METHOD FOR ELECTROCHEMICAL DETECTION OF MYCOBACTERIA
The subject of the present invention is a process or a method for detecting mycobacteria, which is based on measuring acyltransferase activity, in particular the catalytic activity of Antigen 85, with an electrochemical analysis method. The present invention is applicable in human and veterinary medicine, for the diagnostic of human and animal tuberculosis and mycobacteriosis, and also in environmental diagnosis. In the description below, the references between square brackets ([ ]) refer to the list of references presented at the end of the text. Mycobacteria belong to the phylum Actinobacteria and are characterized by a wall rich in mycolic acids giving them particular staining properties associated with resistance of their wall to successive decolorings by an acid and then by 90° alcohol (AAFB: Acid-Alcohol-Fast Bacilli). Approximately 200 species belonging to the Among them, mycobacteria referred to as tuberculous are necessary pathogenic bacteria with predominantly respiratory tropism, that are responsible for tuberculosis in human beings and animals. Non-tuberculous mycobacteria (termed atypical or environmental) group together opportunistic bacteria responsible for mycobacteriosis in human beings and animals. Leprosy is also a disease caused by a Human tuberculosis is mainly due to With regard to animals, tuberculosis affects a very large number of species: bovines, members of the goat family and also numerous wild species, such as small rodents, for example. Atypical mycobacteria, widely found in the environment (soil and water), exhibit a very variable pathogenicity in human beings and animals. In human beings, the incidence of infections associated with atypical mycobacteria appears to increase in industrialized countries. They generally occur where there is a background of local or general immunodepression causing mainly pulmonary infections (for example The detection of mycobacteria in human beings, animals or in the environment is based on the following techniques. The historical detection method is based on a microscopic examination of samples (sputum smear, ground material from lesions, etc.) which makes it possible to demonstrate the presence of acid-alcohol-fast bacilli (AAFBs), a partially specific characteristic of mycobacteria. In human beings, the microscopic examination is carried out on a biological sample smear or the centrifugation pellet obtained after fluidization-decontamination of contaminated pathological products. Two stainings are used: Ziehl-Neelsen staining (conventional microscopy) and auramine staining (fluorescence microscopy). It is a key examination since the majority of cases of tuberculosis in countries where there is a high incidence are diagnosed in this way in peripheral microscopy centers. Microscopic examination also remains the starting point for the diagnosis scheme adopted in diagnostic laboratories in developed countries. Microscopic examination is easy to implement (little material, personnel not highly qualified) with the result being provided rapidly (2 to 3 hours) and at low cost. However, this examination has several drawbacks: operator-dependent implementation, subjective interpretation of the result, lack of sensitivity (detection of 50% to 70% of pulmonary tuberculosis cases), and also a lack of identification of the species involved. The performance levels thereof are even lower in patients infected with HIV and children (specimens with few bacteria). With regard to the bacteriological detection of tuberculosis and of mycobacteriosis in human beings and animals, and also that of environmental contamination, the reference technique remains culture on a suitable medium (solid: Coletsos, Lowenstein-Jensen, Middlebrook 7H11 or liquid: Middlebrook 7H9) optionally supplemented with antibiotics and antifungals. The culturing of the bacterium starting from sputum, ground tissue matter, other biological samples or environmental specimens is commonly used and has the advantage of being sensitive. Automated liquid culture systems of Bactec MGIT™ (Becton Dickinson) or BacT/Alert® (BioMérieux) type combine incubation and spectroscopic measurement of bacterial growth. However, this method allows only a delayed diagnosis since, from a microbiological and culture point of view, tuberculous mycobacteria and some atypical mycobacteria are slow-growing microorganisms: at least 1 to 6 weeks of incubation at 37° C. are necessary in order to observe growth of the bacterium on the culture media. Whatever the nature of the sample, a prior decontamination treatment optionally combined with fluidization with physicochemical agents (N-acetylcysteine—sodium hydroxide, sodium hypochlorite, acids, detergents) is essential before it is cultured, in order to prevent the development of fast-growing microorganisms, reducing the sensitivity of the method. Following the culture, the identification of the species can be carried out by DNA hybridization techniques optionally coupled with PCR, gene sequencing, genotyping (insertion sequences, spoligotyping, etc.), by identification of biomarkers (analysis of mycolic acids by liquid-phase chromatography, protein profile by MALDI-TOF mass spectrometry for example). However, all these techniques require expensive laboratory equipment and highly qualified staff and are not therefore suitable for outsourced and rapid diagnosis of mycobacterial infections. Because of their rapidity (result provided during the day), molecular biology methods are also today widely used in diagnostic laboratories both for human beings and for animals and the environment. Based on the specific amplification of target mycobacterial genes, they allow both the detection of the bacterium and its identification, or even its possible resistance to antibiotics (human diagnosis only). However, their use generally requires suitable infrastructure, expensive equipment and also qualified staff (DNA extraction and interpretation of the results). The GeneXpert® technology developed by the Cepheid laboratory for the molecular diagnosis of human tuberculosis limits the drawbacks mentioned above by virtue of the use of an automated device which carries out all the steps without human intervention. The result obtained in 2 hours makes it possible to detect the presence of a tuberculous mycobacterium and also its potential resistance to rifampicin, a frontline antibiotic used in the treatment of human tuberculosis. However, its price constitutes a curb on its generalization in countries with low revenues. Furthermore, this technology does not make it possible to carry out more than about twenty analyses per day. In animals, the diagnosis of tuberculosis is carried out post-mortem after prophylactic screening or a discovery of lesions in the abattoir. It is carried out on ground material from lesions or from lymph nodes. As in human beings, the samples can be cultured after decontamination and/or can be analyzed by molecular biology methods. The limits of this detection are identical to those mentioned above: delayed production of the result with culture and expensive molecular biology automated devices with qualified staff. Regarding the detection of mycobacteria in the environment, the search for said mycobacteria is not standardized and no standard is currently available. Culture on a suitable medium after chemical decontamination comes up against the same limits as that of biological samples: slow growth with delayed production of the result and contamination of the culture media by fast-growing bacteria in particular. Molecular biology has made it possible to bypass this culture step, but still does not allow suitable quantification. With a view to proposing new methods of identifying mycobacteria, the detection of specific antigens, such as Ag MPT64 or those of the Antigen 85 (Ag85) complex have been envisioned. To do this, an immunochromatographic test based on the identification of Ag MPT64 after culture (present in tuberculous mycobacteria, absent in atypical mycobacteria) has been sold (SD Bioline TB Ag MPT64, Standard Diagnostic, Inc.). ELISA assays used for the detection of Ag85 in liquid culture filtrates, serum or in cerebrospinal fluid (Phunpae et al., Diagn. Microbiol. Infect. Dis., 78(3): 242-248, 2014; Kashyap et al., BMC infectious diseases, 7:74, 2007; Kashyap et al., Clin Diagn Lab Immunol., 12(6):752-758, 2005) are described in the literature. The Antigen 85 (Ag85) complex is composed of three secreted homologous proteins: Antigen 85A (Ag85A), Antigen 85B (Ag85B), and Antigen 85C (85C) which share a high sequence identity (68-79%) in their secreted mature forms. They are mycolyltransferases (enzymes having a molecular weight of approximately 30 000 Da) which are involved specifically in the construction and maintenance of the walls of Corynebacteriales—order to which the The detection and the activity of Ag85 being widely studied for the search for and evaluation of new methods of diagnosis and of monitoring of the efficacy of antitubercular chemotherapy treatments (Elamin et al., J Microbiol Methods, 79(3):672-678, 2002) several spectrophotometric methods (UV-visible, fluorescence, etc.) have been described for the assaying of this protein via the measurement of its acyltransferase activity (Boucau et al., Analytical Biochem., 385: 120-127, 2009; Favrot et al., J. Biol. Chem., 289(36): 25031-25040, 2014). International application WO 2011/030160 describes a method for detecting the presence of mycobacteria in an organism or a biological sample via the demonstration of the catalytic activity of Ag85 during the culture step. To do this, molecular probes consisting of a labelled polysaccharide (trehalose and other saccharide derivatives) (radiotracer, fluorophore, nanoparticles, biotin) have been synthesized and added to the culture medium in order to be incorporated into the bacterial wall during bacterial growth by virtue of the transferase activity of Ag85. At the end of this step, the bacteria are rinsed and isolated from the culture medium and then detected using a suitable technique (scintillation counter, fluorimeter, microscopy, NMR, in vivo imaging techniques, etc.). However, this method, which allows the detection of viable mycobacteria by labeling them, can only be envisaged for the analysis of very contaminated samples (about 107bacilli·ml−1) or after quite a long culture step. Furthermore, several steps of rinsing the bacteria are obligatory in order to remove the excess labeled probe not incorporated. Finally, the detection of the marker is carried out using delicate and expensive laboratory instrumental techniques which require qualified staff to implement them and to interpret the results. Patent application CN102087283 describes a method of electrochemical detection of There is thus a need for a method for detecting mycobacteria that is simple and rapid to carry out and that overcomes the drawbacks of the processes of the prior art. In order to meet this need for a more effective diagnostic test for human, animal and/or environmental tuberculosis for which at the current time there is no suitable solution, the inventors have developed a new electrochemical method capable of rapidly detecting (obtaining the results in approximately 2 to 5 h) the presence or absence of mycobacteria in samples such as, for example, in a culture medium and in human respiratory specimens: the EDMYC (Electrochemical Detection of MYCobacteria) method. Thus, the inventors have developed a new method for electrochemical detection of mycobacteria via the electrochemical measurement of the acyltransferase activity in the mycobacteria, in particular of the catalytic activity of Ag85 in the presence of a substrate of the enzyme, e.g. p-aminophenyl-6-O-octanoyl-β-D-glucopyranoside (p-AP-OG), and of a cofactor or co-substrate, e.g. trehalose. Indeed, since Ag85 is very widely excreted by mycobacteria, e.g. by The principle of the invention is based on the fact that acyltransferases such as Ag85 hydrolyze the ester bond of the substrate, and transfer the acyl group thus released onto the cofactor. The product is then detected by voltammetry. According to one particular embodiment, the present invention is based on the capacity of acyltransferases, in particular of Ag85, to hydrolyze the ester bond of p-AP-OG and to transfer the octanoyl group of p-AP-OG onto a sugar, e.g. trehalose, according to a ping-pong mechanism, in order, respectively, to generate p-aminophenyl-β-D-glucopyranoside (p-AP-G) and to form acyltrehalose ( Thus, the inventors have developed a simple and rapid method for detecting mycobacteria and their viability with or without a prior culture step. To date, the proof of concept of the method has been successfully demonstrated with the detection of several mycobacterial species frequently encountered in pulmonary infections, including In addition, the inventors have demonstrated an improvement in the specificity of the detection method with respect to mycobacteria and Ag85 by proposing 1) the use of a substrate of the enzyme with acyl groups having carbon chains longer than that of p-AP-OG, for example alkyl chains ranging from C7H15to C29H59, and/or 2) a method for extracting and decontaminating actual samples in order to isolate the mycobacteria. A subject of the present invention is thus a process for electrochemical detection of mycobacteria in a biological sample, said process comprising the steps of:
According to one particular embodiment of the detection process of the present invention, the biological sample is chosen from the group consisting of: bacterial cultures, biological specimens of human or animal origin, environmental samples, etc. A bacterial culture may for example be obtained on a nutritive agar or in a liquid culture medium, according to techniques well known to those skilled in the art. A biological specimen of human origin may for example be a sample of pulmonary origin (sputum, bronchial secretions, biopsy), a blood sample, a cerebrospinal fluid sample, a urine sample, a sample of intestinal origin (intestinal biopsy, feces), and also any other tissue sample. A biological specimen of animal origin may for example be a tissue sample (lymph node, lung, liver, spleen, etc.), a sample of feces or a milk sample. A sample of environmental origin may for example be a sample of waste water, or of hospital waste water, a sample of treated waste water, a sample of sludge resulting from the treatment of waste water or a soil sample. According to one particular embodiment of the detection process of the present invention, the acyltransferase substrate is chosen from the group consisting of: p-aminophenyl-6-O-octanoyl-β-D-glucopyranoside, and substrates with acyl groups having alkyl chains ranging from C7H15to C29H59. According to one particular embodiment of the detection process of the present invention, the cofactor is a sugar chosen from the group consisting of: trehalose, D-glucose. Preferably, the substrate is p-aminophenyl-6-O-octanoyl-β-D-glucopyranoside, and the cofactor is trehalose. The product formed after enzymatic hydrolysis by the acyltransferases, in particular by Ag85, is p-aminophenyl-β-D-glucopyranoside. According to one particular embodiment of the detection process of the present invention, the electrochemical detection step c) is carried out by means of an amperometric sensor, which is optionally chemically modified (e.g. with carbon nanotubes, graphene). Preferably, said sensor is a screen-printed sensor, which is preferentially single-use. In accordance with the invention, an electrochemical analysis means for carrying out the invention can be a potentiometric measurement, an impedance measurement, a coulometric measurement or an amperometric measurement. According to one advantageous embodiment of the detection process of the present invention, the electrochemical analysis is carried out by an amperometric measurement. For the purposes of the present invention, the term “amperometric measurement” is intended to mean a measurement of the electric current as a function of a potential difference established between the working electrode and the reference electrode. The measurement of the electric current can be carried out by means of known amperometric techniques, preferentially by potential sweep voltammetry which may be linear, cyclic, or pulse voltammetry or else of the potential step type, such as chronoamperometry. In one particularly advantageous embodiment of the detection process of the present invention, the presence of p-aminophenyl-β-D-glucopyranoside is measured by cyclic or linear voltammetry. The use of these techniques requires an assembly which may be a two-electrode or even three-electrode assembly, that is to say an assembly comprising a working electrode, a reference electrode and optionally an auxiliary electrode (counter electrode). The working electrode, the surface of which serves as a site for electron transfer, can be based on carbon or based on a noble metal or else based on metal oxide. The reference electrode is an electrode of which the potential is constant, which makes it possible to impose a precisely defined potential on the working electrode. The reference electrode may be an Ag/AgCl electrode. The counter electrode, which makes it possible to establish the passage of the electric current with the working electrode, can be fabricated with an inert material, such as platinum or carbon. Those skilled in the art will know how to choose and combine the appropriate electrodes according to their general knowledge. With regard to the method of manufacturing the electrodes, the screen-printing technique is preferable, although other methods of industrial fabrication, such as rotagravure, inkjet printing, 3D printing or optionally photolithography, can be envisioned. Electrodes obtained by screenprinting are particularly well suited because they can be produced in bulk at low cost, and thus can optionally be single-use. Furthermore, their geometric shape and also their size can be easily modulated. These electrodes can be screenprinted in the form of a sensor and optionally integrated into the bottom of the wells of a microplate or of other supports or systems allowing the filtration of the bacterial suspensions and the incubation of p-aminophenyl-6-O-octanoyl-β-D-glucopyranoside and of trehalose. According to one particular embodiment of the detection process of the present invention, the amperometric measurement is carried out with a screen-printed sensor. It makes it possible to perform the measurement in a small volume of solution of about a few microliters. According to one particular embodiment of the detection process of the present invention, the amperometric measurement is carried out with a device involved three electrodes: an Ag/AgCl reference electrode, a carbon working electrode and a carbon counter electrode. According to another particular embodiment of the detection process of the present invention, the amperometric measurement is carried out with a screen-printed sensor comprising an Ag/AgCl reference electrode, a carbon working electrode and a carbon counter electrode. The presence of p-aminophenyl-β-D-glucopyranoside is indicated by the presence of an anodic oxidation current in an interval of potentials and the absence of said current for a control devoid of hydrolyzed p-aminophenyl-6-O-octanoyl-β-D-glucopyranoside. When the p-aminophenyl-β-D-glucopyranoside is subjected to a measurement by cyclic voltammetry, its presence is indicated by an anodic oxidation current peak specific to p-aminophenyl-β-D-glucopyranoside in a determined interval of potentials (+0.3 to +0.5 V vs. Ag/AgCl). Preferably, the biological sample to be tested is prepared so as to isolate the mycobacteria that it contains while at the same time eliminating a maximum amount of contaminants before the contacting and detection steps. To do this, an extraction step with an apolar solvent such as, for example, hexane is necessary in order to selectively isolate the mycobacteria—the wall of which is very hydrophobic—from the sample previously placed in solution in a fluidizing agent such as N-acetylcysteine for a respiratory specimen or in a phosphate buffer with a neutral pH for a soil sample, for example. In the case of complex samples such as soil, where millions of different bacterial species can coexist, the extraction step can be followed by a decontamination of the extract with, for example, an acid (HCl, H2SO4) and/or a base (NaOH) or a quaternary ammonium. Thus, a subject of the present invention is also a process for isolating mycobacteria from a biological sample, said process comprising the steps of:
According to one particular embodiment of the process for isolating mycobacteria of the present invention, the extraction step b) is carried out with a solution of hexane or a hexane-isopropanol mixture. According to one particular embodiment of the process for isolating mycobacteria of the present invention, the process can also comprise a step of decontaminating a′) the biological sample placed in solution at the end of step a) and before step b), and/or a step of decontaminating c′) the filtering membrane at the end of step c) and before step d), with acidic solutions (e.g. solution of hydrochloric acid) and/or basic solutions (e.g. solution of sodium hydroxide or of quaternary ammonium), and/or addition of sodium hypochlorite, and/or with at least one other disinfecting compound (e.g. chlorhexidine or squalamine). According to one particular embodiment of the process for isolating mycobacteria of the present invention, step c′) can be followed, before step d), by a rinsing step c″), for example in the presence of phosphate buffer, in order to remove the sodium hypochlorite (bleach) from the filter (preferably made of Teflon). According to one particular embodiment of the process for isolating mycobacteria of the present invention, step d) of recovering the mycobacteria is carried out by scraping the filter with a loop (wire loop) in order to detach the cells from the filter. The mycobacteria thus recovered are then cultured in a suitable medium (enriched and supplemented medium 7H11), for approximately two months, at 37° C., in order to allow counting thereof. A subject of the present invention is also a kit for carrying out the process for electrochemical detection of mycobacteria in a biological sample according to the present invention, said kit comprising:
For the purposes of the present invention, the term “device” of step a) is intended to mean a sealed container, which is preferably single-use, for example a single-use tube or column equipped with a filtration system in which the steps of diluting the sample, extracting, decontaminating if necessary and recovering the mycobacteria are carried out. For the purposes of the present invention, the term “device” of step b) is intended to mean a sealed container, which is preferably single-use, for example a single-use tube equipped with a filtration system in which the incubation of the mycobacteria with the substrate and the co-substrate is carried out. For the purposes of the present invention, the expression “device for the electrochemical detection” of step c) is intended to mean for example an amperometric sensor, which is preferably screenprinted and single-use, for example those sold by the companies Dropsens and Palmsens. The amperometric sensor can optionally be integrated into the device of step b. By way of example of a suitable reader, mention may be made of portable readers based on the principle of the blood glucose reader, for example those sold by the companies Dropsens and Palmsens and which make it possible to carry out the measurements in a few seconds. 1.1. Reagents and solutions
1.2. Strains and Culture Media
With the aim of promoting the growth of the mycobacteria, the liquid and solid culture media were enriched with 10% of a mixture consisting of oleic acid, albumin, dextrose and catalase (OADC; Table 1). The 7H11 medium was also supplemented with 10% of heat-inactivated bovine serum. Oleic acid and long-chain fatty acids are essential for mycobacteria metabolism. Dextrose is an energy source. Catalase allows neutralization of peroxides, which can be toxic to bacteria. Albumin plays a protective role against toxic agents. 1.3. Sensors and Measurement Apparatus The electrochemical measurements were carried out by linear voltammetry (v=50 mV·s−1) with a 910 PSTAT mini potentiostat (Metrohm, France) powered through the USB connection of the computer and controlled by the PSTAT software (version 1.0). To do this, drops of solution of 30-50 μl were deposited on the surface of single-use screen-printed carbon sensors supplied by Dropsens (DRP-110) and connected to the potentiostat via the connector (DRP-DSC). The amperometric detection of the product generated during the reaction catalyzed by Ag85 was always carried out after a step of filtration of the reaction mixture with a filtration device (Microcon 10 kDa, Millipore). All the potentials are measured relative to the Ag/AgCl reference electrode. 1.4. Detection of Ag85 in the Supernatant of a Liquid Culture of The principle of this 7-step protocol is shown schematically in 1.5. Detection of Ag85 in the Bacterial Pellet Obtained from the Liquid Cultures of Volumes of 1 ml of liquid culture of each mycobacterial species (˜106bacilli·ml−1) were centrifuged at 6 000×g for 6 minutes. Once the supernatant had been removed, the bacterial pellets were rinsed with 1 ml of PBS and then centrifuged at 6 000×g for 6 minutes. After removal of the supernatant, 10 μl of p-AP-OG at 2×10−3M, and 10 μl of trehalose at 5×10−3M are added to the bacterial pellets. After a step of incubation for 4 hours at 37° C. with shaking, the electrochemical measurement of the product of the enzymatic reaction was carried out according to the protocol described in section 1.3. A negative control (enriched 7H9 medium without bacteria) was analyzed in duplicate in the same way. 1.6. Detection of Ag85 in 1.6.1. The Liquid Culture Two samples of 7.5 ml of liquid culture of Analysis of the Bacterial Pellet: Each bacterial pellet was rinsed by adding 100 μl of PBS, then the liquid was removed by turning the tube upside down on an absorbent paper (Whatman). 20 μl of p-AP-OG at 2×10−3M and 20 μl of trehalose at 5×10−3M were added to the tubes and incubated for 4 hours at 37° C. without shaking. Negative controls (PBS) were analyzed in duplicate in the same way. The electrochemical measurement of the product of the enzymatic reaction was carried out according to the protocol described in section 1.3. Analysis of the Supernatant: Six ml of each culture supernatant were centrifuged at 7 000×g for 20 min in an Amicon 50 kDa filtration device (Millipore). The filtered liquid was transferred into an Amicon 10 kDa filtration device and then centrifuged at 7 000×g for 20 min. The filter was then rinsed with 1 ml of PBS and then centrifuged at 7 000×g for 20 min. The volume of residual liquid remaining on the filter was transferred into a tube to which 30 μl of p-AP-OG at 2×10−3M and 30 μl of trehalose at 5×10−3M were added. The reaction mixture was incubated for 4 hours at 37° C. without shaking. A negative control (enriched 7H9 medium without bacteria) was analyzed in duplicate in the same way. The electrochemical measurement of the product of the enzymatic reaction was carried out according to the protocol described in section 1.3. 1.6.2. The Isolated Colonies A few colonies taken from a 7H11 agar were deposited in a 2 ml tube. The colonies were rinsed with PBS, the tubes were centrifuged and the supernatant was removed. 20 μl of p-AP-OG at 2×10−3M and 20 μl of trehalose at 5×10−3M were added to the tubes and incubated for 4 hours at 37° C. without shaking. A negative control (PBS) was analyzed in duplicate in the same way. The electrochemical measurement of the product of the enzymatic reaction was carried out according to the protocol described in section 1.3. 1.7. Extraction and Detection of Respiratory specimens (sputum, tracheal aspiration and bronchial aspiration products) from nontuberculous patients were supplied by the CHU [University hospital center] of Dijon. The samples were fluidized beforehand by the CHU of Dijon with the Digest-EUR kit (Eurobio). Volumes of 1 ml of respiratory sample were dispensed into sterile 15 ml tubes and then incubated with 200 μl of a liquid culture of Extraction of 9 ml of hexane-isopropanol mixture (3:2, v/v) were added to each respiratory specimen tube and stirred for 1 minute. After a centrifugation step at 3 000×g for 2 minutes, the supernatant (that is to say the hexane, and the interface) was removed and then vacuum-filtered on a membrane (Durapore, 25 mm; 0.45 μM). Once rinsed with PBS, the membrane was placed in a small polyethylene bag with welded zip closure having the dimensions of the membrane. Electrochemical Detection of A volume of 100 μl of the mixture of substrate at 2×10−3M and trehalose at 5×10−3M, prepared in PBS, was introduced into the bag before it was closed. After an incubation step at 37° C. for 4 hours, the electrochemical measurement of the product of the enzymatic reaction was carried out according to the protocol described in section 1.3. 1.8. Extraction and Detection of Extraction of The soil microcosms were prepared from a clay-loam soil with pH 7.75, autoclaved twice for 15 min at 121° C. with an interval of 48 h, in order to get rid of the endogenous microflora. Microcosms of 5 g of sterile soil were prepared in 45 ml Falcon tubes, then inoculated with Decontamination of the Extract and Counting This step is very useful, or even essential, for removing the interfering edaphic microbial flora from the soil. To do this, the decontamination protocol combined an acid decontamination (addition of 100 μl of 4% hydrochloric acid, incubation for 20 min) with an estimated pH of 1.3 and a basic decontamination (addition of 200 μl of 4% sodium hydroxide, incubation for 20 min) with an estimated pH of 12.6, then the mixture was neutralized by adding 100 μl of hydrochloric acid before the addition of 1 ml of mixture of sodium hypochlorite and sodium hydroxide. After incubation for 15 minutes, the supernatant was removed, deposited on a new Teflon filtering membrane and rinsed with 20 ml of 0.1 M phosphate buffer in order to remove the bleach. The membrane was then placed in a new pill bottle containing 1 ml of enriched 7H9 and scraped with a loop (wire loop) in order to detach the cells from the filter. For the purpose of counting the mycobacteria extracted, 100 μl of each final suspension were inoculated in triplicate on enriched and supplemented 7H11, and incubated for two months at 37° C. 2.1. Voltammetric Behavior of p-AP-OG and of p-AP-G The voltammograms presented in Indeed, as shown by the scheme in 2.2. Electrochemical Detection of the Acyltransferase Activity of the Ag85 Protein In order to demonstrate the acyltransferase activity of Ag85 in the presence of p-AP-OG and of an acyl group accepter, the reaction mixture containing the Ag85 protein, p-AP-OG and trehalose was incubated in the presence and absence of p-AP-G with stirring for 4 hours at 37° C. A negative control containing only p-AP-OG was analyzed in parallel under the same conditions. As shown by the series of voltammograms presented in A supplementary study (results not presented) showed that the value of the p-AP-OG oxidation peak potential increases as a function of the chloride ion concentration in the solution. Moreover, the information regarding the commercially available Ag85 indicates that the protein was lyophilized from a buffer containing 0.1 M NaCl. Thus, the higher values of the oxidation peak potentials of p-AP-OG and of p-AP-G recorded in the presence of Ag85 are probably due to that of the chlorides in the solution. In order to validate this hypothesis, Ag85 was also incubated in the presence of trehalose and of a mixture of p-AP-OG and p-AP-G. The comparison of the voltammograms (curve as a continuous line and dotted curve) of 2.3. Electrochemical Detection of the Acyltransferase Activity of Ag85 in a Liquid Culture of For the purpose of applying the method for electrochemically detecting Ag85 in order to demonstrate the growth of mycobacteria in a liquid culture medium, the analysis of the supernatant of a culture of 2.3.1. Analysis of the Supernatant 1. It has been demonstrated that Ag85 is a major secretion product of 2.3.2. Analysis of the Bacterial Pellet Since Ag85 is also involved in the repair and construction of the wall of mycobacteria, the protocol represented schematically in The voltammogram (curve as a line ---) presented in Thus, the amperometric response of p-AP-G measured during the analysis of the bacterial pellet of 2.4. Electrochemical Detection of Several Species of Mycobacteria in Liquid Cultures The proof of concept of the electrochemical detection of mycobacteria in the presence of p-AP-OG and trehalose was carried out for the analysis of several species of mycobacteria that can be handled in an L2 containment laboratory. The species selected are those frequently found in respiratory specimens: 2.5. Detection of The electrochemical detection of With an oxidation peak specific for the p-AP-OG hydrolysis product, the series of voltammograms presented in Moreover, the p-AP-G oxidation peaks recorded for the analysis of the bacterial cells ( 2.6. Extraction and Detection of In order to propose a method for direct (that is to say without prior culture) electrochemical detection of mycobacteria in samples of pulmonary origin (sputum, tracheal aspiration and bronchial aspiration products), specimens from nontuberculous patients were inoculated with a known amount of Since the commercially available fluidization-decontamination methods that are of use for the preparation of the samples—which combine N-acetylcysteine and sodium hydroxide—are not compatible with electrochemical detection (poorly defined signals), the development of a protocol for extracting the mycobacteria from specimens of pulmonary origin was envisioned. The method proposed involves a hexane-isopropanol mixture as extraction solvent. By precipitating the constituents of the respiratory specimen, isopropanol makes it possible to get rid of the viscous nature of the specimen, while the apolar solvent, which hexane is, selectively extracts the mycobacteria, the wall of which is very hydrophobic. Once recovered by filtration, the mycobacteria were incubated with the substrate/co-substrate mixture for the purpose of carrying out the electrochemical detection of the acyltransferase activity (Ag85 and other enzymes present in the mycobacterial cell). The voltammograms presented in 2.7. Extraction and Detection of In order to evaluate the impact of the volume of hexane on the extraction yield, sterile soil (5 g) was inoculated with the The particular features of Although the method for extraction-decontamination of the mycobacteria in soil of the invention makes it possible to recover only approximately 2.5% of the bacteria inoculated into sterile soil, this yield is much higher than that described in the literature for the analysis of naturally contaminated environmental samples with a magnetic immunocapture process (0.1%) (Sweeney et al., Lett. Appl Microbiol., 43(4): 364-369, 2006; Sweeney et al., Appl Environ Microbiol., 73(22): 7471-7473, 2007) [10, 11]. 2.8. Quantitative Aspects of the Method In order to take into account all of the random errors during the implementation of the protocols, p-AP-OG was chosen as an internal standard and the analytical response of the method was defined as the ratio of the intensity of the p-AP-G oxidation peak to the intensity of the p-AP-OG oxidation peak: The parameters ip-AP-OGand ip-AP-Ghave been defined on the voltammogram presented in As shown by the values of R collated in table 2 below, the electrochemical method proposed by the inventors made it possible to detect amounts of Finally, a first repeatability study was carried out for the analysis of the pellet of a culture of 2.9. Application of the Method of Electrochemical Detection of the Invention to the Monitoring of 2.9.1. Comparison of the Time to Positivity of a Liquid Culture of To do this, three volumes of medium (volumes, A, B and C) were prepared for testing the detection method described in the invention (Table 3). An The tubes incubated in the BACTEC™ automated device were subjected to an automatic measurement of the fluorescence once an hour. The time to positivity was expressed in days. The tubes were analyzed daily by the method of the invention, by taking a volume of 30 μl of the culture and depositing it at the surface of a screen-printed sensor without prior treatment. The measurements were carried out by linear voltammetry and the positivity of the sample corresponds to the appearance of a p-AP-G oxidation peak at around ˜+0.50 V vs. Ag/AgCl. Since the electrochemical measurements were not carried out continuously (once a day in the best of cases), there is for the moment an uncertainty about the exact moment at which the positivity appears, hence the expression of the results in the form of ≤x days. The results of table 4 indicate that the electrochemical method made it possible to monitor the culture of 2.9.2. Comparison of the Time to Positivity of Four Respiratory Samples Inoculated or not Inoculated with Four respiratory samples were supplied by the CHU Dijon (samples 1 and 2: fibroscopy, sample 3: bronchoalveolar lavage and sample 4: sputum). For each sample, a 3 ml aliquot was contaminated with 500 μl of The tubes incubated in the BACTEC™ automated device were subjected to an automatic measurement of the fluorescence once an hour. The time to positivity was expressed in days. The cultures carried out in the volumes of medium A (table 3) were incubated at 37° C. and analyzed with the electrochemical method of the invention. The electrochemical measurements and the interpretation thereof were carried out as in section 2.9.1. above. The results collated in table 5 show that the electrochemical method was as effective as BACTEC™ for the detection in liquid culture of respiratory samples artificially contaminated with Finally, by combining the results of tables 4 and 5, the electrochemical method of the invention is capable of demonstrating more rapidly the growth of 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) selecting a substrate of at least one acyltransferase and its cofactor; b) bringing said biological sample into contact with said substrate and cofactor; c) electrochemically detecting the product resulting from the catalytic activity of said at least one acyltransferase. 2) The process as claimed in 3) The process as claimed in 4) The process as claimed in 5) The process as claimed in 6) The process as claimed in 7) The process as claimed in A. placing said biological sample in solution; B. treating with an apolar solvent the solution obtained in step A); C. recovering the mycobacteria by filtration or centrifugation of the solution resulting from step B); and D. recovering the mycobacteria from the filtrate or from the centrifugation pellet obtained at the end of step C). 8) The process as claimed in 9) The process as claimed in 10) The process as claimed in 11) The process as claimed in 12) A kit for carrying out the process for electrochemical detection of mycobacteria in a biological sample as defined in i. a device and the reagents for collecting and preparing the biological sample to be tested; ii. a device comprising a substrate of Ag85 and its cofactor for the incubation with Ag85; iii. a device for the electrochemical detection by means of a suitable reader. 13) The kit as claimed in FIELD OF THE INVENTION
PRIOR ART
DESCRIPTION OF THE INVENTION
BRIEF DESCRIPTION OF THE FIGURES
EXAMPLES
Example 1: Materials and Methods
Composition of the OADC enrichment Components Dextrose 20.0 g Bovine albumin 50.0 g Oleic acid* 0.6 Catalase* 0.003 g Water 1 l Example 2: Results
Standardized analytical responses R (R0corresponds to the analytical response registered for the culture medium without bacteria) calculated from the voltammograms recorded for the analysis of according to the protocol described in section 1.4. R/R0 0 1 102 2.9 103 4.5 104 7.4 105 12.2 106 40 Volumes of the various reagents used to prepare the culture media Electrochemical detection Reagents BACTEC ™ Volume A Volume B Volume C BD BACTEC ™ 7 ml 3.5 ml 2 ml 1 ml MGIT ™ medium Growth 0.8 ml 0.4 ml 230 μl 115 μl supplement and BBL MGIT ™ PANTA p-AP-OG 100 μl 57 μl 29 μl 2 × 10−2M Trehalose 100 μl 57 μl 29 μl 5 × 10−1M Final volume 7.8 ml 4.1 ml 2.344 ml 1.173 ml Times to positivity obtained with the BACTEC ™ automated device and with the electrochemical method during the incubation of control tubes and of tubes inoculated with (1 to 2 repetitions per sample) Electrochemical detection (time in BACTEC ™ days) Samples (time in days) Volume A Volume B Volume C Control 1 Negative Negative Negative Negative Control 2 Negative Negative −2 6.09 ≤7 ≤7 ≤4 −2 6.09 ≤7 ≤7 ≤3 −1 4.2 ≤4 ≤3 ≤2 −1 4.21 ≤4 ≤3 ≤2 Time to positivity of four respiratory samples that were contaminated or not inoculated (control) in MGIT tubes obtained with the electrochemical method and the BACTEC automated device Electrochemical BACTEC ™ detection Respiratory (time in days) (time in days) samples Control Inoculated Control Inoculated Sample 1 — 9.15 — ≤7 Sample 2 — 4.16 — ≤5 Sample 3 — 5.07 — ≤5 Sample 4 — 5.20 — ≤5 LIST OF REFERENCES








