	US 11,952,637 B2
	Rapid low-cost detection of SARS-CoV-2 using isothermal amplification and sensing methods
Alexander Green, Boston, MA (US); Keith Pardee, Toronto (CA); Margot Karlikow, Toronto (CA); Kaiyue Wu, Tempe, AZ (US); and Masoud Norouzi, Toronto (CA)
Assigned to ARIZONA BOARD OF REGENTS ON BEHALF OF ARIZONA STATE UNIVERSITY, Scottsdale, AZ (US); and THE GOVERNING COUNCIL OF THE UNIVERSITY OF TORONTO, Toronto (CA)
Appl. No. 18/001,395
Filed by ARIZONA BOARD OF REGENTS ON BEHALF OF ARIZONA STATE UNIVERSITY, Scottsdale, AZ (US); and THE GOVERNING COUNCIL OF THE UNIVERSITY OF TORONTO, Toronto (CA)
PCT Filed Jun. 11, 2021, PCT No. PCT/US2021/037092 § 371(c)(1), (2) Date Dec. 9, 2022, PCT Pub. No. WO2021/252956, PCT Pub. Date Dec. 16, 2021.
Claims priority of provisional application 63/038,609, filed on Jun. 12, 2020.
Prior Publication US 2023/0212698 A1, Jul. 6, 2023
Int. Cl. C12Q 1/70 (2006.01); C12Q 1/6897 (2018.01)

CPC C12Q 1/6897 (2013.01) [C12Q 1/701 (2013.01)]

19 Claims

1. A method of detecting a viral SARS-CoV-2 RNA in a sample from a subject, the method comprising the steps of:

(a) heating the sample, whereby viral RNAs are released from viral particles present in the sample;

(b) amplifying the released viral RNAs, wherein amplifying comprises isothermal amplification and produces a target nucleic acid; and

(i) a first region that is complementary to at least a portion of the target nucleic acid or a reverse complement thereof;

(ii) a second region that encodes a reporter protein and translation regulatory sequences;

wherein the contacting occurs under conditions that allow translation of the reporter protein in the presence of the target nucleic acid or reverse complement thereof, but not in the absence of the target nucleic acid or reverse complement thereof; and

(d) detecting the reporter protein as an indicator that the viral SARS-CoV-2 RNA is present in the subject sample;

wherein each riboregulator first region comprises, independently, one of SEQ ID NOs: 26-51.