Compositions and methods for amplifying or detecting varicella-zoster virus
Disclosed are oligonucleotides, oligonucleotide compositions, kits, methods, formulations, and reaction mixtures that provide for sensitive and specific detection of a target nucleic acid sequence, or amplicon generated from a target nucleic acid sequence, of Varicella-Zoster Virus (VZV1 (if present) in a sample. The oligonucleotides, compositions, kits, methods, formulations, and reaction mixtures can be used to detect the presence of VZV in a sample. The oligonucleotides, compositions, kits, methods, formulations, and reaction mixtures can also be used to amplify specific target nucleic acid regions of VZV.
1 . A composition for amplifying a Varicella-Zoster Virus (VZV) target nucleic acid sequence and detecting an amplified VZV target nucleic acid sequence comprising:
(a) a forward amplification primer 19-50 nucleobases in length and comprising the nucleobase sequence of SEQ ID NO: 1, 2, 3, 4, 5, 6, or 7; and
(b) a reverse amplification primer 19-50 nucleobases in length and comprising the nucleobase sequence of SEQ ID NO: 16, 17, 18, 19, 20, 21, or 22, wherein:
(i) (a) comprises SEQ ID NO: 1 and (b) comprises SEQ ID NO: 16 or 17;
(ii) (a) comprises SEQ ID NO: 2 and (b) comprises SEQ ID NO: 17;
(iii) (a) comprises SEQ ID NO: 3 and (b) comprises SEQ ID NO: 18;
(iv) (a) comprises SEQ ID NO: 4 and (b) comprises SEQ ID NO: 19;
(v) (a) comprises SEQ ID NO: 5 and (b) comprises SEQ ID NO: 20;
(vi) (a) comprises SEQ ID NO: 6 and (b) comprises SEQ ID NO: 21; or
(vii) (a) comprises SEQ ID NO: 7 and (b) comprises SEQ ID NO: 22; and
(c) a detection probe comprising a detectable label.
2 . The composition of claim 1 , wherein the detection probe is 20-50 nucleobases in length and comprises the nucleobase sequence of SEQ ID NO: 8, 9, 10, 11, 12, 13, 14, or 15.
3 . The composition of claim 2 , wherein the forward amplification primer, the reverse amplification primer, and/or the detection probe comprises at least one modified nucleotide.
4 . The composition of claim 3 , wherein the at least one modified nucleotide comprises: a 2′-O-methyl modified nucleotide, a 2′-fluoro modified nucleotide, or a 5-methylcytosine.
5 . The composition of claim 2 , wherein the detectable label is selected from the group consisting of:
(a) a chemiluminescent label;
(b) a fluorescent label;
(c) a quencher;
(d) a combination of two or more of (a), (b), and (c); or
(e) a combination of (b) and (c).
6 . The composition of claim 2 , wherein the detection probe comprises a 5′ non-target-hybridizing sequence that base pairs with the 3′ end of the detection probe or a 3′ non-target-hybridizing sequence that base pairs with the 5′ end of the detection probe.
7 . The composition of claim 6 , wherein the detection probe comprises a molecular beacon or a molecular torch.
8 . The composition of claim 1 , further comprising one or more of: buffer, salt, dNTPs, detergent, and enzyme.
9 . The composition of claim 8 , wherein the enzyme comprises: a thermostable DNA polymerase, a reverse transcriptase, an RNA polymerase, or a combination of any two or more of a thermostable DNA polymerase, a reverse transcriptase, and an RNA polymerase.
10 . The composition of claim 1 , wherein the forward amplification primer and the reverse amplification primer are in aqueous solution, frozen, or lyophilized.
11 . The composition of claim 1 , wherein the composition comprises two or more pairs of amplification primers and/or two or more detection probes, wherein each pair of amplification primers consists of a forward amplification primer and a reverse amplification primer.
12 . The composition of claim 11 , wherein the two or more pairs of amplification primers and/or two or more detection probes amplify target nucleic acid sequences in the same or different organisms.
13 . The composition of claim 1 , further comprising an internal control target nucleic acid sequence, oligomers for amplifying and/or detecting an internal control target nucleic acid sequence, or a combination thereof.
14 . A method for amplifying a VZV target nucleic acid sequence and detecting an amplified VZV target nucleic acid sequence comprising:
(a) obtaining a sample containing or suspected of containing the VZV target nucleic acid sequence;
(b) contacting the sample with the composition of claim 1 ; and
(c) providing conditions sufficient to amplify the target nucleic acid sequence, thereby producing an amplification product of the VZV target nucleic acid sequence if the VZV target nucleic acid sequence is present in the sample.
15 . The method of claim 14 , wherein the detection probe comprises an oligonucleotide comprising the nucleobase sequence of SEQ ID NO: 8, 9, 10, 11, 12, 13, 14, 15, 28, 29, 30, 31, 32, or 33.