Amplification technology using dual enzyme cascade detection

Patent metadata

Field	Value
Publication number	US-11385229-B2
Application number	US-201916541111-A
Country	US
Kind code	B2
Filing date	Aug 14, 2019
Priority date	Feb 20, 2017
Publication date	Jul 12, 2022
Grant date	Jul 12, 2022

Abstract

Diagnostic assays and methods employ a dual-enzyme cascade system comprised of two enzymatic amplifiers and two molecular probes, by (a) incubating a target enzyme with a target enzyme substrate to liberate a thiol; (b) incubating the thiol with a disulfide inactivated amplification enzyme to activate the amplification enzyme in an interchange reaction of the thiol and the disulfide; and (c) incubating the activated amplification enzyme with an amplification enzyme substrate to generate an amplified signal.

First claim

The invention claimed is:

1. An assay method to determine the presence of a β-lactam resistant bacterium in a sample from a subject, the method comprising:
obtaining the sample from the subject;
incubating the sample with reaction components comprising:
(i) a β-lactamase probe having a structure selected from the group consisting of:

(ii) papain-S—S—CH 3 ; and (iii) a papain probe having a structure
selected from the group consisting of:

and determining the generation of a colorimetric signal from incubating the sample with the reaction components by visual inspection of the sample or by absorption spectroscopy, wherein the absorption spectroscopy is carried out at a wavelength of 405 nm;
wherein, the colorimetric signal which has been generated from the incubated sample indicates that the subject's sample comprises a β-lactam resistant bacterium,
wherein the sample that is obtained from the subject is an unprocessed urine sample,
wherein the unprocessed urine sample is obtained from a subject that has, or is suspected of having, a urinary tract infection, and
wherein the sample is incubated with the reaction components at room temperature.

Assignees

• Univ California

Inventors

• Murthy Niren
• Riley Lee W
• Beboer Tara Renee

Classifications

• G01N33/573 Primary

  for enzymes or isoenzymes · CPC title

• C12Q1/34

  involving hydrolase · CPC title

• C12Q1/26

  involving oxidoreductase · CPC title