Mass spectrometric rapid detection of Salmonella

Patent metadata

Field	Value
Publication number	US-10144946-B2
Application number	US-201113102085-A
Country	US
Kind code	B2
Filing date	May 6, 2011
Priority date	May 7, 2010
Publication date	Dec 4, 2018
Grant date	Dec 4, 2018

Abstract

The invention relates to the detection of specified, flagellated bacteria, particularly Salmonella, in food and stool. A single culturing period of about 12 to 24 hours in a liquid nutrient medium without agitation is combined with a position-selective sampling of the flagellated microbes from the liquid of the culture, after which a mass spectrometric detection method is used which recognizes the target bacteria in mixtures. A second culture step is only necessary in exceptional cases. A species-selective or genus-selective culture medium is advantageous. Positional selection becomes possible because these bacteria use their flagella to counteract sedimentation by chemotaxis, and they collect near the surface. This provides a low-cost detection method that is several days faster than conventional methods

First claim

What is claimed is:

1. A method for detecting the presence or absence of flagellated target bacteria in stool or food samples comprising microbes that may or may not comprise flagellated target bacteria, comprising the steps:
(a) introducing a stool or food sample into a liquid nutrient medium;
(b) incubating the liquid nutrient medium with the sample for a predetermined time for culturing the microbes;
(c) removing a sample of liquid from the surface of the liquid nutrient medium, wherein the liquid nutrient medium is not agitated at least during a period of time prior to removing the sample of liquid;
(d) directly after being removed from the liquid nutrient medium, centrifuging, washing and disintegrating the microbes from the removed sample of liquid; and then
(e) detecting the target bacteria by mass spectrometric analysis of the constituents of the disintegrated microbes.

2. The method of claim 1 , wherein the liquid nutrient medium is selective for the flagellated target bacteria.

3. The method of claim 1 , wherein the predetermined time is about 12 to 24 hours.

4. The method of claim 1 , wherein the constituents of the disintegration are ionized by matrix-assisted laser desorption.

5. The method of claim 4 , wherein the constituents of the disintegration are prepared with the matrix substance α-cyano-4-hydroxycinnamic acid (HCCA) on a sample support plate for the mass spectrometer.

6. The method according to claim 1 , wherein the target bacteria are of the genus Salmonella.

7. The method according to claim 1 , wherein the target bacteria are driven to the surface of the liquid nutrient medium by the addition of repellants and/or attractants.

8. The method according to claim 1 , wherein a further selective culture is performed if a mixture mass spectrum acquired in mass spectrometric analysis of step (e) is too densely populated with mass signals.

9. The method according to claim 8 , wherein the further selective culture is performed if more than 50 percent of the mixture mass spectrum is occupied by mass signals.

10. The method according to claim 1 , wherein the presence of the target bacteria in the microbe mixture is determined by the fact that all the mass signals of a reference mass spectrum that are definitely to be expected are also in fact present in the mixture mass spectrum acquired in mass spectrometric analysis of step (e).

11. The method of claim 1 , wherein the absence of the target bacteria in the microbe mixture is determined by the fact that mass signals of a reference mass spectrum that are definitely to be expected are not present in a mixture mass spectrum acquired in mass spectrometric analysis of step (e).

12. A method for detecting the presence of flagellated target bacteria in stool or food samples comprising microbes that may or may not comprise flagellated target bacteria, comprising the steps:
(a) introducing a stool or food sample into a liquid nutrient medium;
(b) incubating the liquid nutrient medium with the sample for a predetermined time for culturing the microbes;
(c) removing a sample of liquid from the surface of the liquid nutrient medium, wherein the liquid nutrient medium is not agitated at least during a period of time prior to removing the sample of liquid;
(d) centrifuging, washing and disintegrating the microbes from the removed sample of liquid without further isolation on agar plates; and
e) detecting the target bacteria by mass spectrometric analysis of the constituents of the disintegrated microbes.

Assignees

• Sparbier Katrin
• Kostrzewa Markus
• Bruker Daltonik Gmbh

Inventors

• Sparbier Katrin
• Weller Ulrich
• Kostrzewa Markus

Classifications

• C12Q1/04 Primary

  Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor {(C12Q1/6897 takes precedence)} · CPC title

• Y02A50/451

  Cross-Sectional Technologies · mapped topic

• G01N2560/00

  Chemical aspects of mass spectrometric analysis of biological material · CPC title

• Y02A50/30

  Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change · CPC title

• G01N33/6848

  Methods of protein analysis involving mass spectrometry · CPC title