METHOD OF QUANTIFYING HIV-1 RNA-DNA HYDRID AND DIAGNOSIS KIT

• US 20070224610A1
• Filed: 01/16/2007
• Published: 09/27/2007
• Est. Priority Date: 11/27/2000
• Status: Abandoned Application

• Current Assignee: Kabushiki Kaisha Kitazato Supply
• Original Assignee: Kabushiki Kaisha Kitazato Supply
• Inventors: KATO, Shingo
• Application Number:
  US11/623,503
• Publication Number:
  US 20070224610A1
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12Q 1/6851 Quantitative amplification
• CPC Class Codes: C12Q 1/703 Viruses associated with AIDS
• CPC Class Codes: C12Q 2521/301 Endonuclease
• CPC Class Codes: C12Q 2545/107 with a competitive internal standard/control
• CPC Class Codes: C12Q 2549/119 using nested primers

Abstract

A diagnostic kit for evaluating the progress of an HIV-1-related disease and/or the efficacy of an anti-HIV-1 treatment using the quantity of an HIV-1 RNA-DNA hybrid in a sample as an indicator, comprising: at least one primer pair consisting of a downstream primer having a sequence complementary to a portion of the nucleotide sequence of the constituent RNA of the HIV-1 RNA-DNA hybrid and an upstream primer having a sequence complementary to a portion of the nucleotide sequence of the constituent DNA of the HIV-1 RNA-DNA hybrid; and restriction enzyme by which double-stranded DNA containing the same nucleotide sequence as DNA extended by the primer pair can be cleaved at any specific site in the nucleotide sequence.

Claims

1-4. -4. (canceled)

5. A method for evaluating the progress of HIV infection and/or the efficacy of an antiretroviral therapy comprising determining the concentration of an HIV-1 RNA-DNA hybrid in a sample as an indicator.

6. The method of claim 5, comprising:

providing at least one primer pair consisting of a downstream primer having a sequence complementary to a portion of the nucleotide sequence of the constituent RNA of the HIV-1 RNA-DNA hybrid and an upstream primer having a sequence complementary to a portion of the nucleotide sequence of the constituent DNA of the HIV-1 RNA-DNA hybrid,

digesting double-stranded DNA present in the sample with a restriction enzyme by which double-stranded DNA containing the same nucleotide sequence as DNA to be amplified by the primer pair can be cleaved at any specific site in the nucleotide sequence,

extending the DNA with the primer pair using the HIV-1 RNA-DNA hybrid as template to achieve DNA amplification, and

detecting and quantifying the amplified DNA to assess the HIV-1 RNA-DNA hybrid concentration in the sample.

7. The method of claim 6, further comprising adding a known quantity of DNA capable of competing with DNA extended by the primer pair.

8. The method of claim 5, wherein the restriction enzyme is at least one of MseI, Tsp5091 or RsaI.