US 2024/0240266 A1Published application

Isothermal gene amplification method, gene detection method, virus detection method, and kit used therefor

AssigneeBioSeeds Corporation

InventorManish BIYANI

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Abstract

An isothermal gene amplification method that allows an RNA virus to be detected easily and inexpensively in a short time is presented. The isothermal gene amplification method involves a target sequence in an RNA genome, and includes: (A) producing an amplification reaction template; (B) DNA amplifying for increasing the amplification reaction template; and (C) RNA amplifying.

Claims15

Dependent claims

An isothermal gene amplification method of a target sequence in an RNA genome, comprising:

(A) producing an amplification reaction template, comprising:
(A1) hybridizing, a first primer having a promoter sequence of an RNA polymerase on a 5′ side of the first primer and a complementary sequence that is complementary to a 5′-side sequence of the target sequence in the RNA genome on a 3′ side of the first primer, to the 3′-side sequence of the target sequence in the RNA genome, and extending a 3′ end of the first primer by reverse transcription activity of a reverse transcriptase so as to produce a double-stranded complex of the RNA genome and an extended DNA strand;
(A2) degrading the RNA genome of the double-stranded complex formed in (A1) by RNA degrading activity of the reverse transcriptase so as to produce a single-stranded DNA having a first primer sequence;
and
(A3) hybridizing a second primer having the same sequence as a 3′-side sequence of the target sequence to a sequence complementary to the target sequence of the single-stranded DNA produced in (A2), and extending a 3′ end of the second primer by DNA synthetic activity of the reverse transcriptase so as to produce a single-stranded DNA having a second primer sequence, thereby producing a double-stranded DNA as the amplification reaction template;
(B) DNA amplifying for increasing the amplification reaction template, comprising:
(B1) hybridizing the second primer to a 3′ side of the single-stranded DNA comprising the first primer and hybridizing the first primer to a 3′ side of the single-stranded DNA having the second primer sequence in the double-stranded DNA as the amplification reaction template produced in (A3) by a recombinase in a presence of a single-stranded DNA-binding protein;
and
(B2) extending the 3′ end of the first primer and the 3′ end of the second primer by strand displacement DNA synthetic activity of a strand displacement DNA polymerase in a presence of the single-stranded DNA-binding protein so as to produce a double-stranded DNA that is the same as the amplification reaction template produced in (A3);
and
(C) RNA amplifying, comprising:
(C1) synthesizing a single-stranded RNA by an RNA synthetic activity of an RNA polymerase with a sequence that is downstream on a 3′-side relative to the promoter sequence of an RNA polymerase of the double-stranded DNA as the amplification reaction template produced in (A3) being used as a template sequence;
(C2) hybridizing the second primer to a 5′ side of the single-stranded RNA synthesized in (C1), and extending the 3′ end of the second primer by the reverse transcription activity of the reverse transcriptase so as to produce a double-stranded complex of the single-stranded RNA and an extended DNA;
(C3) degrading the single-stranded RNA of the double-stranded complex produced in (C2) by the RNA degrading activity of the reverse transcriptase so as to produce a single-stranded DNA;
and
(C4) hybridizing the first primer to a 3′ side of the single-stranded DNA produced in (C3), extending the 3′ end of the first primer by the DNA synthetic activity of the reverse transcriptase, and extending a 3′ end of the single-stranded DNA produced in (C3) with the promoter sequence of the RNA polymerase of the first primer being used as a template so as to produce a double-stranded DNA that is the same as the amplification reaction template produced in (A3).

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Dependencies are inferred from the claim text.

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Abstract

An isothermal gene amplification method of a target sequence in an RNA genome, comprising:

(A) producing an amplification reaction template, comprising:
(A1) hybridizing, a first primer having a promoter sequence of an RNA polymerase on a 5′ side of the first primer and a complementary sequence that is complementary to a 5′-side sequence of the target sequence in the RNA genome on a 3′ side of the first primer, to the 3′-side sequence of the target sequence in the RNA genome, and extending a 3′ end of the first primer by reverse transcription activity of a reverse transcriptase so as to produce a double-stranded complex of the RNA genome and an extended DNA strand;
(A2) degrading the RNA genome of the double-stranded complex formed in (A1) by RNA degrading activity of the reverse transcriptase so as to produce a single-stranded DNA having a first primer sequence;
and
(A3) hybridizing a second primer having the same sequence as a 3′-side sequence of the target sequence to a sequence complementary to the target sequence of the single-stranded DNA produced in (A2), and extending a 3′ end of the second primer by DNA synthetic activity of the reverse transcriptase so as to produce a single-stranded DNA having a second primer sequence, thereby producing a double-stranded DNA as the amplification reaction template;
(B) DNA amplifying for increasing the amplification reaction template, comprising:
(B1) hybridizing the second primer to a 3′ side of the single-stranded DNA comprising the first primer and hybridizing the first primer to a 3′ side of the single-stranded DNA having the second primer sequence in the double-stranded DNA as the amplification reaction template produced in (A3) by a recombinase in a presence of a single-stranded DNA-binding protein;
and
(B2) extending the 3′ end of the first primer and the 3′ end of the second primer by strand displacement DNA synthetic activity of a strand displacement DNA polymerase in a presence of the single-stranded DNA-binding protein so as to produce a double-stranded DNA that is the same as the amplification reaction template produced in (A3);
and
(C) RNA amplifying, comprising:
(C1) synthesizing a single-stranded RNA by an RNA synthetic activity of an RNA polymerase with a sequence that is downstream on a 3′-side relative to the promoter sequence of an RNA polymerase of the double-stranded DNA as the amplification reaction template produced in (A3) being used as a template sequence;
(C2) hybridizing the second primer to a 5′ side of the single-stranded RNA synthesized in (C1), and extending the 3′ end of the second primer by the reverse transcription activity of the reverse transcriptase so as to produce a double-stranded complex of the single-stranded RNA and an extended DNA;
(C3) degrading the single-stranded RNA of the double-stranded complex produced in (C2) by the RNA degrading activity of the reverse transcriptase so as to produce a single-stranded DNA;
and
(C4) hybridizing the first primer to a 3′ side of the single-stranded DNA produced in (C3), extending the 3′ end of the first primer by the DNA synthetic activity of the reverse transcriptase, and extending a 3′ end of the single-stranded DNA produced in (C3) with the promoter sequence of the RNA polymerase of the first primer being used as a template so as to produce a double-stranded DNA that is the same as the amplification reaction template produced in (A3).

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