Composition for increasing microorganism wall permeability and method for detecting said microorganisms on a membrane

Abstract

The present invention relates to a composition for permeabilizing microorganism walls, comprising the combination of polyethyleneimine (PEI) with at least one alcohol, and also to a method using said composition for counting and detecting in a targeted manner the microorganisms on a membrane. The invention also relates to a kit and to probes that are suitable for carrying out said method.

1 . Composition for permeabilizing microorganism walls, comprising the combination of polyethyleneimine (PEI) with at least one alcohol. 
     
     
         2 . The composition according to  claim 1 , wherein the concentration of polyethyleneimine in the composition is between 100 and 900 μg/ml. 
     
     
         3 . Composition according to  claim 1 , wherein the concentration of polyethyleneimine in the composition is between 200 and 800 μg/ml. 
     
     
         4 . Composition according to  claim 1 , wherein said alcohol is a primary alcohol. 
     
     
         5 . Composition according to  claim 1 , wherein said alcohol is ethanol. 
     
     
         6 . Method for counting and/or identifying on a membrane, the microorganisms initially present in a liquid or gaseous medium, comprising the following steps:
 (a) filtering said liquid or gaseous medium through a membrane so as to retain on or within the membrane the microorganisms present in this medium;   (b) contacting said membrane and the microorganisms with a permeabilizing composition comprising polyethyleneimine and at least one alcohol;   (c) fixing the cells to the membrane by means of a crosslinking agent;   (d) contacting said microorganisms with one or more optionally labeled macromolecules capable of crossing the microorganism wall; and   (e) detecting the macromolecules having penetrated into the microorganisms.   
     
     
         7 . Method according to  claim 6 , further comprising, between step a) and step b), an additional step consisting of the culturing of the microorganisms. 
     
     
         8 . Method according to  claim 6 , wherein said agent that serves as a crosslinking agent in step c) is selected from the group consisting of glutaraldehyde, formaldehyde and paraformaldehyde. 
     
     
         9 . Method according to  claim 6 , wherein in step d), the macromolecule used is a PNA-type probe. 
     
     
         10 . Method according to  claim 6 , wherein in step d), the macromolecule used is an oligonucleotide-type probe. 
     
     
         11 . Method according to  claim 9 , wherein the probe used in step d) comprises a sequence exhibiting at least 80% identity with SEQ ID No. 1, SEQ ID No. 2 or SEQ ID No. 3. 
     
     
         12 . Method according to  claim 6 , wherein in step d), the probes or the primers are labeled by coupling with an enzyme that allows the emission of a light signal. 
     
     
         13 . Method according to  claim 12 , wherein the detection of the microorganisms in step e) is carried out by means of a chemiluminescence reaction and recognition of the light signal emitted by means of an appropriate interface. 
     
     
         14 . Method according to  claim 6 , wherein in step d), the probes or the primers are labeled with a fluorescent molecule. 
     
     
         15 . Method according to  claim 14 , wherein the detection of the microorganisms in step e) is carried out by detecting a fluorescence emission signal, corresponding to the labeling of the probe or of the primer, by means of an appropriate interface. 
     
     
         16 . Method according to  claim 6 , further comprising, between step d) and step e), an additional step consisting of specific hybridization of the probes or primers with the nucleic acids of said microorganisms. 
     
     
         17 . Method according to  claim 6 , further comprising, between step d) and step e), an additional step consisting of specific amplification of the nucleic acids present in the microorganisms. 
     
     
         18 . Method according to  claim 6 , wherein the membrane on which the microorganisms are detected is selected from the group consisting of PVDF and Nylon®. 
     
     
         19 . Method according to  claim 6 , wherein the composition used in step b) comprises polyethyleneimine and at least one alcohol. 
     
     
         20 . (canceled) 
     
     
         21 . (canceled) 
     
     
         22 . (canceled) 
     
     
         23 . (canceled) 
     
     
         24 . (canceled) 
     
     
         25 . (canceled) 
     
     
         26 . Hybridization probe comprising a sequence exhibiting at least 80% identity with SEQ ID No. 1, SEQ ID No. 2 or SEQ ID No. 3. 
     
     
         27 . Kit for detecting and counting microorganisms, comprising:
 a membrane for filtering a liquid; and   a composition comprising the combination of polyethyleneimine and alcohol.   
     
     
         28 . Kit according to  claim 27 , wherein the concentration of polyethyleneimine in the composition is between 100 and 900 μg/ml. 
     
     
         29 . Kit according to  claim 27 , further comprising a composition comprising a crosslinking agent selected from the group consisting of glutaraldehyde, formaldehyde and paraformaldehyde. 
     
     
         30 . Kit according to  claim 27 , further comprising at least one probe for the specific detection of microorganisms. 
     
     
         31 . Kit according to  claim 30 , wherein said probe for the specific detection of the microorganism being sought comprises a sequence exhibiting at least 80% identity with SEQ ID No. 1, SEQ ID No. 2 or SEQ ID No. 3. 
     
     
         32 . Kit according to  claim 27 , wherein said membrane is selected from the group consisting of PVDF f and Nylon®.