Digital pcr assay designs for multiple hepatitis b virus gene targets and non-extendable blocker oligonucleotides therefor
AssigneeRoche Molecular Systems, Inc.
InventorsPaul Joshua Dawson, Aaron Thaddeus Hamilton, Calvin Mano, Elizabeth Marie Scott, Jingtao Sun, Wei Yang
Life of this patent
Estimating the term…
Abstract
The present invention provides compositions and methods relating to novel digital PCR (dPCR) assay designs (droplet digital or other systems) for the detection and quantitation of multiple hepatitis B virus (HBV) gene targets. The compositions and method can further include non-extendible blocker oligonucleotides for reduction of non-specific inter-amplicon extension.
Claims22
-4. (canceled)
A method to detect and quantify at least two different Hepatitis B Virus (HBV) target nucleic acids in a sample by Polymerase Chain Reaction (PCR), comprising,
- providing the sample;
- randomly distributing the sample into a plurality of equal sized and independent partitions;
- performing in each partition a PCR assay with at least two forward and reverse primer sets for amplifying each of the HBV target nucleic acids, and with at least two probes, each probe labeled with a fluorescent dye that generates a different signal, for detecting each of the HBV target nucleic acids;
- and
- measuring the amount of signal generated in each of the partitions to calculate the quantity of each of the at least two different HBV target nucleic in the sample.
-16. (canceled)
A method of reducing unwanted amplification in a multiplex Digital PCR (dPCR) assay by the use of a blocker oligonucleotide having a non-extendable 3′ terminus and higher melting temperature (Tm) to template nucleic acid relative to the primers and probes used in dPCR.
A kit for selectively detecting at least two targets in a nucleic acid, the kit comprising:
- a first set of primers to produce a first amplification product if a first portion of a nucleic acid is present in the sample;
- a second set of primers to produce a second amplification product if a second portion of the nucleic acid is present in the sample;
- a blocker oligonucleotide complementary to the nucleic acid intermediate the first portion and the second portion;
- a first detectable probe complementary to the first amplification product;
- and
- a second detectable probe complementary to the second amplification product.
Dependencies are inferred from the claim text.