein the beads are magnetic beads.
- 14. The method of claim 13, wherein the isolating, amplifying and detecting are performed in a single container.
- 15. The method of claim 11, wherein the capture nucleic acids comprise one or more oligonucleotides, wherein each of the oligonucleotides is not more than about 60 nucleotides in length and comprises at least 10 contiguous nucleotides from a sequence selected from the group consisting of SEQ ID NO: 10, SEQ ID NO:11, SEQ ID NO: 12, SEQ ID NO:13 and SEQ ID NO:14.
- 16. The method of claim 15, wherein the capture nucleic acids further comprise a homopolymer chain at either the 3′ or 5′ end of about 15-25 nucleotides in length, wherein the homopolymer chain is selected from the group consisting of polyA, polyT, polyG, polyC, and polyU.
- 17. The method of claim 16, wherein the homopolymer chain is a polyA chain.
- 18. The method of claim 9, wherein amplifying comprises PCR, transcription-mediated amplification (TMA) or TaqMan.
- 19. The method of claim 18, wherein amplifying comprises TMA.
- 20. The method of claim 18, further comprising using a probe oligonucleotide comprising a detectable label for detecting the amplified sequence, wherein the probe oligonucleotide is not more than about 60 nucleotides in length and comprises at least 10 contiguous nucleotides comprising SEQ ID NO: 3.
- 21. The method of claim 20, wherein the probe comprises detectable labels at the 5′-end and at the 3′-end.
- 22. The method of claim 21, wherein the detectable label is a fluorescent label selected from the group consisting of 6-carboxyfluorescein (6-FAM), tetramethyl rhodamine (TAMRA), and ′, 4′, 5′, 7′,-tetrachloro-4-7-dichlorofluorescein (TET).
- 23. A method for detecting Hepatitis A virus (HAV) infection in a biological sample, the method comprising:
(a) contacting a solid support with capture nucleic acids comprising one or more oligonucleotides, wherein the one or more oligonucleotides comprises a sequence selected from the group consisting of SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13 and SEQ ID NO:14, under conditions wherein the capture nucleic acids become associated with the solid support, (b) contacting the solid support of (a) with the biological sample under hybridizing conditions wherein target nucleic acid strands from HAV when present hybridize with the capture nucleic acids; and (c) separating the solid support of (b) from the sample; (d) amplifying the nucleic acids using a sense primer comprising SEQ ID NO: 1 and an antisense primer comprising SEQ ID NO:2, wherein the sense and antisense primers are sufficiently complementary to a portion of the sense and antisense strands, respectively, of the isolated nucleic acid to hybridize therewith; and (e) detecting the presence of the amplified nucleic acids as an indication of the presence or absence of HAV in the sample.
- 24. The method of claim 23, wherein the solid support comprises beads.
- 25. The method of claim 24, wherein the beads are magnetic beads.
- 26. The method of claim 25, wherein the isolating, amplifying and detecting are performed in a single container.
- 27. A kit for detecting Hepatitis A virus (HAV) infection in a biological sample, the kit comprising:
capture nucleic acids comprising one or more oligonucleotides, wherein each of the oligonucleotides is not more than about 60 nucleotides in length and comprises a nucleotide sequence of at least 10 contiguous nucleotides of a sequence selected from the group consisting of SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13 and SEQ ID NO:14; at least two primers wherein (a) each of the primers is not more than about 60 nucleotides in length and one primer comprises a nucleotide sequence of at least 10 contiguous nucleotides from SEQ ID NO: 1 and the other primer comprises a nucleotide sequence of at least 10 contiguous nucleotides from SEQ ID NO:2; and written instructions for identifying HAV infection.
- 28. The kit of claim 27, further comprising a polymerase and buffers.
- 29. The kit of claim 27, further comprising a probe oligonucleotide of not more than about 60 nucleotides in length and at least 10 contiguous nucleotides comprising SEQ ID NO:3.
- 30. The kit of claim 29, wherein the probe further comprises detectable labels at the 5′-end and at the 3′-end.
- 31. The kit of claim 30, wherein the detectable label is a fluorescent label selected from the group consisting of 6-carboxyfluorescein (6-FAM), tetramethyl rhodamine (TAMRA), and 2′, 4′, 5′, 7′,-tetrachloro-4-7-dichlorofluorescein (TET).
CROSS-REFERENCE TO RELATED APPLICATION
[0001] This application is related to provisional application No. 60/388,544 filed Jun. 12, 2002, from which priority is claimed under 35 USC §119(e)(1) and which application is incorporated herein by reference in its entirety.
Provisional Applications (1)
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Number |
Date |
Country |
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60388544 |
Jun 2002 |
US |