Compositions and methods for amplifying, detecting or quantifying human polyomavirus BK virus
Oligomer nucleotides, compositions, methods, kits, and uses are provided for detecting or quantifying a human polyomavirus BK virus (BKV) nucleic acid, e.g., using nucleic acid amplification and hybridization assays.
1 . A combination of oligonucleotides for amplifying a BK polyomavirus (BKV) nucleic acid sequence comprising:
a) one or more first amplification primers wherein each first amplification primer is 18-30 nucleobases in length and comprises a nucleotide sequence having at least 90% identity to the nucleotide sequence selected from the group consisting of: SEQ ID NO: 46, and SEQ ID NO:47, and
b) one or more second amplification primers wherein each second amplification primer is 19-30 nucleobases in length and comprises a nucleotide sequence having at least 90% identity to the nucleotide sequence of: SEQ ID NO: 22.
2 . The combination of claim 1 , wherein the first amplification primer and/or the second amplification primer contains one or more modified nucleotides independently selected from the group consisting of: a 2′-O-methyl modified nucleotide, a 2′-fluoro modified nucleotide, and a 5-methyl-2′-deoxycytosine.
3 . The combination of claim 1 , wherein every cytosine in the first amplification primer and/or the second amplification primer except for a 3′ terminal cytosine, if present, is a 5-methyl-2′-deoxycytosine.
4 . The combination of claim 1 , wherein:
a) the nucleotide sequence of each of the one or more first amplification primers consists of a nucleotide sequence selected from the group consisting of: SEQ ID NO:24, SEQ ID NO:46, and SEQ ID NO:47, and
b) the nucleotide sequence each of the one or more second amplification primers consists of a nucleotide sequence of: SEQ ID NO:22.
5 . The combination of claim 1 , wherein
the first amplification primer comprises the nucleotide sequence of SEQ ID NO: 24 and the second amplification primer comprises the nucleotide sequence of SEQ ID NO: 22.
6 . The combination of claim 1 , wherein:
the one or more first amplification primers comprise at least one amplification primer comprising the nucleotide sequence of SEQ ID NO:46 and at least one amplification primer comprising the nucleotide sequence of SEQ ID NO:47 and the second amplification primer comprises the nucleotide sequence of SEQ ID NO:22.
7 . The combination of claim 1 , further comprising a probe oligonucleotide for detecting an amplified BKV nucleic acid sequence wherein the probe oligonucleotide is 21-30 nucleobases in length and comprises a nucleotide sequence having at least 90% identity to the nucleotide sequence of: SEQ ID NO: 23, and wherein the probe oligonucleotide is attached to at least one detectable label.
8 . The combination of claim 7 , wherein the probe oligonucleotide contains one or more modified nucleotides independently selected from the group consisting of: a 2′-O-methyl modified nucleotide, a 2′-fluoro modified nucleotide, or a 5-methyl-2′-deoxycytosine.
9 . The combination of claim 8 , wherein the probe oligonucleotide contains 2, 3, 4, 5, 6, 7, or 8 5-methyl-2′-deoxycytosines.
10 . The combination of claim 9 , wherein every cytosine in the probe oligonucleotide is a 5-methyl-2′-deoxycytosine.
11 . The combination of claim 7 wherein the nucleotide sequence of the probe oligonucleotide consists of the nucleotide sequence of SEQ ID NO:23.
12 . The combination of claim 7 , wherein the detectable label contains a fluorescent molecule attached at the 5′ or 3′ end of the probe oligonucleotide or a fluorescent molecule attached at the 5′ end of the probe oligonucleotide and a quencher is attached at the 3′ end of the probe oligonucleotide.
13 . The combination of claim 12 wherein the probe oligonucleotide is a hydrolysis probe.
14 . A kit for amplifying a BKV nucleic acid sequence comprising: the amplification primers of claim 1 or the amplification primers of claim 1 and the probe oligonucleotide of claim 7 , wherein the amplification primers and the probe oligonucleotide are provided in a single container or separate containers.
15 . The kit of claim 14 , further comprising at least one reaction mixture wherein the at least one reaction mixture comprises one or more of: DNA polymerase, deoxyribonucleotides, ribonucleotides, positive control nucleic acid, negative control nucleic acid, internal control nucleic acid, control amplification primers, control probe oligonucleotide, KCl, MgCl 2 , potassium acetate, buffer, BSA, sucrose, trehalose, DMSO, betaine, formamide, glycerol, polyethylene glycol, non-ionic detergents, ammonium ions, and EDTA.
16 . The kit of claim 14 , further comprising instructions for use.
17 . A method of amplifying a BKV nucleic acid sequence comprising: contacting a sample containing or suspected of containing the BKV nucleic acid sequence with the combination of claim 1 and performing a nucleic acid amplification reaction, wherein the BKV nucleic acid sequence, if present in the sample, is amplified by the amplification primers to produce a BKV amplicon.
18 . The method of claim 17 , wherein the nucleic acid amplification reaction comprises polymerase chain reaction (PCR).
19 . The method of claim 17 , wherein the method further comprises contacting the sample with a probe oligonucleotide for detecting the amplified BKV nucleic acid sequence wherein the probe oligonucleotide is 21-30 nucleobases in length and comprises a nucleotide sequence having at least 90% identity to the nucleotide sequence of: SEQ ID NO:23, thereby detecting the presence of absence of the BKV amplicon.
20 . A method of claim 19 , wherein the method further comprises quantifying the amount of the BKV nucleic acid sequence or BKV amplicon, if present, in the sample.
21 . The method of claim 20 , wherein the BKV nucleic acid sequence or BKV amplicon is detected and/or quantified in real time.