Method for detecting nucleic acid in sample, method for designing probes, system for designing probes therefor

• US 8,080,372 B2
• Filed: 04/07/2008
• Issued: 12/20/2011
• Est. Priority Date: 04/11/2007
• Status: Expired due to Fees

• Current Assignee: Canon Kabushiki Kaisha (Canon Inc.)
• Original Assignee: Canon Kabushiki Kaisha (Canon Inc.)
• Inventors: Inui, Mimune
• Primary Examiner(s): Whisenant, Ethan C
• Application Number:
  US12/098,994
• Publication Number:
  US 20080254472A1
• Time in Patent Office: 1,352 Days
• Field of Search: 435/6, 536/23.1, 536/24.3
• US Class Current: 435/6
• CPC Class Codes: C12Q 1/689 for bacteria
• CPC Class Codes: G16B 25/00 ICT specially adapted for hybridisation; ICT specially adapted for gene or protein expression
• CPC Class Codes: G16B 25/20 Polymerase chain reaction [PCR]; Primer or probe design; Probe optimisation

Abstract

Accurate detection of a target microorganism from a sample, which may contain a plurality of microorganisms, is intended. This can be realized by eliminating a possibility of cross-hybridization of a microorganism with other probes, which can accelerate the designing speed, detecting microorganisms one-by-one in the designed order, and absorbing all the existing microorganisms.

Claims

1. A method for detecting a nucleic acid in a liquid sample, comprising:

a first capturing step, in which a singularity or a plurality of first probes immobilized on a surface of a carrier and capable of specifically capturing a particular first nucleotide sequence out of a plurality of nucleic acid sequences potentially present in the liquid sample are prepared and the first probes capture the first nucleotide sequence in the liquid sample such that the first nucleotide sequence is practically excluded from the liquid sample;

a separating step, in which the liquid sample is separated from the carrier on which the singularity or plurality of first probes are immobilized;

a second capturing step, in which a singularity or a plurality of second probes immobilized on a surface of a carrier and capable of specifically capturing a particular second nucleotide sequence out of the plurality of nucleic acid sequences excluding the first nucleotide sequence captured by the first probes are prepared and the second probes capture the second nucleotide sequence in the liquid sample having passed through the first capturing step, wherein the liquid sample is not in contact with the carrier on which the singularity or plurality of first probes are immobilized when the second probes capture the second nucleotide sequence; and

detecting the presence or amount of at least one of the first nucleotide sequence captured in the first capturing step and the second nucleotide sequence captured in the second capturing step,

wherein the second probes comprise a probe which can discriminate specifically only when the first nucleotide sequence is practically excluded from the liquid sample.

2. The method according to claim 1, wherein the first capturing step and the second capturing step are carried out in an identical reaction region.

3. The method according to claim 1, wherein the first capturing step is carried out in a first reaction region and the second capturing step is carried out by moving the liquid sample to a second reaction region after the first capturing step.

4. The method according to claim 3, wherein the first reaction region, the second reaction region and a flow channel, through which a fluid can be transported between the reaction regions, are formed on a substrate, on which the transport of the fluid and reactions are carried out.

5. A method according to claim 1, wherein the singularity or plurality of first probes are used in excess of the amount of the first nucleotide sequence in the liquid sample.

6. A method for detecting a nucleic acid in a liquid sample, comprising:

a first capturing step for capturing a first group of target nucleic acids from the liquid sample using a first carrier having a surface on which a plurality of first probes are immobilized,

a separating step, in which the liquid sample is separated from the first carrier;

a second capturing step after the separating step for capturing a second group of target nucleic acids from the liquid sample using a second carrier having a surface on which a plurality of second probes are immobilized,

detecting the presence or amount of at least one of the target nucleic acids captured in the first capturing step and the second capturing step,

wherein the first probes are capable of specifically capturing the first group of target nucleic acids,

wherein the second probes are capable of specifically capturing the second group of target nucleic acids when the first group of target nucleic acids are practically excluded from the liquid sample, and

wherein the second probes comprise a probe which can discriminate specifically only when the first group of target nucleic acids are practically excluded from the liquid sample.

7. The method according to claim 6, further comprising

a third capturing step after the separating step for capturing a third group of target nucleic acids from the liquid sample using a third carrier having a surface on which a plurality of third probes are immobilized,

wherein the third probes are capable of specifically capturing a third group of target nucleic acids when the first group of target nucleic acids and the second group of target nucleic acids are excluded from the liquid sample.

8. A method for detecting a nucleic acid in a liquid sample, comprising:

a first capturing step, in which a singularity or a plurality of first probes immobilized on a surface of a carrier and capable of specifically capturing a particular first nucleotide sequence out of a plurality of nucleic acid sequences potentially present in the liquid sample are prepared and the first probes capture the first nucleotide sequence in the liquid sample such that the first nucleotide sequence is practically excluded from the liquid sample;

a separating step, in which the liquid sample is separated from the carrier on which the singularity or plurality of first probes are immobilized;

a second capturing step, in which a singularity or a plurality of second probes immobilized on a surface of a carrier and capable of specifically capturing a particular second nucleotide sequence out of the plurality of nucleic acid sequences excluding the first nucleotide sequence captured by the first probes are prepared and the second probes capture the second nucleotide sequence in the liquid sample having passed through the first capturing step, wherein the liquid sample is not in contact with the carrier on which the singularity or plurality of first probes are immobilized when the second probes capture the second nucleotide sequence; and

detecting the presence or amount of at least one of the first nucleotide sequence captured in the first capturing step and the second nucleotide sequence captured in the second capturing step,

wherein the second probes are capable of binding to the first and second nucleotide sequences, and the first nucleotide sequence is practically excluded from the liquid sample by the first capturing step so that the second probes bind to the second nucleotide sequence.

9. A method for detecting a nucleic acid in a liquid sample, comprising:

a first capturing step for capturing a first group of target nucleic acids from the liquid sample using a first carrier having a surface on which a plurality of first probes are immobilized,

a separating step, in which the liquid sample is separated from the first carrier;

a second capturing step after the separating step for capturing a second group of target nucleic acids from the liquid sample using a second carrier having a surface on which a plurality of second probes are immobilized,

detecting the presence or amount of at least one of the target nucleic acids captured in the first capturing step and the second capturing step,

wherein the first probes are capable of specifically capturing the first group of target nucleic acids,

wherein the second probes are capable of specifically capturing the second group of target nucleic acids when the first group of target nucleic acids are practically excluded from the liquid sample, and

wherein the second probes are capable of binding to the first and second groups of target nucleic acids, and the first group of target nucleic acids are practically excluded from the liquid sample by the first capturing step so that the second probes bind to the second group of target nucleic acids.