Method for assaying nucleic acid

• US 6,541,205 B1
• Filed: 05/23/2000
• Issued: 04/01/2003
• Est. Priority Date: 05/24/1999
• Status: Active Grant

• Current Assignee: Tosoh Corporation
• Original Assignee: Tosoh Corporation
• Inventors: Yokoyama, Akihiro, Ishiguro, Takahiko, Saitoh, Juichi
• Primary Examiner(s): Horlick, Kenneth R.
• Assistant Examiner(s): WILDER, CYNTHIA B
• Application Number:
  US09/576,537
• Time in Patent Office: 1,043 Days
• Field of Search: 435/91.2, 435/6, 435/91.1, 435/91.5, 435/91.51, 435/91.21, 536/24.2, 536/24.1, 536/24.3, 536/23.1, 536/24.33
• US Class Current: 435/6.16
• CPC Class Codes: C12Q 1/6851 Quantitative amplification
• CPC Class Codes: C12Q 1/6865 Promoter-based amplification, e.g. nucleic acid sequence amplification [NASBA], self-sustained sequence replication [3SR] or transcription-based amplification system [TAS]
• CPC Class Codes: C12Q 2521/101 DNA polymerase
• CPC Class Codes: C12Q 2521/107 RNA dependent DNA polymerase,(i.e. reverse transcriptase)
• CPC Class Codes: C12Q 2527/113 Time
• CPC Class Codes: C12Q 2531/143 Promoter based amplification, e.g. NASBA, 3SR, TAS
• CPC Class Codes: C12Q 2563/173 staining/intercalating agent, e.g. ethidium bromide

Abstract

A method for assaying a target nucleic acid, comprising: providing an RNA amplification system comprising producing a double-stranded DNA using, as a template, a target RNA containing a specific nucleotide sequence in a sample, said double-stranded DNA having a promoter sequence and being capable of transcribing an RNA comprising the specific nucleotide sequence or a sequence complementary to the specific nucleotide sequence, producing an RNA transcription product comprising the specific nucleotide sequence or a sequence complementary to the specific nucleotide sequence in the presence of an RNA polymerase, and producing the double-stranded DNA using the RNA transcription product as a template, in the presence of a probe labeled with an intercalating fluorochrome having a sequence complementary to the RNA transcription product; measuring the fluorescence intensity in the RNA amplification system with time; calculating a time when the fluorescence intensity satisfies a prescribed criterion based on the measured change in the fluorescence intensity with time; and determining a concentration of the target nucleic acid in the sample based on the calculated time.

Claims

1. A method for determining a concentration of a target RNA in a sample consisting of the steps:

providing an RNA amplification system in which;

(a) a double-stranded DNA is produced using, as a template, a target RNA containing a specific nucleotide sequence in a sample, said double-stranded DNA has a promoter sequence and transcribes an RNA comprising the specific nucleotide sequence or a sequence complementary to the specific nucleotide sequence;

(b) an RNA transcription product comprising the specific nucleotide sequence or a sequence complementary to the specific nucleotide sequence is produced in the presence of an RNA polymerase; and

(c) the double-stranded DNA is produced using the RNA transcription product as a template,

 

wherein said steps (a), (b) and (c) are carried out in the presence of a probe labeled with an intercalating fluorochrome having a sequence complementary to the RNA transcription product,

measuring the fluorescence intensity in the RNA amplification system with time;

calculating a time when the fluorescence intensity satisfies a prescribed criterion based on the measured change in the fluorescence intensity with time; and

determining a concentration of the target RNA in the sample based on the calculated time, wherein all steps of;

production of a double-stranded DNA using a target RNA;

production of an RNA transcription product from the double-stranded DNA;

measurement of the RNA transcription product using a probe; and

production of the above double-stranded using the RNA transcription product;

are carried out continuously.

2. The method according to claim 1, wherein the time which satisfies a prescribed criterion is a time to reach a definite fluorescence intensity.

3. The method according to claim 1, wherein the time which satisfies a prescribed criterion is a time when an increasing rate of the fluorescence intensity per unit of time becomes a maximum.

4. The method according to claim 1, wherein the concentration of the target RNA is determined by comparing the time which satisfies a prescribed criterion with the times in at least two RNA samples containing the specific nucleotide sequence at a different concentration.

5. The method according to claim 2, wherein the concentration of the target RNA is determined by comparing the time which satisfies a prescribed criterion with the times in at least two RNA samples containing the specific nucleotide sequence at a different concentration.

6. The method according to claim 3, wherein the concentration of the target RNA is determined by comparing the time which satisfies a prescribed criterion with the times in at least two RNA samples containing the specific nucleotide sequence at a different concentration.

7. The method according to claim 1, wherein, in the RNA amplification system,

a single-stranded DNA is produced in the presence of an RNA-dependent DNA polymerase using a primer having a sequence. complementary to the specific nucleotide sequence and a primer having a homologous sequence of the specific nucleotide sequence, wherein one of the primers is a promoter primer having a promoter sequence of the RNA polymerase at the 5′ -side, and using the target RNA as the template;

the double-stranded DNA is produced in the presence of a DNA-dependent DNA polymerase using the single-stranded DNA as a template;

the RNA transcription product is produced from the double-stranded DNA in the presence of an RNA polymerase; and

the RNA transcription product is subsequently used as the template for producing the single-stranded DNA in the presence of the RNA-dependent DNA polymerase.

8. The method according to claim 1, wherein the probe labeled with an intercalating fluorochrome is complementary to the RNA transcription product so that the intercalating fluorochrome is intercalated between the produced RNA and the probe.

9. The method according to claim 1, wherein a complex formed by a complementary bond between the probe labeled with an intercalating fluorochrome and the RNA transcription product has a fluorescence characteristic that differs from that of probe molecules that have not formed a complex with the RNA transcription product.

10. The method according to claim 9, wherein the probe labeled with an intercalating fludrochrome is complementary to the RNA transcription product so that the intercalating fluorochrome is intercalated between the produced RNA and the probe.