US 12,753,450 B2 · Coronavirus detection

Abstract

Methods for detecting nucleic acid of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are described. The methods comprise amplifying nucleic acid of a sample, or amplifying nucleic acid derived from nucleic acid of the sample, by an isothermal amplification reaction using a forward nucleic acid amplification primer and a reverse nucleic acid amplification primer, wherein each nucleic acid amplification primer hybridises specifically to nucleic acid sequence that is conserved in SARS-CoV-2 nucleic acid, but not in other human coronavirus nucleic acid, or the complement thereof. The methods are particularly useful for point-of-care (ROC) testing. Kits, primers, probes, sets of primers, sets of oligonucleotides, and oligonucleotides, and their use in the methods are also described.

Claims20

Dependent claims

A method for determining whether a sample includes a severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) nucleic acid, wherein the method comprises amplifying a nucleic acid of the sample, or amplifying a nucleic acid derived from the nucleic acid of the sample, by an isothermal amplification reaction using a set of forward and reverse nucleic acid amplification primers selected from (i)-(iii):

(i) at least one forward nucleic acid primer and at least one reverse nucleic acid amplification primer that each hybridize specifically to a nucleic acid sequence that is conserved in the ORF1ab gene of SARS-CoV-2, wherein the at least one forward nucleic acid primer comprises the nucleic acid sequence of:
CTGTTGGTCAACAAGACGGCA (SEQ ID NO:
1), GTCAACAAACTGTTGGTCAA (SEQ ID NO:
2), GTCAACAAACTGTTGGTCAACA (SEQ ID NO:
3), or CATTACAGGTGGTGTTGTTCAGTT (SEQ ID NO:
4);
and
the at least one reverse nucleic acid primer comprises the nucleic acid sequence of:
CAATAGTCTGAACAACTGGTGT (SEQ ID NO:
5), CTGGTGTAAGTTCCATCTCT (SEQ ID NO:
6), or AGGTGACAATTTGTCCACCGAC (SEQ ID NO:
7);
(ii) a forward nucleic acid primer and a reverse nucleic acid primer that each hybridize specifically to a nucleic acid sequence that is conserved in the nucleocapsid gene of SARS-CoV-2, wherein the forward nucleic acid primer comprises the nucleic acid sequence of:
TAGTTGATGACCCGTGTCCT (SEQ ID NO:
14);
and
the reverse nucleic acid primer comprises the nucleic acid sequence of:
TGGGGTCCATTATCAGACAT (SEQ ID NO:
15), CAACACGAACGTCATGATAC (SEQ ID NO:
16), or CATAGAACGAACAACGCAC (SEQ ID NO:
17);
or
(iii) a combination of (i) and (ii).

20 more elements

A kit for determining whether a sample includes a SARS-CoV-2 nucleic acid, which comprises:

a forward nucleic acid amplification primer and a reverse nucleic acid amplification primer, for amplifying a template nucleic acid by an isothermal amplification reaction, wherein each nucleic acid amplification primer hybridises specifically to a nucleic acid sequence that is conserved in the SARS-CoV-2 nucleic acid, but not in other human coronavirus nucleic acid;
a nucleic acid capture probe, wherein the capture probe hybridises specifically to a nucleic acid sequence that is conserved in the SARS-CoV-2 nucleic acid, but not in other human coronavirus nucleic acid, or the complement thereof;
and/or
a nucleic acid detector probe, wherein the detector probe hybridises specifically to a nucleic acid sequence that is conserved in the SARS-CoV-2 nucleic acid, but not in other human coronavirus nucleic acid, or the complement thereof, wherein the detector probe comprises a detectable label for labelling a product of the isothermal nucleic acid amplification, wherein:
the forward and the reverse nucleic acid primers hybridize specifically to a nucleic acid sequence that is conserved in the ORF1ab gene of SARS-CoV-2, wherein the forward nucleic acid primer comprises the nucleic acid sequence of:
CTGTTGGTCAACAAGACGGCA (SEQ ID NO:
1), GTCAACAAACTGTTGGTCAA (SEQ ID NO:
2), GTCAACAAACTGTTGGTCAACA (SEQ ID NO:
3), or CATTACAGGTGGTGTTGTTCAGTT (SEQ ID NO:
4), and the reverse nucleic acid primer comprises the nucleic acid sequence of:
CAATAGTCTGAACAACTGGTGT (SEQ ID NO:
5), CTGGTGTAAGTTCCATCTCT (SEQ ID NO:
6), or AGGTGACAATTTGTCCACCGAC (SEQ ID NO:
7);
and/or
the forward and reverse nucleic acid primers hybridize specifically to a nucleic acid sequence that is conserved in the nucleocapsid gene of SARS-CoV-2, wherein the forward nucleic acid primer comprises the nucleic acid sequence of:
TAGTTGATGACCCGTGTCCT (SEQ ID NO:
14) and the reverse nucleic acid primer comprises the nucleic acid sequence of:
TGGGGTCCATTATCAGACAT (SEQ ID NO:
15), CAACACGAACGTCATGATAC (SEQ ID NO:
16), or CATAGAACGAACAACGCAC (SEQ ID NO:
17).

19 more elements

A set of primers for amplifying SARS-CoV-2 nucleic acid by an isothermal nucleic acid amplification reaction, which comprises a forward nucleic acid amplification primer and a reverse nucleic acid amplification primer, wherein each nucleic acid amplification primer hybridises specifically to a nucleic acid sequence that is conserved in the SARS-CoV-2 nucleic acid, but not in other human coronavirus nucleic acid, wherein the nucleic acid sequence that is conserved in the SARS-CoV-2 nucleic acid is a nucleic acid sequence that is conserved in the ORF1ab gene or the nucleocapsid gene of SARS-CoV-2, wherein:

the forward and the reverse nucleic acid primers hybridize specifically to the nucleic acid sequence that is conserved in the ORF1ab gene of SARS-CoV-2, wherein the forward nucleic acid primer comprises the nucleic acid sequence of:
CTGTTGGTCAACAAGACGGCA (SEQ ID NO:
1), GTCAACAAACTGTTGGTCAA (SEQ ID NO:
2), GTCAACAAACTGTTGGTCAACA (SEQ ID NO:
3), or CATTACAGGTGGTGTTGTTCAGTT (SEQ ID NO:
4), and the reverse nucleic acid primer comprises the nucleic acid sequence of:
CAATAGTCTGAACAACTGGTGT (SEQ ID NO:
5), CTGGTGTAAGTTCCATCTCT (SEQ ID NO:
6), or AGGTGACAATTTGTCCACCGAC (SEQ ID NO:
7);
and/or
the forward and reverse nucleic acid primers hybridize specifically to the nucleic acid sequence that is conserved in the nucleocapsid gene of SARS-CoV-2, wherein the forward nucleic acid primer comprises the nucleic acid sequence of:
TAGTTGATGACCCGTGTCCT (SEQ ID NO:
14), and the reverse nucleic acid primer comprises the nucleic acid sequence of:
TGGGGTCCATTATCAGACAT (SEQ ID NO:
15), CAACACGAACGTCATGATAC (SEQ ID NO:
16), or CATAGAACGAACAACGCAC (SEQ ID NO:
17);
wherein the reverse nucleic acid primer further comprises a promoter sequence for a DNA-dependent RNA polymerase at its 5′-end;
optionally wherein the forward and/or the reverse nucleic acid primer is up to 50 nucleotides long.

17 more elements

A set of oligonucleotides for amplifying SARS-CoV-2 nucleic acid by an isothermal nucleic acid amplification reaction, and for capturing and/or detecting a product of the amplification reaction, wherein the set of oligonucleotides comprises:

(i) a set of primers, wherein the set of primers comprises a forward nucleic acid amplification primer and a reverse nucleic acid amplification primer, wherein each nucleic acid amplification primer hybridises specifically to a nucleic acid sequence that is conserved in the SARS-CoV-2 nucleic acid, but not in other human coronavirus nucleic acid, wherein the nucleic acid sequence that is conserved in the SARS-CoV-2 nucleic acid is the nucleic acid sequence that is conserved in the ORF1ab gene or the nucleocapsid gene of SARS-CoV-2, wherein:
the forward and the reverse nucleic acid primers hybridize specifically to the nucleic acid sequence that is conserved in the ORF1ab gene of SARS-CoV-2, wherein the forward nucleic acid primer comprises the nucleic acid sequence of:
CTGTTGGTCAACAAGACGGCA (SEQ ID NO:
1), GTCAACAAACTGTTGGTCAA (SEQ ID NO:
2), GTCAACAAACTGTTGGTCAACA (SEQ ID NO:
3), or CATTACAGGTGGTGTTGTTCAGTT (SEQ ID NO:
4), and the reverse nucleic acid primer comprises the nucleic acid sequence of:
CAATAGTCTGAACAACTGGTGT (SEQ ID NO:
5), CTGGTGTAAGTTCCATCTCT (SEQ ID NO:
6), or AGGTGACAATTTGTCCACCGAC (SEQ ID NO:
7);
and/or
the forward and reverse nucleic acid primers hybridize specifically to the nucleic acid sequence that is conserved in the nucleocapsid gene of SARS-CoV-2, wherein the forward nucleic acid primer comprises the nucleic acid sequence of:
TAGTTGATGACCCGTGTCCT (SEQ ID NO:
14), and the reverse nucleic acid primer comprises the nucleic acid sequence of:
TGGGGTCCATTATCAGACAT (SEQ ID NO:
15), CAACACGAACGTCATGATAC (SEQ ID NO:
16), or CATAGAACGAACAACGCAC (SEQ ID NO:
17);
(ii) a nucleic acid capture probe, wherein the capture probe hybridises specifically to the nucleic acid sequence that is conserved in SARS-CoV-2 nucleic acid, but not in other human coronavirus nucleic acid;
and/or
(iii) a nucleic acid detector probe, wherein the detector probe hybridises specifically to the nucleic acid sequence that is conserved in SARS-CoV-2 nucleic acid, but not in other human coronavirus nucleic acid, wherein the detector probe comprises a detectable label for labelling a product of the isothermal nucleic acid amplification.

19 more elements

A method for determining whether a sample includes severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) nucleic acid, which comprises amplifying a nucleic acid of the sample, or amplifying a nucleic acid derived from the nucleic acid of the sample, by an amplification reaction using a set of forward and reverse nucleic acid amplification primers, which further comprises detecting a product of the amplification reaction by hybridizing the product to a nucleic acid detector probe, selected from (i)-(iii):

(i) the forward nucleic acid primer, the reverse nucleic acid primer, and the detector probe each hybridizes specifically to the nucleic acid sequence that is conserved in the ORF1ab gene of SARS-CoV-2, but not in other human coronavirus nucleic acid, wherein:
the forward nucleic acid primer hybridizes specifically to the nucleic acid sequence that is conserved in the ORF1ab gene of SARS-CoV-2;
the reverse nucleic acid primer hybridizes specifically to the nucleic acid sequence that is conserved in the ORF1ab gene of SARS-CoV-2;
and
the detector probe hybridizes specifically to the nucleic acid sequence that is conserved in the ORF1ab gene of SARS-CoV-2, wherein the detector probe comprises the nucleic acid sequence of:
GAGGTTCAACCTCAATTAGAGATGG (SEQ ID NO:
12);
(ii) the forward nucleic acid primer, the revers nucleic acid primer, and the detector probe each hybridizes specifically to the nucleic acid sequence that is conserved in the nucleocapsid gene of SARS-CoV-2, but not in other human coronavirus nucleic acid, wherein:
the forward nucleic acid primer hybridizes specifically to the nucleic acid sequence that is conserved in the nucleocapsid gene of SARS-CoV-2;
the reverse nucleic acid primer hybridizes specifically to the nucleic acid sequence that is conserved in the nucleocapsid gene of SARS-CoV-2;
and
the detector probe hybridizes specifically to the nucleic acid sequence that is conserved in the nucleocapsid gene of SARS-CoV-2, wherein the detector probe comprises an oligonucleotide comprising the nucleic acid sequence of:
CTGGTTCTAAATCACCCATTCA (SEQ ID NO:
18);
or
(iii) a combination of (i) and (ii).

13 more elements

Dependencies are inferred from the claim text.

Coronavirus detection

Life of this patent

Coronavirus detection

Life of this patent

Abstract

Claims20