US 11,739,382 B2
Compositions and methods for amplification and detection of hepatitis B virus RNA, including HBV RNA transcribed from cccDNA
Jeffery Fong, Lafayette, CA (US); Aaron T. Hamilton, Mountain House, CA (US); Marintha Heil, Danville, CA (US); Igor Kozlov, Danville, CA (US); Ed Gustavo Marins, Brentwood, CA (US); Elizabeth Marie Scott, San Ramon, CA (US); and Ling Wang, Dublin, CA (US)
Assigned to Roche Molecular Systems, Inc., Pleasanton, CA (US)
Filed by Roche Molecular Systems, Inc., Pleasanton, CA (US)
Filed on Feb. 24, 2020, as Appl. No. 16/798,911.
Claims priority of provisional application 62/892,132, filed on Aug. 27, 2019.
Prior Publication US 2021/0062259 A1, Mar. 4, 2021
Int. Cl. C12Q 1/6876 (2018.01); C07H 21/00 (2006.01); G01N 33/49 (2006.01)
CPC C12Q 1/6876 (2013.01) [G01N 33/49 (2013.01); C12Q 2525/161 (2013.01); C12Q 2545/114 (2013.01); C12Q 2600/112 (2013.01)] 78 Claims
 
1. A method for detecting one or more target nucleic acids of Hepatitis B Virus (HBV) RNA in a sample, the method comprising:
(a) providing a sample;
(b) performing an amplification step comprising contacting the sample with one or more competitive blocking oligonucleotides and one or more set of primers, wherein the one or more set of primers comprises one or more forward primer and one or more reverse transcription (RT) primer that also functions as a reverse primer, to produce an amplification product, if the one or more target nucleic acids of HBV RNA is present in the sample, wherein the one or more competitive blocking oligonucleotides are characterized by being from 24 bases to 61 bases in length and/or by having modified stabilizing bases to increase melting temperature (Tm) and binding strength, and wherein the one or more RT primer contains HBV-specific sequence at its 3′ end to reduce non-specific binding;
(c) performing a hybridization step, comprising contacting the amplification product, if the one or more target nucleic acids of HBV RNA is present in the sample, with one or more probes; and
(d) performing a detection step, comprising detecting the presence or absence of the amplification product, wherein the presence of the amplification product is indicative of the presence of the one or more target nucleic acids of HBV RNA in the sample, and wherein the absence of the amplification product is indicative of the absence of the one or more target nucleic acids of HBV RNA in the sample.