Methods and reagents for the specific and sensitive detection of SARS-CoV-2

1. A multiplexed method for specific detection of the presence or absence of a SARS-CoV-2 RNA in a sample, comprising:

(A) providing a sample;

(B) subjecting the sample to a first reverse transcription reaction with a first reverse primer to generate a first cDNA copy of SARS-CoV-2RNA in the sample, amplifying any resultant first cDNA, and detecting any first amplified product with a first probe,

wherein the first reverse primer comprises the sequence 5′-CTCCCTTTGTTGTGTTGT-3′ (SEQ ID NO: 2), or a variant thereof, and is from 15 to 21 bases in length; and/or

wherein the first forward primer comprises the sequence 5′-ATGAGCTTAGTCCTGTTG-3′ (SEQ ID NO: 1), or a variant thereof, and is from 15 to 21 bases in length; and/or

wherein the first probe comprises the sequence 5′-AGATGTCTTGTGCTGCCGGTA-3′ (SEQ ID NO: 3) or a variant thereof, or the complement thereof of either of these, and is from 18 to 24 bases in length; and

(C) subjecting the sample to a second reverse transcription reaction with a second reverse primer to generate a second cDNA copy of SARS-CoV-2RNA in the sample, amplifying any resultant second cDNA, and detecting any second amplified product with a second probe

wherein the second reverse primer comprises the sequence 5′-CTGGTCAAGGTTAATATAGG-3′ (SEQ ID NO: 5) or a variant thereof, and is from 17 to 23 bases in length; and/or

wherein the second forward primer comprises the sequence 5′-GGTAACTGGTATGATTTCG-3′ (SEQ ID NO: 4), or a variant thereof, and is from 16 to 22 bases in length; and/or

wherein the second probe comprises the sequence 5′-TCATACAAACCACGCCAGG-3′ (SEQ ID NO: 6), or a variant thereof, or the complement of either of these, and is from 16 to 22 bases in length.

2. The method of claim 1 ,

wherein the first reverse primer consists of the sequence 5′-CTCCCTTTGTTGTGTTGT-3′ (SEQ ID NO: 2); and/or

wherein the first forward primer consists of the sequence 5′-ATGAGCTTAGTCCTGTTG-3′ (SEQ ID NO: 1); and/or

wherein the first probe consists of the sequence 5′-AGATGTCTTGTGCTGCCGGTA-3′ (SEQ ID NO: 3), or the complement thereof; and/or

wherein the second reverse primer consists of the sequence 5′-CTGGTCAAGGTTAATATAGG-3′ (SEQ ID NO: 5); and/or

wherein the second forward primer consists of the sequence 5′-GGTAACTGGTATGATTTCG-3′ (SEQ ID NO: 4); and/or

wherein the second probe consists of the sequence 5′-TCATACAAACCACGCCAGG-3′ (SEQ ID NO: 6), or the complement thereof.

3. The method of claim 2 ,

wherein the first reverse primer consists of the sequence 5′-CTCCCTTTGTTGTGTTGT-3′ (SEQ ID NO: 2);

wherein the first forward primer consists of the sequence 5′-ATGAGCTTAGTCCTGTTG-3′ (SEQ ID NO: 1);

wherein the first probe consists of the sequence 5′-AGATGTCTTGTGCTGCCGGTA-3′ (SEQ ID NO: 3), or the complement thereof;

wherein the second reverse primer consists of the sequence 5′-CTGGTCAAGGTTAATATAGG-3′ (SEQ ID NO: 5);

wherein the second forward primer consists of the sequence 5′-GGTAACTGGTATGATTTCG-3′ (SEQ ID NO: 4); and

wherein the second probe consists of the sequence 5′-TCATACAAACCACGCCAGG-3′ (SEQ ID NO: 6), or the complement thereof.

4. The method of claim 1 , wherein a first amplified product is detected in (B).

5. The method of claim 1 , wherein a second amplified product is detected in (C).

6. The method of claim 1 , wherein a first amplified product is detected in (B) and a second amplified product is detected in (C).

7. A kit for detection of SARS-CoV-2 RNA in a sample using a multiplexed amplification method, the kit comprising:

(A) a first reverse primer, a first forward primer, and a first probe for amplifying and detecting a first amplification product,

wherein the first reverse primer comprises the sequence 5′-CTCCCTTTGTTGTGTTGT-3′ (SEQ ID NO: 2), or a variant thereof, and is from 15 to 21 bases in length; and/or

wherein the first forward primer comprises the sequence 5′-ATGAGCTTAGTCCTGTTG-3′ (SEQ ID NO: 1), or a variant thereof, and is from 15 to 21 bases in length; and/or

wherein the first probe comprises the sequence 5′-AGATGTCTTGTGCTGCCGGTA-3′ (SEQ ID NO: 3) or a variant thereof, or the complement thereof of either of these, and is from 18 to 24 bases in length; and

(B) a second reverse primer, a second forward primer, and a second probe for amplifying and detecting a second amplification product,

wherein the second reverse primer comprises the sequence 5′-CTGGTCAAGGTTAATATAGG-3′ (SEQ ID NO: 5) or a variant thereof, and is from 17 to 23 bases in length; and/or

wherein the second forward primer comprises the sequence 5′-GGTAACTGGTATGATTTCG-3′ (SEQ ID NO: 4), or a variant thereof, and is from 16 to 22 bases in length; and/or

wherein the second probe comprises the sequence 5′-TCATACAAACCACGCCAGG-3′ (SEQ ID NO: 6), or a variant thereof, or the complement of either of these, and is from 16 to 22 bases in length;

wherein at least one of the first reverse primer, first forward primer, first probe, second reverse primer, second forward primer, and second probe is labelled.

8. The kit of claim 7 ,

wherein the first reverse primer consists of the sequence 5′-CTCCCTTTGTTGTGTTGT-3′ (SEQ ID NO: 2); and/or

wherein the first forward primer consists of the sequence 5′-ATGAGCTTAGTCCTGTTG-3′ (SEQ ID NO: 1); and/or

wherein the first probe consists of the sequence 5′-AGATGTCTTGTGCTGCCGGTA-3′ (SEQ ID NO: 3), or the complement thereof;

and/or wherein the second reverse primer consists of the sequence 5′-CTGGTCAAGGTTAATATAGG-3′ (SEQ ID NO: 5); and/or

wherein the second forward primer consists of the sequence 5′-GGTAACTGGTATGATTTCG-3′ (SEQ ID NO: 4); and/or

wherein the second probe consists of the sequence 5′-TCATACAAACCACGCCAGG-3′ (SEQ ID NO: 6), or the complement thereof.

9. The kit of claim 7 ,

wherein the first reverse primer consists of the sequence 5′-CTCCCTTTGTTGTGTTGT-3′ (SEQ ID NO: 2);

wherein the first forward primer consists of the sequence 5′-ATGAGCTTAGTCCTGTTG-3′ (SEQ ID NO: 1);

wherein the first probe consists of the sequence 5′-AGATGTCTTGTGCTGCCGGTA-3′ (SEQ ID NO: 3), or the complement thereof;

wherein the second reverse primer consists of the sequence 5′-CTGGTCAAGGTTAATATAGG-3′ (SEQ ID NO: 5);

wherein the second forward primer consists of the sequence 5′-GGTAACTGGTATGATTTCG-3′ (SEQ ID NO: 4); and

wherein the second probe consists of the sequence 5′-TCATACAAACCACGCCAGG-3′ (SEQ ID NO: 6), or the complement thereof.