WO2013135933A1

WO2013135933A1 - Method for the label-free identification of viruses and bacteria, using compact disc technology

Info

Publication number: WO2013135933A1
Application number: PCT/ES2013/070158
Authority: WO
Inventors: Ángel MAQUIEIRA CATALÀ; Sergi Beñat MORAIS EZQUERRO; Rosa PUCHADES PLÀ; Luís Antonio TORTAJADA GENARO
Original assignee: Universitat Politècnica De València
Priority date: 2012-03-13
Filing date: 2013-03-12
Publication date: 2013-09-19
Also published as: ES2425714B1; ES2425714A1

Abstract

The invention relates to a rapid analysis method for the label-free identification and detection of Gram + and Gram - bacteria and/or viruses that may or may not be antibiotic resistant, using audio-video discs (CD, DVD, BD, Lightscribe, Superdisk, etc.) as a support for immobilising specific molecular receptors of biomolecules present on the surface of the bacteria and/or viruses, using a disc player/recorder (CD, DVD, BD, Lightscribe, etc.) as a detector. The invention comprises three main processes: resuspension of the viruses and/or bacteria, incubation on the disc, and label-free detection using a compact disc player/recorder (CD, DVD, BD, etc.). The invention has been designed to identify receptors or bioreceptors immobilised separately on the polycarbonate face of the disc in known ordered positions, forming a micro-array. The specific bioreceptors of biomolecules present on the surface the viruses and/or bacteria can be, for example, proteins, lipids and nucleic acids.

Description

METHOD FOR IDENTIFICATION OF BACTERIA AND VIRUSES WITHOUT MARKING BASED ON COMPACT DISC TECHNOLOGY

DESCRIPTION

SECTOR OF THE TECHNIQUE

The present invention focuses on the biotechnology sector, specifically in the analysis for the identification and detection of bacteria and / or viruses without marking. The main applications of the invention occur in epidemiological surveillance, quality control of water and air, and in general any area that involves the detection of bacteria by biospecific interactions based on the use of separate and ordered molecular receptors on a support.

STATE OF THE PREVIOUS TECHNIQUE

The technology of detection and / or identification of bacteria and viruses using optical discs as supports, and disc readers / recorders as detectors is known in the state of the art.

ES2288429B2 (Polytechnic University of Valencia, 2006) describes a method of chemical modification of a compact disk, intended as a support for the covalent immobilization of biomolecules, while maintaining the optical and mechanical properties of the compact disk. The covalent immobilization of the biomolecules is carried out in an established order and arrangement constituting an array or microarray on the polymer surface of the compact disk. The covalent bonds of the method described in ES2288429B2, in most cases are irreversible. The covalent bond is more durable over time compared to electrostatic interactions as well as hydrophilic / hydrophobic interactions, allowing the type of binding of biomolecules to the polymeric surface to be stronger to resist Better the washes. Biomolecules are labeled for direct or indirect detection by conjugation with a fluorochrome or with an enzyme.

EP1324042A2 (José Reclame, 2001) describes a method for the detection and / or quantification of molecules anchored to capture molecules that are fixed on the surface of a compact disk by expensive and complicated means. The described method allows readings and / or biological detections to be made on one side of the disk, while on the other side, readings or recordings of the obtained fluorescence signals can be made.

ES2304093A1 (Polytechnic University of Valencia, 2006) describes an integrated data storage system for chemical analysis of samples and for detection and storage of analytical results. The described system is capable of capturing the analytical results obtained on one side of a data storage disk and obtaining its transposition into digital data for further processing and / or recording on the other side of the disk. The system comprises a compact disc (CD / DVD) as a support as well as a CD / DVD reader / writer for the detection of fluorescence signals.

There is a growing interest in the methods of characterization of molecules through techniques without labeling. Usually the process of detection or recognition of molecules is carried out by techniques that require labeling elements such as fluorescent dyes, enzymes, radioactive isotopes, etc. However, the protocols associated with the process of mareaje of these molecules for their detection imply greater time and greater economic cost. Therefore, it is expected that detection and identification systems of unlabeled molecules will grow in importance in sectors such as clinical, biopharmaceutical, environmental, security, among others due to its simplicity, sensitivity, saving of reagents and analytes, and ultimately, lower cost per detection.

US2007 / 0026382A1 (ALIX YALE & RISTAS LLP, 2006) describes an alternative method to flow cytometry for detection of phenotypes and determination of functional responses in cells, based on a technology without dizziness, on a biosensitive surface, which can be a chip or a plate (biosensor chip), preferably coated with gold. The biosensitive surface described comprises an array of specific capture ligands for cell surface markers so that a specific phenotype correlates with a specific interaction location on the chip. The type of cells that can be analyzed includes animal, plant and bacterial cells. The interaction between the cells and the specific capture ligands is read by a CCD camera or a CMOS camera.

Despite the different procedures described in the state of the art, there is currently a need to develop a rapid analysis method without marking for the identification and detection of bacteria and viruses, based on compact disk technologies.

BRIEF DESCRIPTION OF THE INVENTION

The present invention comprises a rapid analysis methodology for the identification and detection without detection of Gram + and Gram - viruses and bacteria, resistant or not to antibiotics, using as audio-video discs (CD, DVD, BD, Lightscribe, Superdisk, etc.) where to immobilize specific molecular receptors of biomolecules present on the surface of bacteria and / or viruses, using as a detector a disk reader / writer (CD, DVD, BD, Lightscribe, etc.). The present invention has been designed to identify separate immobilized receptors or bioreceptors on the polycarbonate face of the disk and in known and orderly positions, forming a microarray. The Specific bioreceptors of biomolecules present on the surface of viruses and / or bacteria may be for example polysaccharides, proteins, lipids and nucleic acids.

As a preferred embodiment, the present invention has been applied in the identification and detection of a pneumococcal suspension (Strepcococcus pneumoniae) previously isolated from a clinical sample and grown in solid media, as well as a suspension of Escherichia coli and Salmonella spp. . To this end, the invention has been designed to identify specific polysaccharides present on the surface of said bacteria, using antiserum microarrays.

DETAILED DESCRIPTION OF THE INVENTION

In general, the method of identification and / or detection of viruses and / or bacteria object of the present invention comprises the following essential steps:

- resuspension of bacteria and / or viruses contained in the test sample,

- incubation on compact disk, and

- detection without mareaje using a disc reader / writer.

Resuspension of bacteria and / or viruses contained in the test sample

It is based on a test sample (from now on "sample") already obtained, for example a clinical sample, collected for example in a Petri dish and previously incubated in a solid medium (for example in blood agar, chocolate agar, etc.), for a sufficient time for the bacteria and / or viruses contained therein to grow abundantly (10 ^ -10 ^) on the surface of the plate (for example 16-24 h), until forming colonies.

The samples to be analyzed with the invention can be microbial isolates of diverse origin such as blood (blood culture), pleural fluid, cerebrospinal fluid, saliva, sputum, urine, tear secretion, etc.

Resuspension of the material from the bacteria and / or virus colonies in the sample is carried out, for example, by a 1 [iL planting handle that passes through the surface of the solid medium, to be added, for example, to a 1, 5 tube mL containing an adequate volume (for example between 100-500 μί) of a buffered aqueous solution (for example PBST, TBS-T, etc.) which is characterized by being compatible with the suspension of bacteria and / or viruses for its subsequent incubation on the disk.

A suspension of bacteria and / or viruses is obtained which is preferably homogenized before the next step to eliminate possible aggregates and thus facilitate interactions with the immobilized receptors on the surface of the compact disc.

Incubation on the compact disc

It starts from a compact disk (hereinafter "disk") on whose face of polycarbonate molecular receptors located on the surface of bacteria and / or viruses have been immobilized, being arranged in known and orderly positions, forming a microarray. These receptors make it possible to identify biomolecules from the surface of the virus and / or the bacteria, such as proteins, lipids, nucleic acids, or polysaccharides.

This step is called microarray printing on the disc. The immobilization of the receptors on the disc can be performed by different methods (Hermanson, GT Bioconjugate Techniques. 2 * ⁿ Edition Academic Press, San Diego, CA. 2008), preferably but not limitingly, in a preferred embodiment of the present invention is carried out by immobilizing antibodies contained in antisera by disk adsorption, as described below.

Regardless of the immobilization method used, the surface of the disk with the immobilized receivers must maintain the optical and mechanical properties of the disk, a key aspect for its reading by the disk reader / writer of the next step.

The disk is segmented into independent areas (for example between 6-12), each of which can be used to analyze different samples or replicas of the same sample. Each disc may contain, for example, between 50 and 200 points or "spots" among which are the immobilized receptors (specific, controls, and replicas) to determine exactly each type, factor and / or group of bacteria and / or virus. The suspension of bacteria and / or virus obtained in the previous step is incubated for example 15 minutes, in the disk with the previously immobilized receptors.

During the incubation, the receptor fixes the bacteria and / or the virus in a specific and separate position that defines its type, and / or its factor and / or its group.

To remove bacteria and / or viruses that have not been specifically fixed on the disc, washing and drying can be carried out, for example by washing the disc with water and centrifuging it to dry it quickly.

Detection of bacteria and / or viruses on the disk Finally, a reading of the disk is made with a compact disc reader / writer, which uses the laser of the recorder itself as a questioning beam and the photodiode of the pickup head as the detector element.

The specific interaction receptor / bacteria and / or virus produces a precipitate that absorbs and disperses the laser light beam, attenuating the intensity of the reflected signal detected by the pickup head photodiode. The decrease in reflection signal is directly related to the receptor / bacteria and / or virus interaction. Depending on the type of disc, reading speed, and position of the matrix, the surface of the disc is read or scanned in a few minutes (for example 1-8 min).

Once the disc scan is finished, the image obtained on the computer screen is observed, the brightness and contrast may vary, in order to facilitate the interpretation of the data. The detection can be done visually, using a template that indicates the location of each receptor and a table in which the different combinations of positive signals from the receptors are found, also indicating which type and / or group of bacteria and / or viruses correspond to these signals. With this information, the group and / or type of bacteria and / or viruses are determined exactly based on the signal-to-noise ratio obtained from the simultaneous analysis of, for example, more than one sample.

The disk reading comprises two types of data:

one . Data obtained from the points or spots of positive control used to set the interpretation template. These are points with receivers printed directly on the disc that give a positive signal.

2. Data obtained from the points or spots printed within a certain area where receptors that bind the bacteria and / or the virus have been immobilized, and that consequently absorb and disperse the laser light, attenuating the reflection signal detected by the pickup head. In this pattern, each area can be made up of different receivers with, for example, 3-6 replicas of each.

Alternatively, the bacteria and / or viruses can be stained before or after incubation on the disc by staining techniques described for this purpose (for example Gram + staining for the detection of bacteria), thus increasing the signal to noise ratio.

The main field of application of the present invention is epidemiological surveillance, quality control of water and air, and in general any area that involves the detection of bacteria by biospecific interactions based on the use of separate and ordered molecular receptors on a support.

Therefore, the present invention refers to a method for identifying and / or detecting without type the group and / or group of at least one virus and / or a previously isolated bacterium (of at least one sample) and incubated in a solid medium , characterized by understanding the following essential steps:

a) resuspend the virus and / or bacteria in a buffered aqueous solution, obtaining a suspension of the virus and / or bacteria;

b) immobilize at least one receptor of a molecule from the surface of the virus and / or bacteria, forming a microarray on a compact disk as a support;

c) incubate the suspension obtained in step a) on the compact disc obtained in step b),

d) detect the virus and / or bacteria in the compact disc by reading the disc with a compact disc reader / writer as a detector, obtaining a signal to noise ratio; Y e) determine the type and / or group of the virus and / or bacteria based on the signal to noise ratio obtained in step d).

Preferably, before or after step c) defined above, the method may comprise the following additional step: f) staining the virus and / or bacteria to amplify the signal to noise ratio obtained in step d), by means of a technique of which They are known in the state of the art.

Likewise, in a preferred embodiment, the method described above is characterized in that the sample is selected from the following group: blood (blood culture), pleural fluid, cerebrospinal fluid, saliva, sputum, urine, and tear secretion.

In another preferred embodiment, the method described above is characterized in that the microarray of step b) is immobilized on the polycarbonate face of the disk while maintaining the optical and mechanical properties of the disk.

In another preferred embodiment, the method described above is characterized in that the microarray of step b) comprises between 50-200 points comprising at least one receptor of a molecule from the surface of the virus and / or bacteria and at least one point as a control positive. More preferably, the points are at least 0.3-0.5 mm in diameter and are at least 1.0 mm apart.

In another preferred embodiment, the method described above is characterized in that the compact disc reader / writer defined in step d) uses the laser of the recorder itself as a beam of questioning light and the photodiode of the pickup head as the detector element. More preferably, the laser light beam is absorbed and dispersed by the less a precipitate produced by the specific interaction of the receptor with the virus and / or bacteria, decreasing the intensity of the reflection signal detected by the pickup head photodiode. Even more preferably, the decrease in reflection signal is directly related to the specific interaction of the receptor with the virus and / or bacteria.

In another preferred embodiment, the method described above is characterized in that the determination of the type and / or group of the virus and / or bacteria defined in step e) is performed visually with a template indicating the location of the recipient and a table in that the different combinations of positive signals of the receptor are found, also indicating to what type and / or group of bacteria and / or viruses these signals correspond.

In another preferred embodiment, the method described above is characterized in that step b) is performed by immobilizing antibodies contained in antisera by disk adsorption. More preferably, immobilization of antibodies contained in antisera by disk adsorption comprises the following steps: i) printing the disc with at least one antiserum solution in the presence of a printing buffer

(such as an aqueous 0.1 M carbonate / bicarbonate solution, pH 9.5),

ii) incubate at least 4 ° C for a period of

time of at least 16 hours,

iii) wash with water and dry the disc by

centrifugation

Even more preferably, the antiserum solution of step i) contains at least one virus and / or bacterium antibody.

The present invention also refers to a method for immobilize at least one receptor of a molecule of the surface of at least one virus and / or a bacterium, characterized by using a compact disc as support, said method comprises the following steps:

i) print the polycarbonate side of the disc with at least one antiserum solution in the presence of a printing buffer, ii) incubate at least 4 ° C for a period of

time of at least 16 hours, and

iii) wash with water and dry the disc by centrifugation,

where the optical and mechanical properties of the disk are maintained.

The present invention also refers to a compact disc characterized by comprising at least one immobilized receptor of a molecule of the surface of at least one virus and / or a bacterium, forming a microarray to be read by a compact disc reader / writer. More preferably, the disk is obtained by a method comprising the following steps:

time of at least 16 hours, and

iii) wash with water and dry the disc by centrifugation,

where the optical and mechanical properties of the disk are maintained.

The present invention also refers to the use of the disk defined above, to identify and / or detect without typesetting the type and / or group of at least one virus and / or a bacterium previously isolated from at least one sample and incubated in a solid medium. .

More preferably, said use is characterized in that the identification and / or detection without marking of the type and / or group of at least one virus and / or a bacterium is for epidemiological surveillance.

Alternatively, said use is characterized by the identification and / or Detection without mareaje of the type and / or group of at least one virus and / or a bacterium is for quality control of water and / or air.

To facilitate the understanding of the present invention, the meaning of some terms and expressions as used herein is set forth below.

In the present invention the term: "rapid analysis method" refers to an assay whose total duration is between 1 and 30 minutes.

In the present invention the term: "detection without marking" refers to an assay in which the signal produced by the bioreceptor-cell or virus interaction is directly obtained, without resorting to a third species, such as a fluorescent substance, an enzyme or a particle.

In the present invention the term: "virus" refers to an organism of simple structure, composed of proteins and nucleic acids, and capable of reproducing only within specific living cells, using its metabolism.

In the present invention the term: "bacteria" refers to single-cell prokaryotic microorganisms, whose various species cause fermentation, disease or rot in living beings or in organic matter.

In the present invention the term: "compact disc" refers to a digital optical disk-shaped medium, used to store any type of information (audio, images, video, documents and other data) and with different presentations and capabilities in in terms of size and capacity and speed of storage and reproduction. In the present invention the term: "compact disc reader / writer" refers to the optical device capable of reproducing and recording the compact discs using a laser that allows it to read the information contained therein and record or re-record information.

In the present invention the term: "bioreceptors or receptors" refers to more complex molecules or molecular organizations, produced or present in cells, capable of recognizing with great selectivity and affinity to another substance. For example: antibodies, oligonucleotides, lectins, lipocalins, etc.

It is also understood as bioreceptors and receptors to synthetic or semi-synthetic substances with the characteristics described above.

In the present invention the term: "biomolecules" refers to the minimum unit of a substance of biological origin that retains its chemical properties.

In the present invention the term: "microarray" refers to a micrometric size matrix. Matrix means a set of numbers, symbols, points, etc., placed in a given arrangement (rectangle, square, circle, ellipse, etc.), often in horizontal and vertical lines.

In the present invention the term: "problem sample" refers to the sample to be analyzed and which contains the substance or substances to be identified and / or determined.

In the present invention the term: "microbial isolates" refers to the separation of microorganisms or viruses from the environment where they are originally found.

In the present invention the term: "solid medium" refers to a medium of culture of microorganisms of firm or solid consistency. They differ from semi-solids (fluid consistency) and liquids.

In the present invention the term: "signal to noise ratio" refers to the ratio between the intensity of a signal generated in the measurement process of an analyte and the signal of a blank (sample without analyte) or electronic noise.

In the present invention the term: "type, factor and group of viruses and / or bacteria" refers to:

"Type": each of the large taxonomic groups into which the animal and plant kingdoms are divided, and which, in turn, are subdivided into classes.

"Factor": antigen present on the cell surface.

"Group": set of microorganisms with genetic, pathogenic, etc. similar characteristics.

In the present invention the term: "epidemiological surveillance" refers to a system for collecting and evaluating information on medical-epidemiological events.

In the present invention the term: "quality control of water and / or air" refers to the actions aimed at regulating the quality of air or water within standards.

Throughout the description and the claims the word "comprises" and its variants are not intended to exclude other technical characteristics, additives, components or steps. For those skilled in the art, other objects, advantages and features of the invention will be derived partly from the description and partly from the practice of the invention. The following figures and examples are provided by way of illustration, and are not intended to be limiting of the present invention. DESCRIPTION OF THE FIGURES

FIGURE 1. Schematic representation of the detector system. The laser guide and disk rotation servomechanisms keep the laser beam focused on the spiral path of the disk, allowing its rotation and scanning. The reflected light is transformed into an electrical analog signal. At the same time, the photosensor detects the mark (trigger), beginning the acquisition of data. The amplification / detection card (DAB) is integrated in the recorder unit. The DAQ card digitizes the analog signals from the DAB and transfers them to the computer for processing. The CD player-recorder is controlled by software (BioDisk) and connected to the computer through a USB2.0 interface.

FIGURE 2. Images obtained after reading the disc when analyzing serotypes of Strepcococcus pneumoniae in microbial isolates. Panel I corresponds to the result of identification without marking and panel II to the response obtained after the staining process.

FIGURE 3. Images obtained after reading the disc when analyzing sermon extracts of Salmonella spp. Panel l-V: S. Malmoe 16263.1; S. Typhimurium 15994.3; S. Llandoff; negative control

FIGURE 4. Images obtained after reading the disc when analyzing extracts of E. coli serotypes. Panel l-IV: E. coli 0157: H7; E. coli O103: H2; E. coli 026: H1 1; negative control

EXAMPLES

The following specific examples provided in this patent document serve to illustrate the nature of the present invention. These examples are included for illustrative purposes only and they have not been interpreted as limitations to the invention claimed herein. Therefore, the examples described below illustrate the invention without limiting its scope of application. EXAMPLE 1: Identification of Strepcococcus pneumoniae

In the present example or preferred embodiment said method

It comprises the following steps: 1. Resuspension of bacteria

2. Immobilization of antibodies contained in antisera in microarray format by disk adsorption

3. Incubation on compact disk

4. Detection and determination by a disk reader / writer 5. Signal amplification by staining

6. Determination

Resuspension of bacteria In this identification and detection test without marking, we start from a suspension of pneumococci (Strepcococcus pneumoniae) previously isolated from a clinical sample and grown in solid media. For this, the bacteria, previously grown in solid media, are collected and subsequently resuspended in 200 μΙ_ of buffered aqueous solution (TBS-T).

Immobilization of antibodies contained in antisera in microarray format by disk adsorption. Immobilization of antibodies contained in antiserum in microarray format is preferably performed by adsorption, without being limiting to the invention. In this example, the anchoring of the bacteria to the receptor occurs through the formation of hydrogen bonds, van der Valls forces, hydrophobic bonds between the receptor and the surface. In this case, the receptors react with the surface of the bacteria in buffered aqueous solutions, their pH being a critical variable to obtain a stable immobilization of the antisera.

In this case, six micromatrices of 144 dots each (12 12) are printed on the polycarbonate side of a DVD disc. Each matrix contains 0.3-0.5 mm diameter points 1.0 mm apart, dispensing antiserum solutions (20-50 nl_), containing specific receptors or bioreceptors to detect and identify five serotypes at the type, factor level and partial group and points used as positive control. The printing buffer is an aqueous 0.1 M carbonate / bicarbonate solution, pH 9.5. After printing, the disc is allowed to incubate at 4 ° C. After 16 hours the disc is washed with water and dried by centrifugation. Thus, the six antisera are printed at four different dilutions with six replicates each.

Incubation on compact disk

100 μΙ_ of the bacteria suspension are taken, and dispensed in one of the areas of the disc, allowing to incubate for 15 min at room temperature. Finally, the disc is washed with water and dried by centrifugation.

Detection and determination by a disk reader / writer

Detection is done by scanning the disc with a disc reader / writer. The appearance of points in the image obtained on the computer identifies the type, factor and group of bacteria present in the sample.

Signal extension by staining Alternatively, the bacteria can be stained before or after incubation on the disc by staining techniques described for this (Gram + staining), thereby increasing the signal to noise ratio.

Determination

The results obtained are shown in Table 1. As can be seen, each antiserum immobilized on the disc recognizes the type or group of bacteria unique and specifically with a signal to noise ratio between 51 (s1 1 C) and 152 (s12). The positive control is detected for all strains except s1.

Table 1. Signal to noise ratio obtained by simultaneously analyzing six pneumococcal suspensions (Strepcococcus pneumoniae) on disk

Sample (strain)

Disk antiserum s1 s11 C s + s12 s13 s20 s1 120 4 3 1 1 1 s11 1 51 1 1 2 3 s + 1 49 34 10 21 17 s12 1 3 1 145 7 7 s13 1 4 1 2 69 6 s20 1 2 6 3 1 150

EXAMPLE 2: Identification of Salmonella spp.

In the present example or preferred embodiment said method comprises the following steps:

one . Resuspension of bacteria

3. Incubation on compact disk

4. Detection and determination by a disc reader / writer 5. Signal amplification by staining

6. Determination

Resuspension of bacteria

It starts from a suspension of Salmonella spp. in TBS-T buffer.

In sections 2, 3, 4 and 5 the same procedure described in example 1 is followed with the only difference that specific antisera are used for this bacterium.

Determination

The results obtained are shown in Figure 3. As can be seen in all cases, positive responses were obtained for the control block (matrix on the left). On the other hand, positive results (right matrix) were obtained in the block with specific antibodies for each type of Salmonella (l-lll panels), the results being negative for the control (panel IV).

EXAMPLE 3: Identification of Escherichia coli

one . Resuspension of bacteria

3. Incubation on compact disk

4. Detection and determination by a disc reader / writer

5. Signal amplification by staining

6. Determination Resuspension of bacteria

It starts from a suspension of E. coli in TBS-T buffer.

Immobilization of antibodies contained in antisera in microarray format by disk adsorption

In sections 2, 3, 4 and 5 the same procedure described in example 1 is followed, with the only difference that specific antisera are used for this bacterium.

Determination

The selectivity of the assay was determined using different E. coli serotypes. Figure 4 shows the images obtained after reading the disc when analyzing extracts of pure colony. As can be seen in all cases, positive responses were obtained for the control block (matrix on the left). On the other hand, positive results (right matrix) were obtained in the block with specific antibodies for each E. coli serotype (l-lll panels), the results being negative for the control (panel IV).

Claims

one . A method for identifying and / or detecting without type the group and / or group of at least one virus and / or a bacterium previously isolated and incubated in a solid medium, characterized by comprising the following essential steps:

a) resuspend the virus and / or bacteria;

b) immobilize at least one receptor of a molecule from the surface of the virus and / or bacteria, forming a microarray on a compact disk;

d) detect the virus and / or bacteria on the compact disc by reading the disc with a compact disc reader / writer; and e) determine the type and / or group of the virus and / or bacteria.

2. The method according to claim 1 characterized in that before or after step c) comprises the following additional step:

f) stain the virus and / or bacteria.

3. The method according to claims 1-2, characterized in that the sample is selected from the following group: blood, pleural fluid, cerebrospinal fluid, saliva, sputum, urine, and tear secretion.

4. The method according to claims 1 -3, characterized in that the microarray of step b) is immobilized on the polycarbonate face of the disk while maintaining the optical and mechanical properties of the disk.

5. The method according to claims 1-4, characterized in that the microarray of step b) comprises between 50-200 points comprising at least one receptor of a surface molecule of the virus and / or bacteria.

6. The method according to claim 5, characterized in that the points are at least 0.3-0.5 mm in diameter and at least 1.0 mm apart.

7. The method according to claims 1-6, characterized in that the compact disc reader / writer defined in step d) uses the laser of the recorder itself as a beam of questioning light and the photodiode of the pickup head as the detector element.

8. The method according to claim 7, characterized in that the laser light beam is absorbed and dispersed by at least one precipitate produced by the specific interaction of the receptor with the virus and / or bacteria, decreasing the intensity of the reflection signal which detects the photodiode of the pickup head.

9. The method according to claim 8, characterized in that the decrease in reflection signal is directly related to the specific interaction of the receptor with the virus and / or bacteria.

10. The method according to claims 1-9, characterized in that the determination of the type and / or group of the virus and / or bacteria defined in step e) is performed visually with a template indicating the location of the receptor and a table in which the different combinations of positive signals of the receptor are found, also indicating to what type and / or group of bacteria and / or viruses these signals correspond.

eleven . The method according to claims 1-10, characterized in that step b) is performed by immobilizing antibodies contained in antisera by disk adsorption.

12. The method according to claim 1, characterized in that the immobilization of antibodies contained in antisera by disk adsorption comprises the following steps:

i) print the disc with at least one antiserum solution in the presence of a printing buffer,

ii) incubate at least 4 ° C for a period of at least 16 hours,

iii) wash and dry the disc.

13. The method according to claim 12, characterized in that the antiserum solution of step i) contains at least one virus and / or bacterial antibody.

14. A method for immobilizing at least one receptor of a molecule of the surface of at least one virus and / or a bacterium, characterized by using a compact disc as support, said method comprises the following steps: i) printing the polycarbonate face of the disk with at least one antiserum solution in the presence of a printing buffer,

ii) incubate at least 4 ° C for a period of at least 16 hours, and

iii) wash and dry the disc, where the optical and mechanical properties of the disc are maintained.

15. A compact disc characterized by comprising at least one immobilized receptor of a surface molecule of at least one virus and / or a bacterium, forming a microarray to be read by a compact disc reader / writer.

16. The disk according to claim 15, characterized in that obtained by the method defined in claim 14.

17. Use of the disk defined in claims 15-16, to identify and / or detect without typesetting the type and / or group of at least one virus and / or a bacterium previously isolated and incubated in a solid medium.

18. Use according to claim 17, characterized in that the identification and / or detection without marking of the type and / or group of at least one virus and / or a bacterium is for epidemiological surveillance.

19. Use according to claim 17, characterized in that the identification and / or detection without marking of the type and / or group of at least one virus and / or a bacterium is for quality control of water and / or air.