Compositions and methods for detecting or quantifying hepatitis B virus
This disclosure provides oligomers, compositions, and kits for detecting and quantifying Hepatitis B virus (HBV), including different genotypes and variants thereof, and related methods and uses. In some embodiments, oligomers target the P and/or S open reading frames of HBV and are configured to provide substantially equivalent quantification of different genotypes and variants of HBV.
1 . A method of determining a concentration of Hepatitis B virus in a sample comprising first and second Hepatitis B amplicons in the presence of a first probe oligomer configured to specifically hybridize to the first amplicon and a second probe oligomer configured to specifically hybridize to the second amplicon, wherein the first probe oligomer is attached to a first label and the second probe oligomer is attached to a second label, respectively, wherein at least one of the first label and the second label is a non-nucleotide detectable label, wherein the second amplicon comprises at least 10 contiguous nucleotides of SEQ ID NO: 35, including the inosine at position 30 of SEQ ID NO: 35, the method comprising:
(i) detecting a first signal emitted from the first label;
(ii) detecting a second signal emitted from the second label;
(iii) determining whether the first signal or the second signal is above a predetermined threshold; and
(iv) calculating the concentration of Hepatitis B virus in the sample,
wherein if the first signal or the second signal is above the predetermined threshold, the concentration is calculated from the greater of the first and second signals;
wherein if the first signal and the second signal are below the predetermined threshold, the concentration is calculated from an average of the first and second signals; and
wherein the predetermined threshold corresponds to a concentration in the range of about 10 IU/ml to about 200 IU/ml.
2 . The method of claim 1 , wherein the sample is an in vitro sample.
3 . The method of claim 1 , comprising determining the average of the first and second signals by determining first and second concentrations corresponding to the first and second signals, and arithmetically averaging the first and second concentrations.
4 . The method of claim 1 , wherein one or both of the first and second amplicons comprise sequence from the S ORF of HBV.
5 . The method of claim 1 , wherein one or both of the first and second amplicons comprise sequence from the P ORF of HBV.
6 . The method of claim 1 , wherein one or both of the first and second amplicons comprise sequence from the overlap of the S and P ORFs of HBV.
7 . The method of claim 1 , wherein the first amplicon comprises at least 10 contiguous nucleotides of one of SEQ ID NOs: 2 or 3.
8 . The method of claim 1 , wherein the first amplicon comprises at least 10 contiguous nucleotides of one of SEQ ID NOs: 20, 21, or 22.
9 . The method of claim 1 , wherein the second amplicon comprises at least 10 contiguous nucleotides of SEQ ID NO: 41.
10 . The method of claim 1 , wherein the predetermined threshold is at a value where expected random error is greater than or about equal to expected error due to a point mutation.
11 . The method of claim 1 , wherein the predetermined threshold is at a value where expected error due to a point mutation is greater than or about equal to expected random error.
12 . The method of claim 1 , wherein the predetermined threshold is in the range of about 20 IU/ml to about 40 IU/ml, about 40 IU/ml to about 60 IU/ml, about 60 IU/ml to about 80 IU/ml, or about 80 IU/ml to about 100 IU/ml.
13 . The method of claim 1 , wherein the first label, the second label, or both are fluorescent.
14 . The method of claim 1 , wherein the first probe oligomer, the second probe oligomer, or both comprises a quencher.