Apparatus and methods for detecting a microbe in a sample

US 20050202414A1
Filed: 11/15/2002
Published: 09/15/2005
Est. Priority Date: 11/15/2001
Status: Abandoned Application

First Claim

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1. Apparatus for detecting the presence of a microbe in a sample, comprising:

a substrate having a plurality of microbe identification sites, each microbe identification site having a unique address indicative of the position of that microbe identification site on the substrate; and

groups of nucleic acid probes disposed at the microbe identification sites, each group of nucleic acid probes being complementary to a target nucleic acid so as to provide a detectable signal at one or more microbe identification sites.

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Abstract

Apparatus for detecting one or more microbes in a sample includes a substrate having a plurality of microbe identification sites with nucleic acid probes disposed thereon, each nucleic acid probe having a nucleotide sequences that is complementary to nucleotide sequences of nucleic acids of one or more microbes. Nucleotide sequences for nucleic acid probes and the primers used to generate the probes are disclosed. Methods of detecting a microbe in a sample using nucleic acid probes are also disclosed.

Citations

46 Claims

1. Apparatus for detecting the presence of a microbe in a sample, comprising:
- a substrate having a plurality of microbe identification sites, each microbe identification site having a unique address indicative of the position of that microbe identification site on the substrate; and
  
  groups of nucleic acid probes disposed at the microbe identification sites, each group of nucleic acid probes being complementary to a target nucleic acid so as to provide a detectable signal at one or more microbe identification sites.
- View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31)
- - 2. The apparatus of claim 1, wherein the microbe identification sites comprise pathogen identification sites.
  - 3. The apparatus of claim 1, wherein the microbe identification sites comprise viral identification sites.
  - 4. The apparatus of claim 1, wherein the microbe identification sites comprise bacterial identification sites.
  - 5. The apparatus of claim 1, wherein the microbe identification sites comprise pathogenic and non-pathogenic identification sites.
  - 6. The apparatus of claim 1, wherein the microbe identification sites comprise cellular and acellular pathogen identification sites.
  - 7. The apparatus of claim 1, wherein the nucleic acid probes comprise nucleotide sequences that are complementary to genetic sequences of viruses or viroids.
  - 8. The apparatus of claim 1, wherein the nucleic acid probes comprise nucleotide sequences that are complementary to genetic sequences of respiratory viruses.
  - 9. The apparatus of claim 1, wherein the nucleic acid probes comprise nucleotide sequences that are complementary to a nucleotide sequence of a pathogen selected from the group consisting of:
    - adenoviruses, influenza A virus, influenza B virus, influenza C virus, parainfluenza 1, parainfluenza 2, parainfluenza 3, parainfluenza 4, mumps virus, respiratory syncytial virus, enterovirus, rhinovirus, rubella virus, coronavirus, chlamidia pneumonia, and mycoplasma pneumonia.
  - 10. The apparatus of claim 1, wherein the nucleic acid probes are complementary to a sequence of the target nucleic acid that has at least 80% homology among different types of microbes from a microbe family.
  - 11. The apparatus of claim 10, wherein the nucleic acid probes are complementary to a sequence of the target nucleic acid that has at least 90% homology among different types of microbes from a microbe family.
  - 12. The apparatus of claim 10, wherein the nucleic acid probes are complementary to a sequence of the target nucleic acid that has at least 98% homology among different types of microbes from a microbe family.
  - 13. The apparatus of claim 1, wherein the nucleic acid probes comprise between 65 nucleotides and 80 nucleotides.
  - 14. The apparatus of claim 13, wherein the nucleic acid probes comprise at least 70 nucleotides.
  - 15. The apparatus of claim 13, wherein the nucleic acid probes comprise between 70 nucleotides and 75 nucleotides.
  - 16. The apparatus of claim 1, wherein the nucleic acid probes comprise a nucleotide sequence 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, SEQ ID NO;
      
      17, SEQ ID NO;
      
      18, SEQ ID NO;
      
      19, SEQ ID NO;
      
      20, SEQ ID NO;
      
      21, SEQ ID NO;
      
      22, SEQ ID NO;
      
      23, SEQ ID NO;
      
      24, SEQ ID NO;
      
      25, SEQ ID NO;
      
      26, SEQ ID NO;
      
      27, SEQ ID NO;
      
      28, SEQ ID NO;
      
      29, SEQ ID NO;
      
      30, SEQ ID NO;
      
      31, SEQ ID NO;
      
      32, SEQ ID NO;
      
      33, SEQ ID NO;
      
      34, SEQ ID NO;
      
      35, SEQ ID NO;
      
      36, SEQ ID NO;
      
      37, SEQ ID NO;
      
      38, SEQ ID NO;
      
      39, SEQ ID NO;
      
      40, SEQ ID NO;
      
      41, SEQ ID NO;
      
      42, SEQ ID NO;
      
      43, SEQ ID NO;
      
      44, SEQ ID NO;
      
      45, SEQ ID NO;
      
      46, SEQ ID NO;
      
      47, SEQ ID NO;
      
      48, SEQ ID NO;
      
      49, SEQ ID NO;
      
      50, SEQ ID NO;
      
      51, SEQ ID NO;
      
      52, SEQ ID NO;
      
      53, SEQ ID NO;
      
      54, SEQ ID NO;
      
      55, SEQ ID NO;
      
      56, SEQ ID NO;
      
      57, SEQ ID NO;
      
      58, SEQ ID NO;
      
      59, SEQ ID NO;
      
      60, SEQ ID NO;
      
      61, SEQ ID NO;
      
      62, SEQ ID NO;
      
      63, SEQ ID NO;
      
      64, SEQ ID NO;
      
      65, SEQ ID NO;
      
      66, SEQ ID NO;
      
      67, SEQ ID NO;
      
      68, SEQ ID NO;
      
      69, SEQ ID NO;
      
      70, SEQ ID NO;
      
      71, SEQ ID NO;
      
      72, SEQ ID NO;
      
      73, SEQ ID NO;
      
      74, SEQ ID NO;
      
      75, SEQ ID NO;
      
      76, SEQ ID NO;
      
      77, SEQ ID NO;
      
      78, SEQ ID NO;
      
      79, SEQ ID NO;
      
      80, SEQ ID NO;
      
      81, SEQ ID NO;
      
      82, SEQ ID NO;
      
      83, SEQ ID NO;
      
      84, SEQ ID NO;
      
      85, SEQ ID NO;
      
      86, SEQ ID NO;
      
      87, SEQ ID NO;
      
      88, SEQ ID NO;
      
      89, SEQ ID NO;
      
      90, and SEQ ID NO;
      
      91.
  - 17. The apparatus of claim 1, wherein a group of nucleic acid probes comprises a plurality of nucleic acids, each nucleic acid of a group having an identical nucleotide sequence.
  - 18. The apparatus of claim 1, wherein the nucleic acid probes are printed on the substrate.
  - 19. The apparatus of claim 1, wherein the substrate comprises a glass slide.
  - 20. The apparatus of claim 1, wherein the substrate comprises a polylysine-coated glass slide.
  - 21. The apparatus of claim 1, wherein the substrate comprises at least one pathogen detection region, and the microbe identification sites are located in the at least one pathogen detection region.
  - 22. The apparatus of claim 1, wherein the target nucleic acid comprises a plurality of nucleic acids amplified by a polymerase chain reaction.
  - 23. The apparatus of claim 1, wherein the target nucleic acid comprises a label attached thereto.
  - 24. The apparatus of claim 23, wherein the label comprises a fluorescent label.
  - 25. The apparatus of claim 23, wherein the label comprises Cy3.
  - 26. A kit comprising the apparatus of claim 1, and further comprising a plurality of nucleic acid primers structured to hybridize to different regions of a target nucleic acid of a microbe to form a nucleic acid comprising about 70 to about 75 bases after a polymerase chain reaction.
  - 27. The kit of claim 26, comprising at least one pair of nucleic acid primers structured to hybridize to a target nucleic acid of a single microbe.
  - 28. The kit of claim 26, comprising at least two pairs of nucleic acid primers structured to hybridize to a target nucleic acid of a microbe.
  - 29. The kit of claim 26, wherein at least one of the nucleic acid primers comprise a nucleotide sequence selected from a group consisting of:
    - SEQ ID NO;
      
      92, SEQ ID NO;
      
      93, SEQ ID NO;
      
      94, SEQ ID NO;
      
      95, SEQ ID NO;
      
      96, SEQ ID NO;
      
      97, SEQ ID NO;
      
      98, SEQ ID NO;
      
      99, SEQ ID NO;
      
      100, SEQ ID NO;
      
      101, SEQ ID NO;
      
      102, SEQ ID NO;
      
      103, SEQ ID NO;
      
      104, SEQ ID NO;
      
      105, SEQ ID NO;
      
      106, SEQ ID NO;
      
      107, SEQ ID NO;
      
      108, SEQ ID NO;
      
      109, SEQ ID NO;
      
      110, SEQ ID NO;
      
      111,SEQ ID NO;
      
      112, and SEQ ID NO;
      
      113.
  - 30. The kit of claim 26, further comprising a scanner positioned to receive signals from the apparatus and to scan the microbe detection region for a detectable signal.
  - 31. The kit of claim 26, further comprising an analyzer in communication with the scanner to receive data from the scanner.

32. An apparatus for detecting the presence of a pathogen in a sample, comprising a nucleic acid probe disposed on a substrate, and that hybridizes to a target nucleic acid of a pathogen, the nucleic acid probe comprising a nucleotide sequence selected from a 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, SEQ ID NO;
  
  17, SEQ ID NO;
  
  18, SEQ ID NO;
  
  19, SEQ ID NO;
  
  20, SEQ ID NO;
  
  21, SEQ ID NO;
  
  22, SEQ ID NO;
  
  23, SEQ ID NO;
  
  24, SEQ ID NO;
  
  25, SEQ ID NO;
  
  26, SEQ ID NO;
  
  27, SEQ ID NO;
  
  28, SEQ ID NO;
  
  29, SEQ ID NO;
  
  30, SEQ ID NO;
  
  31, SEQ ID NO;
  
  32, SEQ ID NO;
  
  33, SEQ ID NO;
  
  34, SEQ ID NO;
  
  35, SEQ ID NO;
  
  36, SEQ ID NO;
  
  37, SEQ ID NO;
  
  38, SEQ ID NO;
  
  39, SEQ ID NO;
  
  40, SEQ ID NO;
  
  41, SEQ ID NO;
  
  42, SEQ ID NO;
  
  43, SEQ ID NO;
  
  44, SEQ ID NO;
  
  45, SEQ ID NO;
  
  46, SEQ ID NO;
  
  47, SEQ ID NO;
  
  48, SEQ ID NO;
  
  49, SEQ ID NO;
  
  50, SEQ ID NO;
  
  51, SEQ ID NO;
  
  52, SEQ ID NO;
  
  53, SEQ ID NO;
  
  54, SEQ ID NO;
  
  55, SEQ ID NO;
  
  56, SEQ ID NO;
  
  57, SEQ ID NO;
  
  58, SEQ ID NO;
  
  59, SEQ ID NO;
  
  60, SEQ ID NO;
  
  61, SEQ ID NO;
  
  62, SEQ ID NO;
  
  63, SEQ ID NO;
  
  64, SEQ ID NO;
  
  65, SEQ ID NO;
  
  66, SEQ ID NO;
  
  67, SEQ ID NO;
  
  68, SEQ ID NO;
  
  69, SEQ ID NO;
  
  70, SEQ ID NO;
  
  71, SEQ ID NO;
  
  72, SEQ ID NO;
  
  73, SEQ ID NO;
  
  74, SEQ ID NO;
  
  75, SEQ ID NO;
  
  76, SEQ ID NO;
  
  77, SEQ ID NO;
  
  78, SEQ ID NO;
  
  79, SEQ ID NO;
  
  80, SEQ ID NO;
  
  81, SEQ ID NO;
  
  82, SEQ ID NO;
  
  83, SEQ ID NO;
  
  84, SEQ ID NO;
  
  85, SEQ ID NO;
  
  86, SEQ ID NO;
  
  87, SEQ ID NO;
  
  88, SEQ ID NO;
  
  89, SEQ ID NO;
  
  90, and SEQ ID NO;
  
  91.
- View Dependent Claims (33)
- - 33. The apparatus of claim 32, comprising a plurality of nucleic acid probes arranged in groups on the substrate, each group of nucleic acid probes comprising a nucleotide sequence 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, SEQ ID NO;
      
      17, SEQ ID NO;
      
      18, SEQ ID NO;
      
      19, SEQ ID NO;
      
      20, SEQ ID NO;
      
      21, SEQ ID NO;
      
      22, SEQ ID NO;
      
      23, SEQ ID NO;
      
      24, SEQ ID NO;
      
      25, SEQ ID NO;
      
      26, SEQ ID NO;
      
      27, SEQ ID NO;
      
      28, SEQ ID NO;
      
      29, SEQ ID NO;
      
      30, SEQ ID NO;
      
      31, SEQ ID NO;
      
      32, SEQ ID NO;
      
      33, SEQ ID NO;
      
      34, SEQ ID NO;
      
      35, SEQ ID NO;
      
      36, SEQ ID NO;
      
      37, SEQ ID NO;
      
      38, SEQ ID NO;
      
      39, SEQ ID NO;
      
      40, SEQ ID NO;
      
      41, SEQ ID NO;
      
      42, SEQ ID NO;
      
      43, SEQ ID NO;
      
      44, SEQ ID NO;
      
      45, SEQ ID NO;
      
      46, SEQ ID NO;
      
      47, SEQ ID NO;
      
      48, SEQ ID NO;
      
      49, SEQ ID NO;
      
      50, SEQ ID NO;
      
      51, SEQ ID NO;
      
      52, SEQ ID NO;
      
      53, SEQ ID NO;
      
      54, SEQ ID NO;
      
      55, SEQ ID NO;
      
      56, SEQ ID NO;
      
      57, SEQ ID NO;
      
      58, SEQ ID NO;
      
      59, SEQ ID NO;
      
      60, SEQ ID NO;
      
      61, SEQ ID NO;
      
      62, SEQ ID NO;
      
      63, SEQ ID NO;
      
      64, SEQ ID NO;
      
      65, SEQ ID NO;
      
      66, SEQ ID NO;
      
      67, SEQ ID NO;
      
      68, SEQ ID NO;
      
      69, SEQ ID NO;
      
      70, SEQ ID NO;
      
      71, SEQ ID NO;
      
      72, SEQ ID NO;
      
      73, SEQ ID NO;
      
      74, SEQ ID NO;
      
      75, SEQ ID NO;
      
      76, SEQ ID NO;
      
      77, SEQ ID NO;
      
      78, SEQ ID NO;
      
      79, SEQ ID NO;
      
      80, SEQ ID NO;
      
      81, SEQ ID NO;
      
      82, SEQ ID NO;
      
      83, SEQ ID NO;
      
      84, SEQ ID NO;
      
      85, SEQ ID NO;
      
      86, SEQ ID NO;
      
      87, SEQ ID NO;
      
      88, SEQ ID NO;
      
      89, SEQ ID NO;
      
      90, and SEQ ID NO;
      
      91.

34. A method for detecting the presence of a microbe in a sample, comprising:
- a) identifying a target nucleic acid of a microbe in a sample;
  
  b) labeling the target nucleic acid;
  
  c) providing a substrate that has different groups of nucleic acid probes at different locations on the substrate; and
  
  d) exposing the labeled target nucleic acid to the substrate such that the labeled target nucleic acid will hybridize to nucleic acid probes that have a nucleotide sequence complementary to the nucleotide sequence of the labeled target nucleic acid.
- View Dependent Claims (35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46)
- - 35. The method of claim 34, wherein step (a) comprises identifying a target nucleic acid of a microbe suspected of being present in a biological sample.
  - 36. The method of claim 35, wherein the biological sample comprises a biological fluid.
  - 37. The method of claim 36, wherein the biological fluid is selected from a group consisting of blood, serum, mucus, urine, sputum, saliva, cerebral spinal fluid, and perspiration.
  - 38. The method of claim 34, further comprising a step of amplifying the target nucleic acid using at least one pair of nucleic acid primers and a polymerase chain reaction.
  - 39. The method of claim 34, further comprising a step of amplifying the target nucleic acid using at least two pairs of nucleic acid primers and a polymerase chain reaction.
  - 40. The method of claim 34, further comprising a step of amplifying the target nucleic acid and labeling the target nucleic acid during the amplification step.
  - 41. The method of claim 34, further comprising a step of detecting the label at specific locations on the substrate where the labeled nucleic acids hybridized to the nucleic acid probes.
  - 42. The method of claim 34, further comprising a step of detecting a fluorescent signal at specific locations on the substrate where the labeled nucleic acids hybridized to the nucleic acid probes.
  - 43. The method of claim 34, wherein step (a) comprises identifying a target nucleic acid of a plurality of microbes suspected of being present in a sample.
  - 44. The method of claim 34, wherein step (a) comprises identifying a target nucleic acid of a virus or viral particle.
  - 45. The method of claim 34, wherein step (a) comprises identifying a target nucleic acid of a pathogenic or non-pathogenic bacteria.
  - 46. The method of claim 34, wherein step (a) comprises identifying a target nucleic acid of a cellular or acellular microbe.

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Current Assignee
University of Minnesota
Original Assignee
Regents of the University of California (University of California)
Inventors
Jia, Xi Yu, Berger, Martina M., Tilles, Jeremiah G.

Application Number

US10/295,787
Publication Number

US 20050202414A1
Time in Patent Office

Days
Field of Search
US Class Current

435/5
CPC Class Codes

C12Q 1/689 for bacteria

C12Q 2600/16 Primer sets for multiplex a...

Apparatus and methods for detecting a microbe in a sample

First Claim

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Abstract

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46 Claims

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Apparatus and methods for detecting a microbe in a sample

First Claim

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Abstract

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