US Patent No. 10,920,288

DETECTION OF PARTICLE-CONTAINED REVERSE TRANSCRIPTASE ACTIVITY


Patent No. 10,920,288
Issue Date February 16, 2021
Title Detection Of Particle-contained Reverse Transcriptase Activity
Inventorship Joel R. Haynes, Bozeman, MT (US)
Evelyn Benson, Bozeman, MT (US)
Assignee Takeda Vaccines, Inc., Cambridge, MA (US)

Claim of US Patent No. 10,920,288

1. A method for detecting the presence of virus particle-contained reverse transcriptase activity in a test sample comprising steps of:(1) adding to the test sample a protease and a detergent in an amount that is insufficient to disrupt an intact virus particle and incubating the resultant solution under conditions that allow the protease to digest any soluble reverse transcriptase present in the resultant solution, thereby producing a digested solution;
(2) inactivating the protease in the digested solution, thereby producing an inactivated protease solution;
(3) adding a detergent in an amount that is sufficient to disrupt an intact virus particle, thereby producing a detergent-containing solution;
(4) adding into the detergent-containing solution or into a fraction of the detergent-containing solution (i) an isolated RNA molecule, (ii) a first primer that hybridizes to a nucleic acid sequence corresponding to a first part of the isolated RNA molecule, (iii) a second primer that hybridizes to the complement of a second part of the isolated RNA molecule, and (iv) a DNA polymerase, thereby preparing a PCR-based reverse transcriptase (PBRT) assay solution;
(5) incubating the PBRT assay solution under conditions that allow a reverse transcription product to be synthesized from the isolated RNA molecule and a PCR-amplified product from the reverse transcription product if a virus particle-contained reverse transcriptase is present in the test sample; and
(6) identifying the PCR-amplified product, thereby detecting the presence of the virus particle-contained reverse transcriptase in the test sample.