Method for detecting microorganisms on a membrane
View Patent ↗The present invention relates to a method using a composition for permeabilizing microorganism walls 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 the method.
1. Method for counting and/or identifying on a membrane gram-negative bacteria 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 bacteria present in this medium; (b) contacting said membrane and said bacteria with a permeabilizing composition comprising polyethyleneimine and ethanol; (c) fixing said bacteria to the membrane by means of a crosslinking agent; (d) contacting said bacteria with one or more macromolecules capable of crossing the bacterial walls of gram-negative bacteria fixed to the membrane in step (c), said macromolecules being labeled with a member selected from the group consisting of an enzyme that allows the emission of a light signal and a fluorescent molecule; (e) hybridizing said one or more macromolecules with the nucleic acids of said gram-negative bacteria; (f) detecting the one or more macromolecules having penetrated into said gram-negative bacteria by causing a light signal or fluorescence signal to be generated and detecting said signal; and (g) counting and/or identifying the gram-negative bacteria initially present in said liquid or gaseous medium.
2. Method according to claim 1 , further comprising, between step a) and step b), an additional step consisting of the culturing of said gram-negative bacteria.
3. Method according to claim 1 , wherein said crosslinking agent in step c) is selected from the group consisting of glutaraldehyde, formaldehyde and paraformaldehyde.
4. Method according to claim 1 , wherein in step d), the one or more macromolecules is a PNA-type probe.
5. Method according to claim 4 , 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.
6. Method according to claim 5 , wherein the membrane is selected from the group consisting of PVDF and Nylon®.
7. Method according to claim 1 , wherein in step d), the one or more macromolecules is an oligonucleotide-type probe.
8. Method according to claim 1 , wherein said one or more macromolecules comprise probes or primers, and wherein in step d), the probes or the primers are labeled by coupling with an enzyme that allows the emission of a light signal.
9. Method according to claim 8 , wherein the detection of the one or more macromolecules in step (f) is carried out by means of a chemiluminescence reaction and recognition of the light signal emitted by means of a video interface.
10. Method according to claim 1 , wherein said one or more macromolecules comprise probes or primers, and wherein in step d), the probes or the primers are labeled with a fluorescent molecule.
11. Method according to claim 10 , wherein the detection of the macromolecules in step (f) is carried out by detecting a fluorescence emission signal, corresponding to the labeling of the probes or of the primers, by means of a video interface.
12. Method according to claim 1 , further comprising, between step d) and step e), an additional step consisting of amplification of nucleic acids present in said gram-negative bacteria.