IP Library › Granted Patent US 12,416,052
Granted Patent B2
US 12,416,052 · App. 18/752,431 · Granted Sep 16, 2025

Capturing, concentrating, and detecting microbes in a sample using magnetic ionic liquids and recombinase polymerase amplification

Inventors: Jared L. Anderson (Ames, IA); Byron F. Brehm-Stecher (Ames, IA); Kevin D. Clark (Ames, IA); Stephanie A Hice (Ames, IA)
Assignee: Iowa State University Research Foundation, Inc.
C12Q1/689C07F5/003C07F13/005C07F15/045C07F15/065C12N1/02C12N1/20C12N15/1013C12Q1/04C12Q1/24C12Q1/6806
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Quick Facts
Patent No.
US 12,416,052
App. No.
18/752,431
Granted
Sep 16, 2025
Kind
B2
Abstract

In this disclosure, a method or kit for using the method of extracting, concentrating, and detecting microbes from a sample is disclosed. The method disclosed herein use transition or rare earth metal-based magnetic ionic liquids (MILs) to extract viable microbes from a sample and to detect them via an amplification-based method and/or a non-amplification-based methods. The method and kit can be used in-field and on-site for detection of viable microbes in a sample within about an hour, without using any powered heat source or powered tool.

Claims (32)

1. A kit for extracting, concentrating, and detecting viable microbes in a sample, comprising:

a magnetic ionic liquid (MIL);

an extracting medium;

an enzyme for Recombinase Polymerase Amplification (RPA); and

a power-free heat source comprising a sodium acetate heat pack.

2. The kit according to claim 1 , wherein the MIL comprises a paramagnetic anionic component and a cationic component, or a paramagnetic cationic component and an anionic component.

3. The kit according to claim 1 , wherein the MIL comprises a paramagnetic anionic component and a cationic component, wherein the cationic component has a general formula (I), (IV), or (V)

[(PR 1 R 2 R 3 R 4 ) + ]  (I)

wherein each of the R 1 , R 2 , R 3 , and R 4 is independently an unsubstituted or substituted alkyl;

wherein the paramagnetic anionic component has the following general formula (II),

[M(Y) x − ]  (II)

wherein M is transition metal or rare earth metal ion; and

Y is a chelating agent having the general formula (III),

each of the R 10 and R 11 are independently a substituted or unsubstituted methyl, phenyl, thiophenyl, napthyl, alkyl, or aryl group; and x is 3 or 4

wherein R is one or more of the following:

and wherein Ti comprises Co, Ni, Mn or a combination thereof and Ln comprises Dy, Gd, Ho, or a combination thereof.

4. The kit according to claim 3 , wherein the MIL has the anionic component of [Co(hfacac) 3 − ], [Ni(hfacac) 3 − ], ([Mn(hfacac) 3 − ]), ([Dy(hfacac) 4 − ]), ([Gd(hfacac) 4 − ]), ([Nd(hfacac) 4 − ]), or combination thereof; wherein hfacac is

and has the cationic component of [(PR 1 R 2 R 3 R 4 ) + ], wherein each of the R 1 , R 2 , R 3 , and R 4 is independently a C 2 -C 20 unsubstituted alkyl.

5. The kit according to claim 1 , wherein the magnetic ionic liquid comprises a paramagnetic cationic component and an anionic component, wherein the anionic component has a general formula (VI) or (VII)

wherein R is one or more of the following:

and wherein Ti comprises Co, Ni, Mn or a combination thereof and Ln comprises Dy, Gd, Ho, or a combination thereof.

6. The kit according to claim 1 , wherein the extracting medium is a Luria-Bertani-derived nutrient broth comprising tryptone, yeast extract, NaCl, or a combination thereof.

7. The kit according to claim 6 , wherein the extracting medium comprises more than 10 g/L of tryptone, more than 5 g/L of yeast extract, more than 10 g/L of NaCl, or a combination thereof.

8. The kit according to claim 1 , wherein a volume ratio between the magnetic ionic liquid and the extracting medium is from about 1:5 to 1:15.

9. The kit according to claim 1 , wherein the enzyme for RPA comprises a recombinase, a single-stranded DNA-binding protein, and a DNA polymerase.

10. The kit according to claim 9 , wherein the enzyme for RPA further comprises a reverse transcriptase.

11. The kit according to claim 1 , further comprising a primer set specific for the microbes.

12. The kit according to claim 11 , wherein the microbes are gram-negative bacteria selected from the group consisting of Cronobacter sakazakii, E. coli, Klebsiella aerogenes, Pantoea eucalypti, Pantoea stewartii, Pectobacterium carotovorum, Salmonella bongori, Salmonella enterica, Serratia marcescens , and Yersinia enterocolitica.

13. The kit according to claim 11 , wherein the microbes are Salmonella.

14. The kit according to claim 13 , wherein the primer set is for the dienelactone hydrolase enzyme of Salmonella.

15. The kit according to claim 13 , wherein the primer set comprises SEQ ID NO: 1 and SEQ ID NO: 2.

16. The kit according to claim 1 , wherein the sample is a food sample.

Assignments (1)
ASSIGNMENT OF ASSIGNOR'S INTEREST Recorded Jun 24, 2024
From: ANDERSON, JARED L.; BREHM-STECHER, BYRON F.; CLARK, KEVIN D.; HICE, STEPHANIE A.
To: IOWA STATE UNIVERSITY RESEARCH FOUNDATION, INC.
Reel/Frame 067819/0217 →
Continuity (3)
Division 16792734 · Feb 17, 2020
Provisional Application 62834169 · Apr 15, 2019
Related Publication 20240336982A1 · Oct 10, 2024
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