IT202100005831A1 - IN VITRO PROCEDURE FOR THE DETECTION OF SARS-COV-2 IN AN ORAL SAMPLE USING A COLORIMETRIC IMMUNOSENSOR AND RELATIVE COLORIMETRIC IMMUNOSENSOR - Google Patents

Description

Description of the industrial invention entitled: ?In vitro process for the detection of SARS-CoV-2 in an oral sample using a colorimetric immunosensor and related colorimetric immunosensor?

DESCRIPTION

FIELD OF THE INVENTION

The present invention relates to an in vitro process for detecting SARS-CoV-2 in an oral biological sample through the use of a colorimetric immunosensor and the related colorimetric immunosensor.

STATE OF THE ART

In the field of research and diagnostics, optical biosensors based on colorimetry are becoming increasingly important due to their versatility, ease of of? use and capacity? to achieve an extremely low limit of detection (LOD) of the analyte.

Among the optical biosensors, the colorimetric biosensors based on gold nanoparticles are known, which exploit a property physics of gold nanoparticles, called Localized Surface Plasmon Resonance (LSPR), to monitor the color change when, following the interaction between the analyte to be detected and the gold nanoparticles, aggregates of different sizes are formed.

Optical transducers are particularly interesting for the direct detection (label free) of microorganisms. These sensors are designed to detect minute conversions in refractive index or thickness that occur as cells attach to receptors immobilized on the transducer surface, correlating changes in concentration, mass, or number of molecules to direct changes in light characteristics . The sensing mechanism is based on the conversion of signals that derive from the link with the target to be detected (target) into physical signals that can be amplified and detected. Nanomaterials, which are characterized by extremely small dimensions and to which appropriate surface modifications can be made, allow highly specific interaction with biomolecular targets, showing enormous potential in the field of biological surveys.

Pi? in general, gold nanoparticles find applications in a wide range of disciplines, including magnetic fluids, catalysis, biotechnology/biomedicine, magnetic resonance imaging and environmental remediation. In most of these applications, there is improved functionality? of these nanoparticles when their size ? lower than a critical value, which depends on the material but typically ? around 10-20 nm.

Biosensors that are based on the specificity? of interaction between an antigen and the corresponding antibody for the determination of the analyte of interest are commonly referred to as immunosensors. The antibody immobilization procedure represents a crucial step in the realization of these devices because the orientation of the antibody molecules on the electrode surface significantly influences the performance of a biosensor. Indeed, the formation of a layer (layer) of antibodies with well-oriented binding sites facing the antigen improves the efficiency of the biosensor, making the choice of the immobilization method one of the most straightforward. important aspects to take into consideration in the realization of an immunosensor. Generally, the antibody immobilization methods involve the physical or chemical adsorption of these molecules.

Among the most adsorption procedures? simple, we mention the method that exploits ionic or electrostatic bonds, hydrophobic interactions and van der Waals bonds between the antibody and the surface ( 2016; Um et al.

2011) and does not require chemical modifications of the protein. The main drawback of this method is that the antibodies are randomly oriented and therefore may not correctly expose the antigen binding sites.

More methods effective for the immobilization of antibodies are based on the formation of covalent bonds between the antibody and the gold surface ( 2012; al.

2010; to the. 2011; Rahman et al. 2007). For example, biotinylated antibodies can be immobilized on surfaces modified with streptavidin or avidin (Barton et al. 2009; Ouerghi et al. 2002) or antibodies can be immobilized on surfaces modified with proteins such as protein A or protein G ( 2013;

2019; Sharafeldin and Rusling 2019; Fowler, Stuart, and Wong 2007). Finally, methods of immobilization of antibodies involving trapping in polymeric matrices have been developed in the last decade (Sun et al. 2011; Bereli et al. 2013; Moschallski et al.

2013; Yamazoe 2019).

Among the possible immobilization strategies, the formation of self assembled monolayers (SAMs) represents one of the most widely used methods for the production of immunosensors. For example, directed immobilization of an antibody on the gold surface of an electrode can be achieved by exploiting the formation of SAMs of thiolated carboxylic acids (Barreiros dos Santos et al. 2013; Malvano, Pilloton, and Albanese 2018; Wan et al. 2016) or by immobilizing the antibodies on electrochemically deposited cysteamine layers (Malvano, Pilloton, and Albanian 2018). Furthermore, ? The use of cross-linking agents such as gluteraldehyde has also recently been reported, specifically for the immobilization of anti-E antibodies. coli on a polyaniline substrate with interesting results in the detection of this bacterium (Chowdhury et al. 2012). Therefore, SAMs are widely used as linker agents for the immobilization of antibodies in an oriented manner on a gold surface but, despite the many advantages they present in various applications, there are still several aspects that should be taken into account to know and check their properties? physical and chemical (Vericat et al. 2010; Mandler and Kraus-Ophir 2011; Chaki and Vijayamohanan 2002). A self assembled monolayer on gold surfaces is commonly represented as a perfect monolayer in which the molecules are in a perfectly packed configuration. Actually, this idea is far from reality and quality control? of a SAM? a crucial point in many applications. The realization of a well-assembled monolayer strongly depends on the purity of the chemical reagents and solutions used and the presence of even a minimal quantity of contaminants, such as thiolated molecules which are typical impurities in thiol compounds, can lead to a non-uniform and therefore non-ideal layer (C. Y. Lee et al. 2005).

In recent years, different types of immunosensors have been described in studies published in the literature.

(2018) (?Colorimetric Immunosensor by Aggregation of Photochemically Functionalized Gold Nanoparticles? ACS Omega 3, 4, 3805? 3812) describes a colorimetric immunosensor which uses the phenomenon of surface plasmon resonance of gold nanoparticles as well as the application of this system for the detection of human immunoglobulin IgG.

(2015) (?Colorimetric detection of influenza A virus using antibody-functionalized gold nanoparticles? Analyst 140(12)3989-3995) investigated the use of a colorimetric immunosensor based on gold nanoparticles modified with anti-haemagglutinin monoclonal antibodies at the in order to determine the influenza A virus. However, no demonstration of the efficacy of the described immunosensor in a clinical setting is provided in this document.

The researches described in Della Ventura B. et al (2020) (?Colorimetric Test for Fast Detection of SARS-CoV-2 in Nasal and Throat Swabs? MedRixv doi: https://doi.org/10.1101/2020.08.15.20175489) concern the use of a colorimetric immunosensor for the detection of the SARS-CoV-2 coronavirus in a nasopharyngeal swab.

The ongoing severe pandemic caused by the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has posed major health challenges. public of many countries. Due to the lack of specificity? symptoms of the serious illness caused by the new coronavirus, called COVID-19, confirming the diagnosis requires laboratory testing of respiratory samples and/or serum samples from patients. Large-scale diagnostic tests also play a vital role in isolating asymptomatic COVID-19 patients, in an attempt to stem the spread of the infection.

Among the procedures currently used for the diagnosis of SARS-CoV-2 coronavirus infection, the method based on the reverse transcription reaction of the chain polymerase (RT-PCR) plays a primary role. This method allows the identification of the viral genome in samples of the upper respiratory tract, in particular in samples taken using nasopharyngeal swabs. However, the diagnostic application of PCR has important limitations due to the complexity of the PCR. of execution and the timing, as well as? to the need? of dedicated instrumentation and trained personnel.

The immunological approaches that have been developed for the diagnosis of COVID-19 also present significant problems. For example, lateral flow assays, while allowing rapid analysis of many samples, are characterized by low sensitivity.

Further problems associated with methodologies aimed at diagnosing SARS-CoV-2 infection concern the choice of the best samples? suitable to operate the search for viral particles, pi? specifically the intact particles. Among these, the samples of choice are currently represented by respiratory tract samples, in particular the nasopharyngeal swab. This levy requires for? a precise operating procedure, i.e. to have value the nose sample must be performed by pushing the swab downwards, to reach the pharynx, and not towards the cavities? nasals.

there ? so the need? to make available diagnostic assays that allow for the rapid identification of the SARS-CoV-2 virus through simple procedures, but which at the same time maintain high parameters of specificity? and sensitivity.

These and other objects are achieved thanks to the in vitro process and the relative kit defined in the annexed independent claims, suitable for the detection of the SARS-CoV-2 virion in an oral biological sample of a subject.

Further characteristics of the invention are identified in the dependent claims, which form an integral part of the description.

How will it turn out? clearly from the detailed description that follows, the in vitro process according to the invention allows the diagnostic result to be obtained in a very short time, in the order of a single minute, and requires minimal quantities of the sample to be analysed, in the order of a volume of about one milliliter, thus facilitating its transport. The particular simplicity? of the procedure, which does not require any sophisticated instrumentation, also allows you to significantly reduce costs.

A first object of the present invention ? then, an in vitro procedure for the detection of the SARS-CoV-2 virion in an oral biological sample of a subject, comprising the steps of: a) placing said biological sample in contact with a colloidal suspension of capture gold nanoparticles bearing on its surface at least one antibody capable of binding a surface antigen of SARS-CoV-2, the antigen being selected from the group consisting of membrane protein (M), envelope protein (E), spike protein (S) , and any combination thereof, thus obtaining? a reaction mixture;

(b) determining the formation in the reaction mixture of an aggregate of gold nanoparticles on the surface of the SARS-CoV-2 virion, said aggregate resulting from the interaction between the aforementioned antibody and the aforementioned antigen, the determination being carried out by the detection the variation of an optical parameter of the reaction mixture,

said variation of an optical parameter of the reaction mixture being indicative of the SARS-CoV-2 virion in the oral sample, wherein the variation of the optical parameter is:

(i) an increase in the transmittance value of the reaction mixture measured at a wavelength of 530 nm;

(ii) a reduction in the absorbance value of the reaction mixture measured at a wavelength of 530 n; and/or

(iii) the reduction of the area under the absorption spectrum of the reaction mixture in a wavelength range between 200 nm and 700 nm.

In the context of this description, with the term ?virion? means the mature viral particle, complete with genome, nucleocapsid and envelope.

The process according to the invention is based on the physical principle of localized surface plasmon resonance (LSPR), which consists in the occurrence of coherent and non-propagating oscillations of free electrons in metal particles following irradiation with an electromagnetic wave whose frequency ? in resonance with the surface plasmon. The surface plasmon resonance, which gives the color to the colloidal solution, depends on various factors, such as the size of the nanoparticles, and can change significantly when the nanoparticles are in contact with each other, or at a distance much more? small of their diameter. In general, the coupling between metal nanoparticles, for example gold nanoparticles, present in a colloidal suspension occurs through the formation of dimers, trimers or more chains. large up to the formation of aggregates, and involves a variation of the plasmon resonance and, therefore, of the color of the solution that can? be detected even with the naked eye.

According to the invention, the aggregation between the gold nanoparticles present in the reaction mixture is mediated by a biological mechanism which consists in the specific interaction between the antibodies immobilized on the surface of said capture nanoparticles and the corresponding antigens present on the surface of the SARS-CoV-2 virus. Therefore, the aggregation phenomenon occurs only if in the sample to be analyzed ? Is the SARS-CoV-2 viral particle present, giving it extreme specificity? to the process of the invention.

The oral samples suitable for use in the process according to the invention are preferably selected from saliva and sputum.

In a preferred embodiment, the oral biological sample contacted with the colloidal suspension of capture gold nanoparticles in step a) is on a swab stick. In this embodiment, in the aforementioned method step, a portion of the biological sample is released from the stick into the colloidal suspension.

Swab sticks suitable for taking an oral biological sample, for example saliva or sputum, are known and described in the state of the art, so that their choice and use fall within the capabilities of the average technician in the sector. Preferably, in the swab stick, one of the two ends? or both ends? they are covered with wadding or cotton.

As illustrated more? in detail in the experimental part that follows, the present inventors have surprisingly found that the process of the invention allows to carry out the detection of the SARS-CoV-2 virus in the saliva or sputum of a subject. Despite the? undoubted advantage of facility? collection of this type of samples, their use in a procedure as previously defined? a result that has so far been particularly difficult, if not impossible, due to the high salt concentration of these samples which causes the non-specific aggregation of the gold nanoparticles, making the biosensor unusable. In addition, saliva contains a high concentration of proteins that can interfere with the antigen-antibody interaction.

As previously indicated, the metal nanoparticle used in the process according to the invention is a gold nanoparticle.

Preferably, the gold nanoparticle has a diameter between 1 nm and 100 nm, more than preferably between 2 nm and 40 nm. Most favorite? a gold nanoparticle having a diameter of 20 nm.

In the process according to the invention, the at least one antibody on the surface of the capture gold nanoparticle is ? capable of binding a surface antigen of SARS-CoV-2 selected from the group consisting of membrane protein (M), envelope protein (E), spike protein (S), and any combination thereof.

As ? known in the art, the previously mentioned viral proteins contribute together to the formation of the outer viral envelope. Among these, the spike protein ? responsible for the binding of SARS-CoV-2 to the host cell favoring the fusion of the viral envelope with the cell membrane.

Among the antibody molecules suitable for use in the process according to the invention, mention is made by way of non-limiting example of monoclonal or polyclonal antibodies, monomeric (Fab) or dimeric (F(ab')2) antibody fragments, single chain antibody fragments ( scFv) or any binding protein derived from an antibody scaffold.

According to the invention, ? it is anticipated that anti-SARS-CoV-2 antibodies as defined above may be present on the capture gold nanoparticle in any possible combination.

Suitable methods to achieve the immobilisation of one or more? Antibody molecules on the surface of a gold nanoparticle are known in the art. To this end can For example, a photochemical technique could be employed in which the immobilization of antibodies on the gold surface in the correct orientation is achieved by irradiation of these molecules with UV light

The selection of the antibody immobilization method is more adequate to be employed in the scope of the present invention falls well within the capabilities? of the average technician in the sector.

Preferably, the quantity? of capture gold nanoparticles as defined above in the colloidal suspension ? included in the range from 1 to 10<20 >nanoparticles (np)/ml on the total volume of the suspension, plus? preferably from 10 to 10<15 >np/ml In an even more? preferred, the quantity? of capture gold nanoparticles ? of 10<10 >np/ml on the total volume of the suspension.

Advantageously, the process according to the invention makes it possible to detect the SARS-CoV-2 viral particle in its entirety in the sample analysed, by means of the ability of gold nanoparticles, bearing antibodies directed specifically against the surface proteins of the virus, to aggregate on the surface of the virion forming a layer around it. Therefore, thanks to the specific determination of active viral particles, the process according to the invention is particularly suitable for identifying cases in which the SARS-CoV-2 infection is ? still in progress.

As illustrated above, in the process according to the invention the formation of an aggregate of gold nanoparticles on the surface of the SARS-CoV-2 virion is determined by detecting the variation of an optical parameter of the reaction mixture using three different methods.

In a first way? of detection, the variation of the detected optical parameter ? an increase in the transmittance value of the reaction mixture measured at a wavelength of 530 nm.

According to the previous modality, the measurement of the transmittance value of the reaction mixture can be carried out using a photometer or colorimeter instrument, preferably calibrated with a standard solution having a transmittance value of 100%.

In a second mode? of detection, in the process of the invention the variation of the optical parameter detected ? a reduction of the absorbance value of the reaction mixture measured at a wavelength of 530 nm. Instrumentation suitable for carrying out the above absorbance measurement ? for example, a spectrophotometer that can? be portable or over the counter.

According to a third modality? of detection, the variation of the detected optical parameter ? the reduction of the area under the absorption spectrum of the reaction mixture in a wavelength range between 200 nm and 700 nm.

In the context of this description with the expression ?wavelength in the visible range? it means a wavelength between about 390 nm and about 760 nm.

In this third mode? of detection, the method according to the invention allows to carry out a quantitative measurement which ? indicative of the viral load of SARS-CoV-2 present in the sample analysed. According to this method, the addition of a standard curve allows to obtain an ?absolute? of the viral load.

? understood that in accordance with the? invention the different modalities? detection of the variation of the optical parameter as previously illustrated can be used in the process object of the invention in combination or alternatively according to the needs, and the choice of modality? detection falls largely within the capabilities? of the branch technician.

In one embodiment of the process of the invention, the variation of the optical parameter detected consists of a color variation of the reaction mixture which is ? detectable with the naked eye.

In this embodiment, a further optional step consists in comparing the detected color of the reaction mixture with a color scale. This passage allows to increase the quality? interpretation of the result.

? otherwise? It is important to note that in the embodiment illustrated above advantageously it is not necessary the use of any instrumentation.

As indicated above, ? otherwise? included within the scope of the present invention is a kit including the means suitable for carrying out the process according to the invention.

Therefore, a second aspect of the present invention ? a diagnostic kit for the detection of the SARS-CoV-2 virion in an oral sample of a subject, comprising a colloidal suspension of capture gold nanoparticles bearing on their surface at least one antibody capable of binding a SARS-CoV surface antigen -2, the antigen being selected from the group consisting of membrane protein (M), envelope protein (E), spike protein (S), and any combination thereof, the kit being characterized by further comprising one or more? swab sticks suitable for taking an oral biological sample.

According to the invention, the one or more swab sticks contained in the kit are as previously defined in relation to the method.

In one embodiment, the diagnostic kit of the invention also comprises a support containing a color scale, such as a color strip.

In another embodiment, the diagnostic kit of the invention also comprises a portable colorimeter or photometer.

Preferably, the portable photometer ? equipped with a tungsten lamp and a monochromator capable of isolating the wavelength at 530 nm.

Preferably, the portable colorimeter ? equipped with a diode capable of emitting at 530 nm.

Among the portable instruments suitable for use in the kit of the invention, the portable photometer model Genotrix Cube 1 from the Genotrix company can be mentioned by way of example .

In the diagnostic kit of the invention, the colloidal suspension comprising the capture gold nanoparticles can be distributed in a plurality? of single disposable tubes.

Alternatively, said colloidal suspension can be supplied in a single package, for example in a dedicated dropper device.

The following experimental examples are provided for illustrative purposes only. In them, reference is made to the accompanying drawings, in which:

- Figure 1 ? a schematic representation of the process of functionalization of the surface of gold nanoparticles with type G immunoglobulins (?Photochemical Immobilization Technique?, PIT). The IgG antibodies are irradiated with UV rays using the ?trylight? lamp, resulting in the reduction of disulfide bonds in specific positions in the light chain, the constant part, of the antibody. The production of thiols allows the formation of a covalent bond between the antibody and the surface of the gold nanoparticle, leaving free one of the two antibody portions for recognizing the antigen.

- Figure 2 illustrates a schematic representation of the process of the invention. The swab stick used for taking the oral biological sample is immersed in the colloidal solution of functionalized gold nanoparticles having an OD value preferably equal to 1.5. Any virions present on the stick are only partially released into the suspension. Both the released virions and the virions trapped in the cotton of the stick contribute to ?subtract? functionalized gold nanoparticles free from suspension, binding them, and what? results in a variation of the absorption spectrum. Pi? precisely, a reduction of the area of the spectrum of the free nanoparticles and the formation of a spectrum due to the aggregation of the nanoparticles around the virions in suspension is observed. All of that? involves a reduction of the absorbance in correspondence with the absorption peak of the ?free? functionalized gold nanoparticles, which is observed at 530 nm when the diameter of the latter? of 20 nm.

Experimental section

Example 1: Preparation of colloidal gold solution (synthesis of nanoparticles)

For their experiments, the present inventors have obtained the synthesis of gold nanoparticles having a diameter of about 20 nm using a variant of a protocol known in the art (Turkevich method). According to this protocol, tetrachloroauric acid is first solubilized in water and with the addition of sodium citrate there is a reduction of the gold with the production of a gold seed (seed) and subsequently the growth of the gold is promoted around the it. The synthesis reaction? consisted of mixing 1mL of HAuCl4 (10mg/ml) and 2ml of sodium citrate dihydrate (10mg/ml) in 100mL of milliQ water (ultra pure). The operating temperature? been maintained at 90°C, with slow stirring. The formation of gold nanoparticles ? was identified by a drastic change in the color of the solution from yellow to orange.

At the end of the synthesis, the solution? was subjected to centrifugation at 6 G for 30 minutes, obtaining the gold nanoparticles ready to be functionalized.

Example 2: Functionalization

For the surface functionalization of gold nanoparticles ? the mechanism known as ?Photochemical Immobilization Technique? (PIT), described in Figure 1.

Briefly, IgG antibodies directed against the membrane protein (Membrane, M), envelope protein (Envelope, E), and spike protein (S) of the SARS-CoV-2 virus were used (0.1 mg/ml ).

A quartz cuvette containing the antibody solution at a concentration of 1 ?g/ml ? was inserted into the Trylight lamp and irradiated with UV rays for 30 seconds in order to obtain the reduction of some disulphide bridges in specific positions of the antibody. Subsequently, gold nanoparticles having a size diameter of 20 nm were functionalized obtaining a concentration of nanoparticles with anti-envelope antibody of 10<10 >nanoparticles (np)/ml, a concentration of nanoparticles with anti-spike antibody of 10 <10 >np/ml and an anti-membrane antibody nanoparticle concentration of 10<10 >np/ml. Any remaining empty spaces on the gold nanoparticles were then blocked using a solution containing BSA (50 ?g/ml).

Finally, the colloidal suspensions containing the three different antibodies were mixed together in the ratio of 1:1:1 in order to obtain a single suspension of gold nanoparticles carrying the three anti-SARS-CoV-2 antibodies, increasing what? significantly the specificity? of the system.

The purification of the samples obtained ? was carried out by centrifugation at 7000 g for 10 minutes.

Example 3: Sample preparation

Saliva sample? was taken with a cotton swab and immediately transferred to a volume of 0.5 ml of the colloidal suspension of functionalized gold nanoparticles. The virions possibly present on the cotton stick are partly released in the suspension, by vigorously rotating the stick around its own axis for about 10 seconds, and partly remain immobilized inside the cotton stick. The viral particles released in the colloidal suspension are recognized by the functionalized gold nanoparticles which aggregate around the virions while other gold nanoparticles recognize the virions trapped in the cotton, aggregating around them. Both modes? of binding of the nanoparticles to the virions, i.e. in suspension and on the stick, lead to a significant reduction of the nanoparticles in solution as a final result. Therefore, the reading of the absorbance value of the reaction mixture at 530 nm, pi? precisely the reduction of the absorbance value at 530 nm, ? the ?sensing? parameter. From there? it follows that, if a person tests positive, the absorbance value at 530 nm tends to zero; otherwise the absorbance value of the reaction mixture does not change with respect to the absorbance value of the starting nanoparticles.

The present inventors have observed that, unlike the assays of the prior art, the process according to the invention surprisingly does not require that the saliva sample, after collection, be resuspended in a buffer solution. The present inventors have also observed that other methods of shaking, such as shaking in an uncoordinated way up and down? the test tube, do not achieve the same result in the same times, also accompanied by a lack of repeatability? and reproducibility.

Example 4: Discovery

For the SARS-CoV-2 virus detection experiments, capture gold nanoparticles prepared and functionalized as previously described were used.

In short, after mixing the saliva or sputum sample with the colloidal suspension of the capture gold nanoparticles, it is possible to occurred a color change of the reaction mixture that ? went from orange to blue via purple. In particular, the blue shift ? major status as a function of increasing virus concentration.

As a negative control, ? was used the simple colloidal solution of nanoparticles of gold capture in which? Absorbance reduction was observed at 530 nm.

For the analysis of transmittance or absorbance, the entire volume of the colloidal solution that has come into contact with the swab stick carrying the oral biological sample is injected into a microcuvette to facilitate reading by the spectrophotometer, as well as better reading with the naked eye.

Subsequently, the test tube or cuvette were housed in the portable reader provided, which provided the values of absorbance or transmittance indicative of positivity/negativity? of the sample, i.e. the presence or absence of SARS-CoV-2 viral particles.

In the experiments conducted by the present inventors ? A portable photometer equipped with a tungsten lamp and a monochromator capable of isolating the wavelength at 530 nm was used, after appropriate calibration with a standard. As standard ? been employed a simple? white?, cio? a sample to which ? 100% transmittance or 0% absorbance was assigned. Disposable cuvettes containing the reaction mixture were used for the photometer analysis.

In their experiments, the present inventors have alternatively used a colorimetric device equipped with a diode capable of emitting at 530 nm and of returning, similarly to the previously indicated device, a transmittance value. In this mode? procedural, saliva or sputum sample after resuspension ? was dispensed and analyzed directly in the disposable reaction cuvette prepackaged with the colloidal suspension of capture gold nanoparticles.

The transmittance values measured in the previously described experiments are indicative of the quantity? of SARS-CoV-2 viral particles present in the sample under examination.

In order to carry out the measurement of the absorbance of the reaction mixture, the inventors have also made use of a bench spectrophotometer instrument which is ? capable of emitting in a broad spectrum of wavelengths, including, for example, between 200 and 700 nm. The absorbance measurements taken at the different wavelengths made it possible to calculate the area under the absorption spectrum, providing a ?relative? quantitative determination? of the viral load. The measurement of the viral load can become ?absolute? by means of a calibration of the described technique.

Table 1 below shows the results obtained on 6 patients (3 positive and 3 negative) using the PCR method and the process according to the invention in parallel. For PCR analysis, the data is expressed in cycle threshold (Ct), which corresponds to the cycle of the PCR reaction in which the emitted fluorescence exceeds the threshold. For the analysis according to the process of the invention, the data are expressed in absorbance values (Abs). The term ?x? represents a negative sample. Table 1

Table 1 shows that negative samples produce an absorbance value of 1.5?0.2. Even considering the 3SD criterion, the sample with the highest viral load? low (Ct=30) clearly distinguishes itself from negative samples. Values more? highs of 30 for the PCR threshold are in fact considered negative.

Claims (8)

1. In vitro procedure for the detection of the SARS-CoV-2 virion in an oral biological sample of a subject, including the steps of: a) placing said biological sample in contact with a colloidal suspension of capture gold nanoparticles bearing on their surface at least one antibody capable of binding a surface antigen of SARS-CoV-2, the antigen being selected from the group consisting of the membrane protein (M), envelope protein (E), spike protein (S), and any combination thereof, thus obtaining a reaction mixture; And (b) determining the formation in the reaction mixture of an aggregate of gold nanoparticles on the surface of the SARS-CoV-2 virion, said aggregate resulting from the interaction between the aforementioned antibody and the aforementioned antigen, the determination being carried out by the detection the variation of an optical parameter of the reaction mixture, said variation of an optical parameter of the reaction mixture being indicative of the SARS-CoV-2 virion in the oral biological sample, where the variation of the optical parameter ?: (i) an increase in the transmittance value of the reaction mixture measured at a wavelength of 530 nm; (ii) a reduction in the absorbance value of the reaction mixture measured at a wavelength of 530 nm; and/or (iii) the reduction of the area under the absorption spectrum of the reaction mixture in a wavelength range between 200 nm and 700 nm. 2. Process according to claim 1, wherein the oral biological sample placed in contact in step a) with the colloidal suspension of capture gold nanoparticles ? onto a swab stick, the biological sample in said step a) being partially released from the swab stick into the colloidal suspension. 3. Process according to claim 1 or 2, wherein the oral biological sample is chosen between saliva and sputum. 4. Process according to any one of claims 1 to 3, wherein the variation of the optical parameter ? detected with the help of a colorimeter or a photometer. 5. Diagnostic kit for the detection of the SARS-CoV-2 virion in an oral biological sample of a subject, comprising a colloidal suspension of capture gold nanoparticles bearing on their surface at least one antibody capable of binding a SARS surface antigen -CoV-2, the antigen being selected from the group consisting of membrane protein (M), envelope protein (E), spike protein (S), and any combination thereof, the kit being characterized by further comprising one or more swab sticks suitable for taking an oral biological sample. 6. Diagnostic kit according to claim 5, wherein the colloidal suspension is distributed in a plurality? of single disposable tubes. The diagnostic kit according to claim 5 or 6, further comprising a holder containing a colorimetric scale. The diagnostic kit according to any one of claims 5 to 7, further comprising a portable colorimeter or photometer.