METHODS AND MICROARRAYS FOR DETECTING ENTERIC VIRUSES

• US 20090136916A1
• Filed: 08/13/2008
• Published: 05/28/2009
• Est. Priority Date: 08/13/2007
• Status: Abandoned Application

• Current Assignee: Trustees of Tufts College
• Original Assignee: Trustees of Tufts College
• Inventors: Brown, David W., Herrmann, John E., Tzipori, Saul, Gunning, Kerry B.
• Application Number:
  US12/191,132
• Publication Number:
  US 20090136916A1
• Time in Patent Office: Days
• US Class Current: 435/5
• CPC Class Codes: B01J 2219/00387 Applications using probes
• CPC Class Codes: B01J 2219/00529 DNA chips
• CPC Class Codes: B01J 2219/00608 DNA chips
• CPC Class Codes: B01J 2219/00612 the surface being inorganic
• CPC Class Codes: B01J 2219/00637 by coating it with another layer
• CPC Class Codes: B01J 2219/00659 Two-dimensional arrays
• CPC Class Codes: C12Q 1/6881 for tissue or cell typing, e.g. human leukocyte antigen [HLA] probes
• CPC Class Codes: C12Q 1/701 Specific hybridization probes
• CPC Class Codes: C12Q 2531/107 Probe or oligonucleotide ligation

Abstract

The present invention relates to methods, microarrays and kits for detecting one or more human astrovirus serotypes in a sample (e.g., a fecal sample) from an individual. The method includes amplifying nucleic acid molecules of the sample with one or more primers, to thereby obtain an amplified nucleic acid product; contacting the amplified nucleic acid product with one or more serotype specific probes having a nucleic acid sequence that is specific for only one astrovirus serotype in the group of astroviruses being assessed, wherein the nucleic acid sequence includes between about 9 and 25 nucleic acid bases (e.g., SEQ ID NO: 5-24); and detecting the hybridization complex. The presence of hybridization complexes with a serotype specific probe indicates the presence of one or more specific astrovirus serotypes, and the absence of hybridization complexes with a serotype specific probe indicates the absence of the specific astrovirus serotype. Identification of the astrovirus serotypes allows for one to diagnose an individual infected with the serotype. The present invention further includes microarrays having any one of the astrovirus specific probe, or kits having microarrays and reagents for carrying out the assay.

Claims

1. A method of detecting one or more human astrovirus serotypes in a group of astroviruses in a sample from an individual comprising:

(a) amplifying nucleic acid molecules of the sample with one or more primers specific to a conserved region of the astrovirus serotypes being assessed to obtain an amplified nucleic acid product;

(b) contacting the amplified nucleic acid product with one or more serotype specific probes having a nucleic acid sequence specific for a single astrovirus serotype in the group of astroviruses being assessed, the nucleic acid sequence having from about 9 o about 25 nucleic acid bases; and

(c) detecting hybridization between the amplified nucleic acid product the serotype specific probe, the presence of absence of hybridization indicating the presence or absence of one or more specific astrovirus serotypes in the sample.

2. The method of claim 1, wherein the amplification of the nucleic acid molecules is obtained using RT-PCR.

3. The method of claim 1, further including contacting the amplified nucleic acid product with one or more conserved sequence probes having a nucleic acid sequence that is specific for a conserved region shared by all astroviruses in the group of astroviruses being assessed.

4. The method of claim 3, the conserved sequence probes having a nucleic acid sequence of SEQ ID NO:

3 or 4 or a complement thereof, a nucleic acid sequence having from about 40% to about 100% of contiguous nucleotides of SEQ ID NO;

3 or 4 or a complement thereof, or a nucleic acid having from about 9 to about 25 contiguous nucleotides of SEQ ID NO;

3 or 4 or a complement thereof.

5. The method of claim 1, wherein the amplified nucleic acid product of step (a) comprises a detectable label.

6. The method of claim 1, wherein primers of step (a) comprise the nucleic acid sequences of SEQ ID NO:

1 or 2.

7. A method of detecting one or more human astroviruses in a sample from an individual, the method comprising:

(a) amplifying nucleic acid molecules of the sample with one or more primers specific to a conserved region of the astroviruses to produce an amplified nucleic acid product;

(b) contacting the amplified nucleic acid product under suitable hybridization conditions with one or more nucleic acid probe having a nucleic acid sequence of any one of SEQ ID NO;

5-24 or a complement thereof, a nucleic acid sequence having from about 40% to about 100% of contiguous nucleotides of any one of SEQ ID NO;

5-24 or a complement thereof, or a nucleic acid having from about 9 to about 25 contiguous nucleotides of SEQ ID NO;

5-24 or a complement thereof; and

.

(c) detecting the presence or absence of hybridization of the amplified nucleic acid product to the probe, the presence or absence of hybridization being indicative of the presence or absence of one or more serotype specific astroviruses.

8. The method of claim 7, further comprising contacting the amplified nucleic acid product with one or more conserved sequence probes having a nucleic acid sequence conserved sequence probes having a nucleic acid sequence of SEQ ID NO:

3 or 4 or a complement thereof, a nucleic acid sequence having from about 40% to about 100% of contiguous nucleotides of SEQ ID NO;

3 or 4 or a complement thereof, or a nucleic acid having from about 9 to about 25 contiguous nucleotides of SEQ ID NO;

3 or 4 or a complement thereof.

9. The method of claim 7, wherein the primers of step (a) comprise SEQ ID NO:

1 or 2.

10. The method of claim 7, wherein amplification of the nucleic acid molecules is obtained using RT-PCR.

11. The method of claim 7, wherein amplification of the nucleic acid molecules is obtained using asymmetric PCR.

12. A method for identifying an astrovirus serotype in a sample from an individual, the method comprises:

(a) reverse transcribing RNA from the sample to using one or more primers specific to a conserved region of the astrovirus serotypes to obtain DNA;

(b) optionally amplifying the DNA by PCR;

(c) labeling the DNA, prior to and/or during step (a) and/or step(b);

(d) contacting DNA of step (c) under conditions suitable for hybridization with one or more nucleic acid molecules having a nucleic acid sequence of any one of SEQ ID NO;

5-24 or complements thereof, or a nucleic acid sequence having between about 40% and about 100% of any contiguous nucleotides of SEQ ID NO;

5-24 or complements thereof, or a nucleic acid sequence having between about 9 and about 25 contiguous nucleotides of SEQ ID NO;

5-24 or complements thereof; and

(e) detecting the presence or absence of the hybridization, the presence of a complex indicates the presence of the serotype and the absence a complex indicates the absence of the serotype, wherein the serotype is astrovirus 1, astrovirus 2, astrovirus 3, astrovirus 4, astrovirus 5, astrovirus 6, astrovirus 7, or astrovirus 8.

13. A method of detecting one or more human astroviruses in a sample from an individual, the method comprises:

(a) isolating viral nucleic acid molecules from the sample;

(b) contacting one or more primers with the sample, the primers comprising nucleic acid sequence of SEQ ID NO;

1 or 2 and at least one primer comprising a tag, under conditions suitable for amplifying nucleic acid molecules of the sample, to obtain an amplified nucleic acid product having a labeled nucleic acid strand and an unlabeled nucleic acid strand;

(c) digesting the unlabeled nucleic acid strand to thereby obtained an amplified labeled single stranded nucleic acid product;

(d) contacting the amplified nucleic acid product under stringency conditions suitable for hybridization with one or more nucleic acid molecules having a nucleic acid sequence of any one of SEQ ID NO;

5-24 or a complement thereof, or a nucleic acid sequence having between about 40% and about 100% contiguous nucleotides of any one of SEQ ID NO;

5-24, or a complement thereof, or a nucleic acid sequence having between about 9 and about 25 contiguous nucleotides of SEQ ID NO;

5-24 or a complement thereof; and

(e) detecting hybridization of the amplified nucleic acid product to one or more nucleic acid molecules, the presence or absence of hybridization being indicative of the presence or absence of one or more specific astroviruses.

14. A method for diagnosing an individual having a disease or condition associated with an astrovirus, the method comprising:

determining the presence or absence of one or more nucleic acid molecules from a sample from the individual that hybridize to one or more nucleic acid molecules having a nucleic acid sequence of any one of SEQ ID NO;

5-24 or complements thereof, a nucleic acid sequence having between about 40% and about 100% of contiguous nucleotides of any of SEQ ID NO;

5-24 or complements thereof, a nucleic acid sequence having between about 9 and about 25 contiguous nucleotides or complements thereof, the presence or absence of one or more complexes indicates the presence or absence of the disease or condition.

15. A method for monitoring treatment or efficacy of therapy for an individual having a disease or condition associated with an astrovirus, the method comprising:

(a) determining the presence or absence of one or more nucleic acid molecules from at least two samples taken from the individual at different time points that hybridize to one or more nucleic acid molecules having a nucleic acid sequence one or more nucleic acid molecules having a nucleic acid sequence of any one of SEQ ID NO;

5-24 or complements thereof, a nucleic acid sequence having between about 40% and about 100% of contiguous nucleotides of any of SEQ ID NO;

5-24 or complements thereof, a nucleic acid sequence having between about 9 and about 25 contiguous nucleotides or complements thereof,

(b) comparing the hybridization of the nucleic acid molecules in the samples, wherein said comparison indicates the efficacy of therapy.

16. A device for the identification of one or more astrovirus serotypes, the device comprising a support having at least one array comprising a plurality of nucleic acid molecules deposited on the support in spatially distinct domains, the nucleic acid molecules having a nucleic acid sequence of any one of SEQ ID NO:

5-24 or complements thereof, a nucleic acid sequence having between about 40% and about 100% of contiguous nucleotides of any of SEQ ID NO;

5-24 or complements thereof, a nucleic acid sequence having between about 9 and about 25 contiguous nucleotides or complements thereof.

17. The device of claim 16, wherein the support comprises at least one of glass, silica chips, nylon membrane, polymer, plastic, ceramic, metal, and optical fiber.

18. The device of claim 17, wherein the solid support has from about one to about 48 different arrays.

19. The device of claim 18, wherein the device comprises the same array duplicated two or more times.

20. The device of claim 19, wherein more than one nucleic acid molecule is used to identify a serotype.

21. A kit comprising:

(a) the device of claim 20; and

(b) one or more reagents used for carrying out a nucleic acid hybridization assay.

22. The kit of claim 21, wherein the regents include at least one of a compound used to detect hybridization, unlabeled primers specific to a conserved region of human astrovirus serotypes, a labeled primers having a sequence specific to a conserved region of human astrovirus serotypes, washing solutions, hybridization buffers, amplification buffers, or exonuclease reaction buffers.

23. An isolated nucleic acid molecule from one or more astrovirus serotypes having a nucleic acid sequence of any one of SEQ ID NO:

5-24 or complements thereof, a nucleic acid sequence having between about 40% and about 100% of contiguous nucleotides of any of SEQ ID NO;

5-24 or complements thereof, a nucleic acid sequence having between about 9 and about 25 contiguous nucleotides or complements thereof.

24. The isolated nucleic acid molecule of claim 23, wherein the nucleic acid molecule is a DNA or RNA molecule.

25. The isolated acid molecules of claim 23, wherein the molecule is a probe that binds to a nucleic acid from an astrovirus serotype.

26. A method of making a device for the identification of an astrovirus serotype comprising:

depositing on a support an array of spatially arranged domains one or more nucleic acid molecules having a nucleic acid sequence of any one of SEQ ID NO;

5-24 or complements thereof, a nucleic acid sequence having between about 40% and about 100% of contiguous nucleotides of any of SEQ ID NO;

5-24 or complements thereof, a nucleic acid sequence having between about 9 and about 25 contiguous nucleotides or complements thereof.

27. The method of claim 26, wherein the nucleic acid molecules are deposited in a solution having a concentration of between about 1 μ

M and 200 μ

M.

28. The method of claim 26, wherein the support is a glass slide, and wherein from about 1 to about 16 arrays are deposited on the slide.

29. The method of claim 28, wherein the same array is duplicated 2 or more times.

30. The method of claim 26, further comprising synthesizing the nucleic acid molecule.

31. The method of claim 26, wherein the nucleic acid molecules are inserted or integrated within the solid support.