METHOD FOR DETECTING NUCLEIC ACID IN SAMPLE, METHOD FOR DESIGNING PROBES, SYSTEM FOR DESIGNING PROBES THEREFOR

• US 20080254472A1
• Filed: 04/07/2008
• Published: 10/16/2008
• Est. Priority Date: 04/11/2007
• Status: Active Grant

• Current Assignee: Canon Kabushiki Kaisha (Canon Inc.)
• Original Assignee: Canon Kabushiki Kaisha (Canon Inc.)
• Inventors: Inui, Mimune
• Granted Patent: US 8,080,372 B2
• Time in Patent Office: Days
• 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 sample, comprising:

a first step, in which a singularity or a plurality of first probes capable of specifically capturing a particular nucleotide sequence out of a plurality of nucleic acid sequences potentially present in the sample are prepared and the first probes capture the nucleotide sequence in the sample;

a second step, in which a singularity or a plurality of second probes capable of specifically capturing a particular nucleotide sequence out of the plurality of nucleic acid sequences excluding the nucleic acid captured by the first probes are prepared and the second probes capture the nucleotide sequence in the sample having passed through the first step; and

detecting the presence or amount of at least one of nucleic acid sequences captured in the first step and the second step.

2. The method according to claim 1, wherein the probes are immobilized on a surface of a carrier.

3. The method according to claim 1, wherein the plurality of first probes are supported in a form of a probe carrier having the plurality of probes immobilized on a surface of a carrier.

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

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

6. The method according to claim 5, 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 liquid and reactions are carried out.

7. A probe design method for designing one-by-one a partial sequence specific to a particular nucleotide sequence out of a plurality of target nucleotide sequences, comprising:

selecting a first sequence out of the plurality of target nucleotide sequences;

determining out of partial sequences of the first sequence, a first partial sequence, which is not identical to any part of the partial sequences of the target nucleotide sequences excluding the first sequence, thereby determining the first partial sequence for a first probe;

excluding the first sequence from the target nucleotide sequences and selecting a second sequence out of the target sequences; and

determining out of partial sequences of the second sequence, a second partial sequence, which is not identical to any part of the partial sequences of the target nucleotide sequences excluding the second sequence, thereby determining the second partial sequence for a second probe.

8. The probe design method according to claim 7, further comprising repeating, after determining the second probe, excluding an (n−

1)^th sequence (n=3 to N) from the target nucleotide sequences and selecting an n^th sequence out of the target nucleotide sequences, putting N as a number of the target sequences (N is an integer); and

determining out of partial sequences of the n^th sequence an n^th partial sequence, which is not identical to any part of the partial sequences of the target nucleotide sequences excluding the n^th sequence, and determining the n^th partial sequence for an n^th probe, until n=N.

9. The probe design method according to claim 7, wherein an order of selections of the first sequence to the n^th sequence is determined according to a descending order of a degree of specificity of a sequence.

10. The probe design method according to claim 7, wherein an order of selections of the first sequence to the n^th sequence is determined from a most frequently detected sequence to a least frequently detected sequence.

11. The probe design method according to claim 7, wherein an order of selections of the first sequence to the n^th sequence is determined according to seriousness of microorganisms from which the sequences are originated.

12. A probe design system for detecting microorganisms individually in a sample potentially containing n-kinds of microorganisms (where, n is an integer 2 or higher), wherein the system comprises a detection target microorganism table storing data of nucleotide sequences corresponding to a first to n^th microorganism and a probe table registering determined probes;

and

selecting a first microorganism from the detection target microorganisms read out of the detection target microorganism table;

determining out of partial nucleotide sequences of nucleotide sequences of a gene of the first microorganism, a first nucleotide sequence, which is not identical to any part of partial nucleotide sequences of genes of (n−

1)-kinds of microorganisms excluding the first microorganism, and determines the first nucleotide sequence for a probe for the first microorganism; and

excluding the first microorganism from the n-kinds of detection target microorganisms and selects a microorganism from the (n−

1)-kinds of detection target microorganisms;

is repeated for the first microorganism to an n^th microorganism to design respective probes for respective microorganisms.