Granted Patent B2

US 12,735,735 · App. 18/628,860 · Granted Sep 15, 2026

Inventors: Christophe Quiring (Marnes la Coquette, FR); Christine Favier (Marnes-la-Coquette, FR); Patrice Sarfati (Marnes la Coquette, FR); Jean Francois Mouscadet (Marnes la Coquette, FR); Ronald Lebofsky (Sceaux, FR); Rebecca Dievart (Marnes la Coquette, FR)

Assignee: BIO-RAD EUROPE GMBH

C12Q1/06C12N9/2428C12N9/2445C12N9/2465C12N9/2471C12N9/2474C12N9/485C12Q1/04C12Q1/045C12Y302/01003C12Y302/01022C12Y302/01035C12Y304/11

Abstract

Claims (23)

1 . A method for determining the presence or absence of a microorganism in a sample, the method comprising:

i) encapsulating a sample in a plurality of water-in-oil emulsion droplets wherein the water-in-oil emulsion droplets further encapsulate a microbiological growth medium;

ii) incubating the plurality of water-in-oil emulsion droplets at a temperature permissive of microbiological growth, and for a period of time sufficient to allow the target microorganisms to go through 5 to 45 doubling times;

iii) measuring autofluorescence, turbidity, an amount of a labeled microorganism binding agent and/or an intercalating dye in each of the water-in-oil emulsion droplets and identifying water-in-oil emulsion droplets comprising target microorganisms on the basis of autofluorescence, turbidity, signal of binding agent bound to the microorganism, and/or the presence of intercalating dye in the nucleic acids of the microorganism; and

iv) responsive to identifying the target microorganism in at least one water-in-oil emulsion droplet, determining that the target microorganism is present in the sample, wherein the target microorganisms are selected from bacteria, yeasts and molds and wherein the incubating step is performed for a period of time corresponding to:

at least about 4 hours and no more than about 16 hours when the target microorganisms are bacteria;

at least about 6 hours and no more than about 12 hours when the target microorganisms are yeasts; and

at least about 8 hours and no more than about 36 hours when the target microorganisms are molds.

2 . The method of claim 1 , wherein the incubating step is performed for a period of time corresponding to:

at least about 4 hours and no more than about 8 hours when the target microorganisms are bacteria;

at least about 8 hours and no more than about 12 hours when the target microorganisms are yeasts; and

at least about 8 hours and no more than about 24 hours when the target microorganisms are molds.

3 . The method of claim 1 , wherein the oil phase of the water-in-oil emulsion droplets comprises an antifungal agent.

4 . The method of claim 3 , wherein the antifungal agent is selected from the group consisting of 2,6-dichloro-4-nitroaniline, 4,5,6,7-tetrachloro-2′,4′,5′,7′-tetraiodofluorescein, (RS)-1-[2-(allyloxy)-2-(2,4-dichlorophenyl)ethyl]-1H-imidazole, and chitosan.

5 . The method of claim 1 , wherein the presence of microorganisms in the sample is determined by the detection of autofluorescence in the water-in-oil emulsion droplets comprising said sample.

6 . The method of claim 1 , wherein the presence of microorganisms in the sample is determined by the detection of turbidity in the water-in-oil emulsion droplets comprising said sample.

7 . The method of claim 1 , wherein the presence of microorganisms in the sample is determined by the detection of intercalated dye within the nucleic acids of microorganisms in the water-in-oil emulsion droplets comprising said sample.

8 . The method of claim 1 , wherein the presence of microorganisms in the sample is determined by the detection of labeled binding agent bound to microorganisms in the water-in-oil emulsion droplets comprising said sample.

9 . The method of claim 1 , wherein the sample is a food matrix, an environmental sample, or a clinical sample from a subject.

10 . The method of claim 9 , wherein the sample is an environmental sample.

11 . The method of claim 9 , wherein the sample is a clinical sample from a subject.

12 . The method of claim 11 , wherein the clinical sample is a blood sample, a plasma sample, a serum sample, a tissue sample, a urine sample, a sample of saliva, a pericardial sample, a pleural sample, a spinal fluid sample, a sputum sample, a bone marrow stem cell concentrate sample, a platelet concentrate sample, a nasal sample, a rectal sample, a vaginal sample, an inguinal swab, a sample from a wound, a skin sample, a sample from a mouth, tongue, or throat, an ascites sample, or a stool sample.

13 . The method of claim 10 , wherein the environmental sample is a sample from a surface or a water sample.

Assignments (1)

Continuity (4)

References Cited (51)