Methods and kits for the detection of SARS-CoV-2
Methods, kits, and oligonucleotides used in the detection of the coronavirus strain, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), are disclosed. In some aspects, the oligonucleotides are primers or probes used in the described methods or kits. The oligonucleotide consists of 40 or less nucleotides and has a nucleotide sequence that consists essentially of, or is a variant of, the nucleotide sequence of: SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7. In some embodiments, the oligonucleotide is modified with an internal spacer or a detectable label. For example, the 5′ terminus is labeled with a fluorophore and the 3′ terminus is complexed to a quencher of fluorescence of said fluorophore. In some embodiments, the nucleotide sequence of the oligonucleotide further comprises a universal tail sequence.
1 . Two pairs of oligonucleotides for amplifying SARS-CoV-2 N and S proteins, each oligonucleotide having a 5′ terminus and a 3′ terminus, wherein the nucleotide sequence of each oligonucleotide consists of 40 or less nucleotides; and the nucleotide sequence of each oligonucleotide comprises:
a nucleotide sequence that binds to a viral genome, said nucleotide sequence for the first primer pair consisting of the nucleotide sequence of: SEQ ID NO: 1 and SEQ ID NO: 2, and said nucleotide sequence for the second primer pair consisting of the nucleotide sequence of: SEQ ID NO:5 and SEQ ID NO:6; and
a universal tail sequence, wherein the universal tail sequence is 5′ to the nucleotide sequence that binds to a viral genome, does not bind to the viral genome, and is capable of acting as a template for amplification with a primer complementary to the universal tail sequence.
2 . A method of detecting the presences of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) polynucleotides in a biological sample in vitro, comprising:
(a) mixing the biological sample in vitro with two primer pairs, each capable of amplifying a SARS-CoV-2 amplicon product, if the SARS-CoV-2 polynucleotide is present in the biological sample, wherein each primer of each primer pair consists of 40 or less nucleotides and the nucleotide sequence of each primer comprises:
a nucleotide sequence capable of detecting SARS-CoV-2, wherein the first primer pair amplifies a nucleocapsid protein amplicon product of SARS-CoV-2 and consists of the nucleotide sequence of: SEQ ID NO: 1 and SEQ ID NO:2, and the second primer pair amplifies a spike protein gene amplicon product of SARS-CoV-2 and consists of the nucleotide sequence of: SEQ ID NO:5 and SEQ ID NO:6; and
a universal tail sequence, wherein the universal tail sequence is 5′ to the nucleotide sequence, does not bind to the SARS-CoV-2 genome, and is capable of acting as a template for amplification with a primer complementary to the universal tail sequence;
(b) amplifying the SARS-CoV-2 amplicon products;
(c) sequencing the amplicon products; and
(d) detecting whether SARS-CoV-2 polynucleotides are present in the biological sample by analyzing the sequencing reads.
3 . The method of claim 2 , wherein the amplicon product has a nucleotide sequence that consists essentially of SEQ ID NO:4 or SEQ ID NO:8.
4 . The method of claim 2 , wherein the biological sample comprises a nasopharyngeal swab sample or sputum.
5 . The method of claim 2 , wherein:
the sequence of one primer of the first primer pair consists of:
SEQ ID NO:9;
the sequence of the other primer of the first primer pair consists of:
SEQ ID NO:10;
the sequence of one primer of the second primer pair consists of:
SEQ ID NO:11; and
the sequence of the other primer of the second primer pair consists of:
SEQ ID NO:12.