Loop-mediated isothermal amplification (LAMP) based assay for detecting microbes

Patent metadata

Field	Value
Publication number	US-11299789-B2
Application number	US-201816237458-A
Country	US
Kind code	B2
Filing date	Dec 31, 2018
Priority date	Jan 2, 2018
Publication date	Apr 12, 2022
Grant date	Apr 12, 2022

Abstract

A method and system are disclosed for detecting microbial pathogens in a sample suspected of containing the pathogens. The method includes combining loop-mediated isothermal amplification (LAMP) reagents and a polymer gel, such as a hydrogel, together with the sample to form a mixture. The gel polymerizes over a short time to immobilize the viral particles within the mixture. If target DNA/RNA are present in the sample, amplicons are produced. The target microorganisms are detected by visually detecting the presence or absence of the amplicons. The target microorganism concentrations may be determined based on the number of fluorescent amplicon dots after the reaction using a smartphone or a fluorescent microscope. The method may be employed for rapidly and inexpensively quantifying microbial pathogens in environmental water samples with high sensitivity.

First claim

What is claimed is:

1. A system for detecting a target microorganisms in a sample suspected of containing the target microorganism, comprising:
reaction agents including:
loop mediated isothermal amplification (LAMP) reagents comprising a plurality of primers including a fluorophore labeled primer; and
a polyethylene glycol (PEG) hydrogel at a concentration of 10% (w/v) of the reaction agents, the polyethylene glycol hydrogel being formed through Michael addition between a four-arm PEG acrylate having a molecular weight (MW) of 10K and thiol-PEG-thiol having a MW 3.4K, at mole ratio of 1:2, wherein crosslinking of the PEG hydrogel is tuned to form a predetermined mesh size of the PEG hydrogel and a predetermined molecular weight between crosslinkers in the PEG hydrogel that restricts diffusion of DNA/RNA templates of the target microorganism within the PEG hydrogel while allowing free diffusion within the PEG hydrogel of molecules having a MW<100 kDa;

a chamber for combining the reaction agents and the sample to form a mixture;
an incubator configured to heat the mixture;
a slide configured to receive the mixture to allow visual detection of the presence or absence of one or more amplicons that are produced as a result of a LAMP reaction amplifying the DNA/RNA templates of the target microorganism if the target microorganism is present in the mixture, wherein the presence of the amplicons is indicative of the presence of the target microorganism in the sample and the absence of the amplicons is indicative of the absence of the target microorganism in the sample; and
a camera configured to visually detect the presence or absence of amplicons in the mixture received on the slide.

2. The system of claim 1 , wherein the primers include a primer-dye and primer-quencher duplex.

Assignees

• California Inst Of Techn

Inventors

• Huang Xiao
• Hoffmann Michael R

Classifications

• C12Q1/70

  involving virus or bacteriophage {(immunoassay for viruses G01N33/56983)} · CPC title

• C12Q1/689 Primary

  for bacteria · CPC title

• G02B21/34

  Microscope slides, e.g. mounting specimens on microscope slides · CPC title

• C12Q1/6844

  Nucleic acid amplification reactions · CPC title

• G02B21/16

  adapted for ultraviolet illumination {; Fluorescence microscopes (G02B21/0076 takes precedence)} · CPC title