Method for electrochemical detection of mycobacteria
The present invention relates to a novel process for biological detection of mycobacteria via electrochemical analysis methods of the catalytic activity of antigen 85.
1. A process for electrochemical detection of mycobacteria in a biological sample, said process comprising the steps of:
a) providing p-aminophyenyl-6-O-ocatanoyl-β-glucopyranoside as a substrate for acyltransferase Antigen 85 (Ag85) and a sugar selected from trehalose and D-glucose wherein said sugar is a cofactor for the acyltransferase;
b) bringing said biological sample into contact with said substrate and cofactor; and
c) electrochemically detecting the product resulting from the catalytic activity of said acyltransferase.
2. The process as claimed in claim 1 , wherein the biological sample is selected from bacterial cultures, biological specimens of human or animal origin, and environmental samples.
3. The process as claimed in claim 1 , wherein the electrochemical detection step c) is carried out by an amperometric sensor.
4. The process as claimed in claim 1 , wherein said mycobacteria has been isolated from the biological sample comprising the steps of:
A) placing said biological sample in solution;
B) contacting the solution obtained in step A) with an apolar solvent;
C) recovering the mycobacteria by membrane filtration or centrifugation of the solution resulting from step B); and
D) recovering the mycobacteria from the resultant filtrate or from the resultant centrifugation pellet obtained at the end of step C).
5. The process as claimed in claim 4 , further comprising a step of A′) decontaminating the biological sample placed in solution at the end of step A) and before step B), and/or a step C′) of decontaminating the membrane filter at the end of step C) and before step D).
6. The process as claimed in claim 5 , wherein step C′) is carried out by the addition of one or more of acidic solutions, basic solutions, sodium hypochlorite, or at least one disinfecting compound.
7. The process as claimed in claim 5 , further comprising a step C″) of rinsing the membrane filter at the end of step C′) and before step D).
8. The process as claimed in claim 7 , wherein the rinsing step C″) is carried out with a phosphate buffer.
9. A kit for carrying out the process for electrochemical detection of mycobacteria in a biological sample as defined in claim 1 , comprising:
i) a device and the reagents for collecting and preparing the biological sample to be tested;
ii) a device comprising p-aminophyenyl-6-O-ocatanoyl-β-glucopyranoside as a substrate of Ag85 and a sugar selected from trehalose and D-glucose wherein said sugar is a cofactor for Ag85; and
iii) a device for the electrochemical detection.
10. The kit as claimed in claim 9 , wherein the device for the electrochemical detection is an amperometric sensor.
11. The kit as claimed in claim 10 , wherein the amperometric sensor is a screen printed sensor.