PORTABLE DIAGNOSTIC DEVICE IN THE SHAPE OF A CYLINDRICAL CASE AND ITS USES

Abstract

TITLE: PORTABLE DIAGNOSTIC DEVICE IN THE SHAPE OF A CYLINDRICAL HOUSING AND ITS USES The present invention relates to a portable device for diagnosing pathogenic agents (viruses, bacteria, microorganisms, etc.) by rapidly detecting their nucleic acids in a biological sample to be tested. The present invention also relates to the uses of the diagnostic device of the invention and the methods that it allows to be implemented. Abstract figure: Fig 4

Description

Title of the invention: PORTABLE DIAGNOSTIC DEVICE IN THE SHAPE OF A CYLINDRICAL HOUSING AND ITS USES FIELD OF THE INVENTION

[0001] The present invention relates to a portable device for performing the diagnosis of pathogenic agents (viruses, bacteria, microorganisms, etc.) by rapidly detecting their nucleic acids in a biological sample to be tested. The present invention also relates to the uses of the diagnostic device of the invention and the methods which it allows to be implemented. CONTEXT OF THE INVENTION

[0002] Nucleic acid amplification tests (NAAT) detect, by amplification, nucleic acids in the infected sample. NAATs have the ability to detect pathogens from the first days of infection, i.e. well before the development of the immune response. The technique is characterized by high sensitivities (10 - 100 particles) and excellent specificities, due to a primer hybridization step. Due to its performance, the PCR (Polymerase Chain Reaction) based NAAT is considered, without doubt, the reference in the field.

[0003] Although extremely useful, the disadvantage of PCR-based NAAT is the cost and use of complex equipment. Many commercial instruments are now commercially available (ThermoFisher®, Roche®, Genexpert®, etc.). However, the cost of the equipment varies from €30k to €200k. In many cases, they require highly trained people, and clean, temperature- and humidity-controlled spaces to run the tests. Such equipment is located in a limited number of locations, hospitals and laboratories, requiring, in most cases, the transport of samples to be analyzed, or the transport of patients to reach these locations, with significant risks of contamination. Thus, the cost and complexity of PCR-based NAAT hinder the possibility of performing massive quantities of tests, or limit it to wealthy countries.

[0004] In France, at the beginning of the COVID-19 epidemic linked to the SARS-CoV-2 virus, in France, a few thousand tests were carried out each day, a figure to be compared to the tens of thousands of people infected. It took a lot of time and resources to increase to 10 - 20,000 tests per day, during April 2020, the number was still insufficient. In the case of this disease, some countries such as South Korea implemented a significant testing approach, which, coupled with the isolation of positive patients, has led to a significant decrease in the spread of the pathogen. However, the deployment of mass testing requires costly and complex logistics, which cannot be easily deployed in middle- and low-income countries.

[0005] In recent years, with the advent of isothermal amplification technologies (Rolling Circle Amplification [RCA], Loop Mediated Amplification [LAMP], Recombinase Polymerase Amplification [RPA], etc.), which do not require thermocycling to amplify nucleic acids, a new field of research has opened up, raising the hope of performing NAAT on-site, using devices much simpler and cheaper than PCR platforms. Today, there are about twenty isothermal amplification techniques (A. Niemz, T. Ferguson, D. Boyle. “Point of care nucleic acid testing for infections diseases". Trends in bio-technology, 29, 240, 2011.).Among them, LAMP isothermal amplification, which operates at 65°C, should be highlighted, due to its performance, in terms of amplification rate (twice as fast as PCR), sensitivity and specificity (similar to PCR), as well as large quantities of cDNA (Complementary DeoxyriboNucleic Acid) produced during the reaction, facilitating reading.

[0006] Recently, NAATs based on RPA and LAMP have been carried out, most often inspired by the ideas developed in recent years in the field of “paper microfluidics” (S. Vella et al., “Measuring markers of liverfunction using a micropatterned paper device designed for blood from a fingerstick”, Analytical Chemistry, vol. 84, pp. 2883-2891, 2012; J. Linnes et al., “Paper based molecular diagnostic for Chlamydia trachomatis, RSC Advances, vol. 4, pp. 42245-42251, 2014; L. Magro et al., “Paper microfluidics for nucleic acid amplification testing (NAAT) of infections”, Lab on a Chip, 14, 2347-2371, 2017; L. Magro et al., “Paper-based RNA detection and multiplexed analysis for Ebola virus diagnostics”, Scientific Reports 7, 1347, 2017). Without any degradation in quality, these tests replace the PCR technique, in terms of simplicity, compactness and costs, which makes it portable, usable in medical offices.However, in practice, the types of existing tests do not allow these uses to be developed at "Point of Care", or "in the field", for various reasons (no freeze-drying of products, no RNA extraction, complicated system to use for medical personnel, too complex surrounding instrumentation, etc.). BRIEF OVERVIEW OF THE INVENTION

[0007] In response to current (i.e. COVID-19 pandemic) and future health needs, the inventors have developed a new portable and easily deployable diagnostic device on a large scale allowing the diagnosis of pathogenic agents (viruses, bacteria, microorganisms, etc.) based on the detection of nucleic acids. This new device allows for rapid, reliable and safe testing, and also allows for testing one or more samples. Furthermore, this new device, which can be single-use and / or disposable, has the advantage of being inexpensive and usable locally. Thus, thanks to the diagnostic device of the invention, diagnoses could be carried out at the doctor's office, in the hospital emergency department, at the workplace, in pharmacies or even at home.

[0008] One of the first objects of the invention is therefore a portable diagnostic device in the form of a cylindrical housing. A second object of the invention relates to the various possible uses of the diagnostic device of the invention. Another object of the invention relates to methods easily carried out by a user, which allow the rapid diagnosis of pathogenic agents (viruses, bacteria, microorganisms, etc.) in a reliable and safe manner. Another object of the invention also relates to the methods for manufacturing the diagnostic device of the invention. LIST OF FIGURES

[0009] The following figures illustrate the invention, without limiting its scope.

[0010] [fig.l]

[0011] Figure 1 shows a perspective view of the diagnostic device of the invention.

[0012] (1) diagnostic device; (3) upper disc; (5) lower disc; (8) membrane; (12) reaction zone and (13a and 13b) pellets.

[0013] [fig.2]

[0014] Figure 2 represents a schematic top view of the diagnostic device of the invention.

[0015] (3) upper disc; (8) membrane; (9) window and (13a and 13b) pellets.

[0016] [fig.3]

[0017] Figure 3 shows a schematic bottom view of the diagnostic device of the invention when the latter comprises a recess.

[0018] (5) lower disc; (6) recess and (13a and 13b) pellets.

[0019] [fig.4]

[0020] Figure 4 shows an exploded perspective view of the diagnostic device of the invention illustrated in Figures 1, 2 and 3.

[0021] (1) diagnostic device; (3) upper disc; (5) lower disc; (7) opening receiver; (8) membrane; (9) window; (11) optical filter; (13a and 13b) pellets and (15) absorbing material.

[0022] [fig.5]

[0023] Figure 5 represents a vertical broken sectional view of the diagnostic device of the invention along the line VV (A) of Figure 2 (i.e. without recess at the level of the lower disc (5)) and (B) of Figure 3 (i.e. with recess at the level of the disc lower (5)).

[0024] (1) diagnostic device; (3) upper disc; (5) lower disc; (6) recess; (7) receiving opening; (8) membrane; (9) window; (10) waterproof material; (11) optical filters; (13b) pellet and (15) absorbent material.

[0025] [fig.6]

[0026] Figure 6 represents a perspective top view of a first variant implementation of the diagnostic device of the invention making it possible to carry out eight tests on the same device.

[0027] (2) diagnostic block (black triangle); (3) upper disc; (4) detection unit (white arc); (7) receiving opening and (9a and 9b) windows.

[0028] [fig.7]

[0029] Figure 7 represents a perspective top view of a second variant implementation of the diagnostic device of the invention making it possible to carry out twelve tests on the same device.

[0030] (3) upper disc; (8) membranes; (12a, 12b) reaction zones and (13a and 13b) lozenges.

[0031] [fig.8]

[0032] Figure 8 shows a bottom view of the upper disc of the diagnostic device of the invention, which comprises three-dimensional elements for assisting rotational movement.

[0033] (3) upper disc; (8) membranes; (11) optical filter; (16) groove; (17) skirt and (18) slide.

[0034] [fig.9]

[0035] Figure 9 shows a schematic view of the plans for printing the upper disc of the diagnostic device of the invention using a 3D printer. The figures and numbers correspond to dimensions in millimeters (mm).

[0036] [fig.10]

[0037] Figure 10 shows a schematic view of the plans for printing the lower disc of the diagnostic device of the invention using a 3D printer. The figures and numbers correspond to dimensions in millimeters (mm).

[0038] [fig.ll]

[0039] Devices used for the detection of DENV2 and SARS-CoV-2 RNA in biological samples, (a) Device for the detection of DENV2, sample containing DENV2 RNA (positive sample). Black bar: fluorescence intensity of the internal control measured at t = 45 minutes after subtraction of the initial fluorescence. Gray hatched bar: fluorescence intensity of the DENV2 RT-LAMP pellet measured at t = 45 min after subtraction of the initial fluorescence, (b) Device for the detection of DENV2, negative sample. Black bar: fluorescence intensity of Fluorescence of the internal control measured at t = 45 minutes after subtraction of the initial fluorescence. White bar: Fluorescence intensity of the RT-LAMP DENV2 reaction pellet measured at t = 45 min after subtraction of the initial fluorescence, (c) Image of the DENV2 pellets, positive sample on the left, negative on the right. Photograph taken with a smartphone, (d) Device for the detection of SARS-CoV-2, sample containing SARS-CoV-2 RNA (positive sample). Black bar: Fluorescence intensity of the internal control measured at t = 45 minutes after subtraction of the initial fluorescence. Hatched gray bar: Fluorescence intensity of the RT-LAMP SARS-CoV-2 pellet measured at t = 45 min after subtraction of the initial fluorescence, (e) Device for the detection of DENV2, negative sample. Black bar: Fluorescence intensity of the internal control measured at t = 45 minutes after subtraction of the initial fluorescence.White bar: Fluorescence intensity of the RT-LAMP SARS-CoV-2 pellet measured at t = 45 min after subtraction of the initial fluorescence, (f) images of the SARS-CoV-2 pellets, positive sample on the left, negative on the right. Photograph taken with a digital camera. DETAILED DESCRIPTION

[0040] The invention consists in coupling an extraction unit and an amplification / detection unit in a portable device, which comprises reagents lyophilized in situ. Examples of this device and its variants are illustrated in Figures 1 to 10.

[0041] A first aspect of the invention relates to a portable diagnostic device in the form of a cylindrical housing including means:

[0042] • extraction of nucleic acids (DNA and / or RNA) from a sample to be tested, • isothermal amplification (eg (RT)-LAMP, (RT)-LAMP-QUASR [Quenching of Unincorporated Amplification Signal Reporters]) of at least one nucleic acid sequence of interest likely to be present in the sample to be tested, and • detection of amplicons if said at least one nucleic acid sequence of interest is present in the sample to be tested.

[0043] A first very general embodiment of the invention according to this first aspect concerns a portable diagnostic device (1) in the form of a cylindrical case comprising two superimposed coaxial discs (3, 5) mounted to rotate relative to each other and defining a closed volume, namely:

[0044] • an upper disc (3) comprising:

[0045] - at least one receiving opening (7) for a biological sample to be tested which is provided with a membrane (8) which is biologically compatible and capable of allowing the extraction and retention of nucleic acids from this sample biological in the presence of appropriate reagents, and at least one window (9) which is either equipped with an optical filter (11) integrated into the mass of the upper disc, or capable of receiving an optical filter (11) positionable on said window in a removable manner,

[0046] this receiving opening (7) and this window (9) being angularly spaced apart by a

[0047]

[0048] central angle of at least 30°, • a lower disc (5) comprising: - an absorbent material (15) capable of containing a volume of liquid, and at least one reaction zone (12) comprising at least one, preferably at least two, biologically compatible, adjacent, independent pellets (13a, 13b) capable of enabling the amplification and detection of at least one nucleic acid sequence of interest likely to be present in the above-mentioned biological sample in the presence of appropriate reagents,

[0049] the receiving opening (7), the window (9), the absorbent material (15) and the reactive zone tional (12) forming a detection unit (4) such that, by rotation, the upper disc can successively

[0050] move from a first position in which, for this detection unit (4), its receiving opening (7) is located in line with its absorbent material (15), so as to allow the extraction and retention of the above-mentioned nucleic acids, and its window (9) is located in line with its reaction zone (12), at a second position in which, for this detection unit (4), its receiving opening (7) is located in line with its reaction zone (12) and covers it in part or in full so as to allow the elution of the above-mentioned nucleic acids, and position itself, by rotation, on the aforementioned first position, so that, for this detection unit (4), its window (9) is located at the right of its reaction zone (12), so as to allow the amplification and detection of the aforementioned at least one nucleic acid sequence of interest likely to be present in the biological sample.

[0051] As described, the transition from the above-mentioned second position to the above-mentioned first position can therefore be done either by a rotation opposite to that allowing the passage from the above-mentioned first position to the above-mentioned second position, or by a rotation in the same direction as the latter. Advantageously, this is done by a rotation opposite, which allows to return to the initial position more directly, by a shorter path, avoiding that the observation window (9) approaches the absorbent material (15). The invention then relates to the diagnostic device (1) as described above in which the passage from the above-mentioned second position to the above-mentioned first position is done by a rotation opposite to that allowing the passage from the above-mentioned first position to the aforementioned second position. In other words, a very general advantageous embodiment of the invention relates to a portable diagnostic device (1) in the form of a cylindrical housing comprising two superimposed coaxial discs (3, 5) mounted to rotate relative to each other and defining a closed volume, namely:

[0052] • an upper disc (3) comprising:

[0053] - at least one receiving opening (7) for a biological sample to be tested which is provided with a membrane (8) which is biologically compatible and capable of allowing the extraction and retention of nucleic acids from this biological sample in the presence of appropriate reagents, and - at least one window (9) which is either equipped with an optical filter (11) integrated into the mass of the upper disc, or capable of receiving an optical filter (11) positionable on said window in a removable manner,

[0054] this receiving opening (7) and this window (9) being angularly spaced apart by a central angle of at least 30°, in particular by a central angle of between 30° and 320°,

[0055] • a lower disc (5) comprising:

[0056] - an absorbent material (15) capable of containing a volume of liquid, and - at least one reaction zone (12) comprising at least one, preferably at least two, biologically compatible, adjacent, independent pellets (13a, 13b) capable of enabling the amplification and detection of at least one nucleic acid sequence of interest likely to be present in the above-mentioned biological sample in the presence of appropriate reagents,

[0057] the receiving opening (7), the window (9), the absorbent material (15) and the reaction zone (12) forming a detection unit (4) such that, by rotation, the upper disc can successively

[0058] • move from a first position in which, for this detection unit (4), its receiving opening (7) is located in line with its absorbent material (15), so as to allow the extraction and retention of the above-mentioned nucleic acids, and its window (9) is located in line with its reaction zone (12), • in a second position in which, for this detection unit (4), its receiving opening (7) is located at the right of its reaction zone (12) and covers it in part or in full so as to allow the elution of the above-mentioned nucleic acids,

[0059] the arrangement of the absorbent material (15) being such that, during rotation, the window (9) cannot come to the right and into contact with it, and

[0060] • return, by an inverse rotation, to the aforementioned first position, so that, for this detection unit (4), its window (9) is located at the right of its reaction zone (12), so as to allow the amplification and the detection of the at least one nucleic acid sequence of interest likely to be present in the biological sample,

[0061] the arrangement of the absorbent material (15) being such that, during reverse rotation, the window (9) cannot come to the right and into contact with it.

[0062] Another very general embodiment of the invention relates to the diagnostic device (1) as described above in which the lower disc (5) further comprises at least one recess (6) which is either equipped with a waterproof material (10) integrated into the mass of the lower disc, or capable of receiving a waterproof material (10) positionable on said recess in a removable manner, and

[0063] which is positioned in line with said at least one reaction zone (12) and integral with it, and in particular allowing the light to reach said at least one, preferably at least two, biologically compatible pellets (13a and 13b) of said at least one reaction zone (12).

[0064] In other words, this other general embodiment concerns the portable diagnostic device (1) in the form of a cylindrical case as described above comprising two superimposed coaxial discs (3, 5) mounted to rotate relative to each other and defining a closed volume, namely:

[0065] • an upper disc (3) comprising:

[0066] - at least one receiving opening (7) of a biological sample to be tested which is provided with a membrane (8) which is biologically compatible and capable of allowing the extraction and retention of nucleic acids from this biological sample in the presence of appropriate reagents, and - at least one window (9) which is either equipped with an optical filter (11) integrated into the mass of the upper disc, or capable of receiving an optical filter (11) positionable on said window in a removable manner,

[0067] this receiving opening (7) and this window (9) being angularly spaced apart by an angle at the center of at least 30°,

[0068] • a lower disc (5) comprising:

[0069] - an absorbent material (15) capable of containing a volume of liquid, - at least one reaction zone (12) comprising at least one, preferably at least two, biologically compatible, adjacent, independent pellets (13a, 13b) capable of enabling the amplification and detection of at least one nucleic acid sequence of interest likely to be present in the above-mentioned biological sample in the presence of appropriate reagents, and - at least one recess (6) which is either equipped with a waterproof material (10) integrated into the mass of the lower disc, or capable of receiving a waterproof material (10) positionable on said recess in a removable manner, and

[0070] which is positioned to the right of said at least one reaction zone (12) and integral therewith, and in particular allowing light to reach said at least one, preferably at least two, biologically compatible pellets (13a and 13b) of said at least one reaction zone (12);

[0071] the receiving opening (7), the window (9), the absorbent material (15), the reaction zone (12) and the recess (6) forming a detection unit (4) such that, by rotation, the upper disc can successively

[0072] move from a first position in which, for this detection unit (4), its receiving opening (7) is located in line with its absorbent material (15), so as to allow the extraction and retention of the above-mentioned nucleic acids, and its window (9) is located in line with its reaction zone (12), at a second position in which, for this detection unit (4), its receiving opening (7) is located in line with its reaction zone (12) and covers it in part or in full so as to allow the elution of the above-mentioned nucleic acids, and position itself, by rotation, on the aforementioned first position, so that, for this detection unit (4), its window (9) is located at the right of its reaction zone (12), so as to allow the amplification and detection of the aforementioned at least one nucleic acid sequence of interest likely to be present in the biological sample.

[0073] Similarly, the passage from the above-mentioned second position to the above-mentioned first position can therefore be achieved either by a rotation opposite to that allowing the passage from the above-mentioned first position to the above-mentioned second position, or by a rotation in the same direction as the latter. Advantageously, this other general embodiment concerns the portable diagnostic device (1) in the form of a cylindrical housing as described above comprising two superimposed coaxial discs (3, 5) mounted to rotate relative to each other and defining a closed volume, namely:

[0074]

[0075] an upper disc (3) comprising: at least one receiving opening (7) for a biological sample to be tested which is provided with a membrane (8) which is biologically compatible and capable of allowing the extraction and retention of nucleic acids from this biological sample in the presence of appropriate reagents, and at least one window (9) which is either equipped with an optical filter (11) integrated into the mass of the upper disc, or capable of receiving an optical filter (11) positionable on said window in a removable manner,

[0076] this receiving opening (7) and this window (9) being angularly spaced apart by a central angle of at least 30°, in particular a central angle of between 30° and 320°,

[0077] a lower disc (5) comprising:

[0078] an absorbent material (15) capable of containing a volume of liquid, at least one reaction zone (12) comprising at least one, preferably at least two, biologically compatible, adjacent, independent pellets (13a, 13b) capable of allowing the amplification and detection of at least one nucleic acid sequence of interest likely to be present in the above-mentioned biological sample in the presence of appropriate reagents, and at least one recess (6) which is either equipped with a waterproof material (10) integrated into the mass of the lower disc, or capable of receiving a waterproof material (10) positionable on said recess in a removable manner, and

[0079] which is positioned in line with said at least one reaction zone (12) and integral with it, and in particular allowing the light to reach said at least one, preferably at least two, biologically compatible pellets (13a and 13b) of said at least one reaction zone (12);

[0080] the receiving opening (7), the window (9), the absorbent material (15), the reaction zone (12) and the recess (6) forming a detection unit (4) such that, by rotation, the upper disc can successively

[0081] • move from a first position in which, for this detection unit (4), its receiving opening (7) is located at the right of its absorbent material (15), so as to allow the extraction and retention of the above-mentioned nucleic acids, and its window (9) is located at the right of its reaction zone (12), to a second position in which, for this detection unit (4), its receiving opening (7) is located at the right of its reaction zone (12) and covers it in part or in full so as to allow the elution of the above-mentioned nucleic acids,

[0082] the arrangement of the absorbent material (15) being such that, during rotation, the window (9) cannot come to the right and into contact with it, and

[0083] return, by a reverse rotation, to the aforementioned first position, so that, for this detection unit (4), its window (9) is located at the right of its reaction zone (12), so as to allow the amplification and detection of the aforementioned at least one nucleic acid sequence of interest likely to be present in the biological sample,

[0084] the arrangement of the absorbent material (15) being such that, during reverse rotation, the window (9) cannot come to the right and into contact with it.

[0085] By "a portable diagnostic device (1) in the form of a cylindrical case" is meant a device substantially the size of a hand (e.g. from 4.00 cm to 20.00 cm in diameter, i.e. 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 cm) which is easily graspable and ergonomic. Due to the materials chosen, it is inexpensive to produce. It is also light which facilitates its use. In addition, and due to its size, it is also easily transportable from one room to another, e.g. a laboratory or a hospital. According to another embodiment, the invention therefore relates to the diagnostic device (1) as described above in which the upper (3) and lower (5) discs are made of a material chosen from: polylactic acid (PLA) and polycarbonate (CO-O-pPh-C(CH3)2-pPh-O)n). In particular, it should be noted that the materials used to manufacture these discs can be opaque or transparent (i.e. let light pass through). According to another embodiment, the invention also relates to the diagnostic device (1) as described above, the diameter to height ratio of the device being comprised from 5 to 30. In particular, the diameter to height ratio of the device is comprised from 5 to 10, from 10 to 15, from 15 to 20, from 20 to 25, from 25 to 30, from 10 to 20 or from 15 to 25.In particular, the diameter to height ratio of the device is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30.

[0086] Advantageously, the diagnostic device (1) of the invention also comprises three-dimensional structures which help to guide the rotations of the upper disc on the lower disc and vice versa, thus making it more ergonomic and even easier to use. According to another embodiment, the invention therefore relates to the diagnostic device (1) as described above in which said rotations are guided by a system of groove(s) (16) and lug(s), also called cleat(s).

[0087] Alternatively, the diameter of the upper (3) and lower (5) discs may be different. The system of groove(s) and lug(s) may then be replaced or supplemented by a system of skirt(s) (17) and slide(s) (18). According to this embodiment, the invention therefore relates to the diagnostic device (1) as described above in which the upper disc (3) is larger than the lower disc (5) or in which the lower disc (5) is larger than the upper disc (3). In particular, it should be noted that the skirt(s) are arranged on the larger disc and the slide(s) (18) on the upper (3) and lower (5) discs to guide the rotations of the device. Therefore, according to another embodiment, the invention relates to the diagnostic device (1) as described above in which said rotations are guided by a system of skirt(s) (17) and slide(s) (18).

[0088] Advantageously, the diagnostic device (1) of the invention also comprises three-dimensional structures which assist in the assembly of the upper (3) and lower (5) discs onto each other. For example, the diagnostic device (1) of the invention comprises a stud in the center of the lower disc (5), or of the upper disc (3), which fits into a circular recess hollowed out (cut out) in the center of the upper disc (3), or of the lower disc (5). This can also be a ring (i.e. a sup additional) on which the upper (3) and lower (5) discs fit (clip), said ring comprising means allowing the discs to turn on each other (eg slides, grooves, lugs or stops, etc.).

[0089] Advantageously, the diagnostic device (1) of the invention also comprises parts, molded or not with (on) the discs, allowing the introduction of the biological sample to be tested and the reagents (or buffers) without loss of liquid.

[0090] By "biological sample to be tested" is meant any type of biological sample likely to contain nucleic acids. This may include, but is not limited to, blood, urine, sweat, saliva, secretions from the ENT sphere (e.g. nose, ears, throat), cerebrospinal fluid, lymphatic fluid, amniotic fluid, etc. It may also include nasopharyngeal swabs, cervicovaginal swabs, etc. The biological sample to be tested may also come from a mammal. In particular, it comes from a mammal chosen from: humans (i.e. children, adults, women and men), monkeys, felines, canines, equines, deer, cattle, sheep and poultry. Preferably, it is a human sample.

[0091] By "nucleic acids" is meant deoxyribonucleic acid (DNA) and / or ribonucleic acid (RNA). The invention, due to its circular configuration, also advantageously makes it possible to carry out the elution of nucleic acids, and the amplification and detection of at least one nucleic acid sequence (DNA and / or RNA) of interest likely to be present in a biological sample at the same location of the device, thus avoiding too many manipulations. Similarly, the invention is configured so that the window (9) equipped with an optical filter (11) can never be at the right angle and in contact with the absorbent material (15), called capillary buffer, thus avoiding contamination of the reaction zone (12) by the absorbents caught in the absorbent material (15).

[0092] By "nucleic acid sequence of interest" is meant a nucleic acid sequence present in the genome of a pathogen (virus, bacterium, microorganism, etc.) and the detection of which in a biological sample allows the diagnosis of a pathology. It may therefore be, for example, a DNA sequence contained in the genomic plasmid of a bacterium (e.g. Salmonella, Pseudomonas, Staphylococcus, Escherichia, Streptococcus, Helicobacter, etc.) or an RNA sequence contained in the transcriptome of said bacterium. It may also be a DNA and / or RNA sequence contained in the genome of a virus (e.g. SARS-CoV-2, Dengue virus, human immunodeficiency virus [HIV], etc.). The invention being a diagnostic device, it makes it possible to test a biological sample to find out whether it presents, for example, an infection (viral, bacterial or another microorganism, etc.).), hence the expression “likely to be present in the aforementioned biological sample”. In other words, in the current health context (i.e. . COVID-19 pandemic), the diagnostic device of the invention makes it possible to test a patient, or even the population, to find out whether or not they have an infection (i.e. SARS-CoV-2 viral infection) and to take appropriate measures (medical treatment, confinement, etc.). By "nucleic acid sequence of interest", we also mean a nucleic acid sequence present in the genome (DNA) or transcriptome (RNA) of a mammal (e.g. humans) whose presence would be linked to a predisposition to a pathology or to the diagnosis of a pathology. The diagnostic device of the invention as described therefore offers the advantage of being versatile and of easily adapting to the diagnosis sought and / or the health context.

[0093] By “detection unit” is meant the minimum assembly which makes it possible to implement the diagnosis of a biological sample to be tested. As indicated, this is provided with:

[0094] • a biologically compatible membrane (8), which receives the biological sample to be tested, and which makes it possible to extract the nucleic acids (DNA and / or RNA) from the sample to be tested and to retain them by affinity in the presence of the appropriate reagents when it is at the level of the absorbent material; • an absorbent material (15), which collects and stores the liquids which pass through the aforementioned membrane (8); • a reaction zone (12) comprising at least one, preferably at least two, biologically compatible pellets (13a and 13b) capable of allowing, after elution of the nucleic acids (DNA and / or RNA) from the above-mentioned membrane (8), the amplification and detection of at least one nucleic acid sequence of interest likely to be present in the biological sample in the presence of appropriate reagents; • a window (9) equipped with an optical filter (11) on the upper disc (3) and capable of being positioned in line with the reaction zone (12); and • possibly a recess (6) equipped with a waterproof material (10) on the lower disc (5) below the reaction zone (12) and in particular allowing the light to reach the at least one, preferably at least two, pellets (13a and 13b).

[0095] By "window" is meant an opening cut into the material of the upper disc (3). However, in order to ensure the hermeticity and sealing of the diagnostic device (1) of the invention, the window(s) are provided with an optical filter (11). This optical filter can be positioned in a removable manner (e.g. adhesive film, flap, shutter, etc.) or be integrated into the mass of the upper disc (3), or even placed on an external device. By "optical filter" is meant a device which allows all or part of the light radiation to pass through and which makes it possible to analyze the light ray at the output of the test. Thus, by "optical filter" is meant materials having specific optical properties (eg bandpass, bandstop, highpass, lowpass), colored filters (eg photographic gelatins, etc.), devices such as optical cubes allowing the reading of a fluorescent signal. Furthermore, and in the context of a colorimetric test or a turbidimetric test (or any other amplification visualization method) it may be a filter helping to read the test. In particular, the invention relates to the diagnostic device (1) as described above in which the optical filter is chosen from: a bandpass filter, a bandstop filter, a highpass filter, a lowpass filter, photographic gelatin and an optical cube.

[0096] By "recess" is meant an opening cut into the material of the lower disc (5). However, in order to ensure the hermeticity and sealing of the diagnostic device (1) of the invention, the recess(es) are provided with a waterproof material (10). This waterproof material can be positioned in a removable manner (e.g. adhesive film, valve, flap, etc.) or be integrated into the mass of the lower disc (5), or even placed on an external device. By "waterproof material" is meant any type of plastic or other material, impermeable and compatible with amplification reactions. It can be transparent and allow light to pass through in the context of a fluorescence reading by transmission. It can also be opaque in the context of a colorimetric reading or a fluorescence reading by reflection.The invention therefore relates to the diagnostic device (1) as described above in which the waterproof material is chosen from: a band-pass filter, a band-stop filter, a high-pass filter, a low-pass filter, photographic gelatin and an optical cube.

[0097] By "absorbent material", also called capillary pad or absorbent sponge, is meant a material which, at the time of extraction of the nucleic acids contained in the sample to be tested at the membrane (8) in the presence of the appropriate reagents [also called buffers], is capable of absorbing, containing and storing the liquids which pass through the membrane (8). This material, capable of containing a volume of at least 1 mL (1 cm3), can also be placed on a receptacle designed to accommodate it. This absorbent material can in particular be an absorbent pad made of cellulose, fiberglass or cotton wool, any other absorbent material or even it can be, for example, an absorbent pad usable for a conventional immunochromatographic test.According to another embodiment, the invention therefore relates to the diagnostic device (1) as described above in which the absorbent material (15) is arranged in a receptacle which is positioned either on the upper surface of the lower disc or integrated into the mass of the latter. According to another embodiment, the invention also relates to the diagnostic device (1) as described above in which the absorbent material (15) is capable of containing a volume of at least 1 mL (1 cm3). For the purposes of the invention, "at . less than 1 mL” means a volume of 1 mL to 2 mL or 1 mL to 1.50 mL. “At least 1 mL” also means that the volume absorbable by the absorbent material (15) is 1 mL; 1.10 mL; 1.15 mL; 1.20 mL; 1.25 mL; 1.30 mL; 1.35 mL; 1.40 mL; 1.45 mL; 1.50 mL; 1.55 mL; 1.60 mL; 1.65 mL; 1.70 mL; 1.75 mL; 1.80 mL; 1.85 mL; 1.90 mL; 1.95 mL or 2.00 mL. In particular, the invention relates to the diagnostic device (1) as described above in which the absorbent material (15) is arranged in a receptacle which is positioned either on the upper surface of the lower disc or integrated into the mass of the latter, and said absorbent material (15) is preferably capable of containing a volume of at least 1 mL.In particular, the invention also relates to the diagnostic device (1) as described above in which the absorbent material (15) is chosen from: an absorbent pad made of cellulose, glass fiber or cotton wool and an absorbent pad usable for an immunochromatographic test.

[0098] By "biologically compatible membrane capable of allowing the extraction and retention of nucleic acids... in the presence of appropriate reagents", also called capture membrane, is meant a membrane capable of receiving a biological sample to be tested, of withstanding the addition of reagents (or buffers) allowing the extraction of nucleic acids (DNA and / or RNA) contained in said sample and of having physicochemical properties capable of retaining (e.g. by affinity) the extracted nucleic acids before their elution. This membrane may in particular be made of cellulose, silica, fiberglass, or any other porous material allowing the physicochemical retention of nucleic acids.According to another embodiment, the invention therefore relates to the diagnostic device (1) as described above in which the membrane (8) biologically compatible and capable of allowing the extraction and retention of nucleic acids is in a material chosen from: cellulose, silica and fiberglass.

[0099] By "biologically compatible pellets ... capable of allowing the amplification and detection of at least one nucleic acid sequence of interest ... in the presence of appropriate reagents", we mean pellets made of a material capable of collecting the nucleic acids (DNA and / or RNA) extracted from the biological sample to be tested after their elution from the membrane (8). This material, permeable to light, also allows, in the presence of appropriate reagents (e.g. primers, fluorophores, enzymes, dNTPs [any deoxyribonucleoside triphosphate], etc.), to support and ensure the amplification then the visual detection (fluorescence, colorimetry, etc.) of at least one nucleic acid sequence likely to be present in the biological sample to be tested.These pellets can be made of fiberglass (containing or not an adjuvant binder), cellulose, or any other porous material that is biocompatible or made biocompatible by prior treatment. Also note . that these pellets are adjacent, i.e. side by side, while being independent, i.e. that they do not touch each other, thus avoiding contamination between adjacent pellets. In addition, these pellets per detection unit are at least one in number, preferably at least two in number, i.e. a detection unit may comprise one, two, three, four, five or six pellets. According to another embodiment, the invention therefore relates to the diagnostic device (1) as described above in which the at least two biologically compatible amplification pellets (13a, 13b) are made of a material chosen from: glass fiber (containing or not an adjuvant binder) and cellulose.

[0100] Advantageously, these suitable reagents allowing the amplification and detection of at least one nucleic acid sequence (DNA and / or RNA) can be lyophilized in situ on the surface (or in) the at least one, preferably at least two, biologically compatible pellets (13a and 13b) of the reaction zone (12). Since lyophilization is one of the best ways to preserve the integrity of these reagents over the long term, it is possible, after having manufactured the diagnostic device (1) of the invention, to lyophilize these reagents therein. The diagnostic device (1) of the invention can then be prepared in advance on a large scale, stored over the long term and be deployed quickly in the event of a health crisis. It can also be used immediately after the lyophilization step described above.Advantageously, the invention therefore relates to the diagnostic device (1) as described above in which at least one reaction zone (12) comprises at least one, preferably at least two, biologically compatible, adjacent and independent amplification pellets (13a, 13b), at least one containing lyophilized reagents capable of allowing the amplification and detection of at least one nucleic acid sequence of interest likely to be present in the above-mentioned biological sample.

[0101] In other words, the invention also relates to the portable diagnostic device (1) in the form of a cylindrical housing as described above comprising two superimposed coaxial discs (3, 5) mounted to rotate relative to each other and defining a closed volume, namely:

[0102] • an upper disc (3) comprising:

[0103] - at least one receiving opening (7) of a biological sample to be tested which is provided with a membrane (8) which is biologically compatible and capable of allowing the extraction and retention of nucleic acids from this biological sample in the presence of appropriate reagents, and - at least one window (9) which is either equipped with an optical filter (11) integrated into the mass of the upper disc, or capable of receiving an optical filter (11) positionable on said window in a removable manner,

[0104] this receiving opening (7) and this window (9) being angularly spaced apart by a central angle of at least 30°,

[0105] • a lower disc (5) comprising:

[0106] - an absorbent material (15) capable of containing a volume of liquid, - at least one reaction zone (12) comprising at least one, preferably at least two, biologically compatible, adjacent and independent amplification pellets (13a, 13b), at least one containing lyophilized reagents capable of allowing the amplification and detection of at least one nucleic acid sequence of interest likely to be present in the aforementioned biological sample, and - possibly at least one recess (6) which is either equipped with a waterproof material (10) integrated into the mass of the lower disc, or capable of receiving a waterproof material (10) positionable on said recess in a removable manner, and

[0107] which is positioned in line with said at least one reaction zone (12) and integral with it, and in particular allowing the light to reach said at least one, preferably at least two, biologically compatible pellets (13a and 13b) of said at least one reaction zone (12);

[0108] the receiving opening (7), the window (9), the absorbent material (15), the reaction zone and possibly the recess (6) forming a detection unit (4) such that, by rotation, the upper disc can successively

[0109] • move from a first position in which, for this detection unit (4), its receiving opening (7) is located in line with its absorbent material (15), so as to allow the extraction and retention of the above-mentioned nucleic acids, and its window (9) is located in line with its reaction zone (12), • in a second position in which, for this detection unit (4), its receiving opening (7) is located at the right of its reaction zone (12) and covers it in part or in full so as to allow the elution of the above-mentioned nucleic acids, and • position itself, by rotation, on the aforementioned first position, so that, for this detection unit (4), its window (9) is located at the right of its reaction zone (12), so as to allow the amplification and detection of the aforementioned at least one nucleic acid sequence of interest likely to be present in the biological sample.

[0110] In the same way, the passage from the above-mentioned second position to the above-mentioned first position can therefore be done either by a rotation opposite to that allowing the passage from the above-mentioned first position to the above-mentioned second position, or by a rotation in the same direction as the latter. Advantageously, the invention also relates to the portable diagnostic device (1) in the form of a cylindrical housing as described above. top comprising two superimposed coaxial discs (3, 5) mounted to rotate relative to each other and defining a closed volume, namely: [YES]

[0112] an upper disc (3) comprising: at least one receiving opening (7) for a biological sample to be tested which is provided with a membrane (8) which is biologically compatible and capable of allowing the extraction and retention of nucleic acids from this biological sample in the presence of appropriate reagents, and at least one window (9) which is either equipped with an optical filter (11) integrated into the mass of the upper disc, or capable of receiving an optical filter (11) positionable on said window in a removable manner,

[0113] this receiving opening (7) and this window (9) being angularly spaced apart by a central angle of at least 30°, in particular a central angle of between 30° and 320°,

[0114]

[0115] a lower disc (5) comprising: an absorbent material (15) capable of containing a volume of liquid, at least one reaction zone (12) comprising at least one, preferably at least two, biologically compatible, adjacent and independent amplification pellets (13a, 13b), at least one containing lyophilized reagents capable of allowing the amplification and detection of at least one nucleic acid sequence of interest likely to be present in the aforementioned biological sample, and possibly at least one recess (6) which is either equipped with a waterproof material (10) integrated into the mass of the lower disc, or capable of receiving a waterproof material (10) positionable on said recess in a removable manner, and

[0116] which is positioned in line with said at least one reaction zone (12) and integral with it, and in particular allowing the light to reach said at least one, preferably at least two, biologically compatible pellets (13a and 13b) of said at least one reaction zone (12);

[0117] the receiving opening (7), the window (9), the absorbent material (15), the reaction zone and possibly the recess (6) forming a detection unit (4) such that, by rotation, the upper disc can successively

[0118] • move from a first position in which, for this detection unit (4), its receiving opening (7) is located in line with its absorbent material (15), so as to allow the extraction and retention of the above-mentioned nucleic acids, and its window (9) is located in line with its reaction zone (12), at a second position in which, for this detection unit (4), its receiving opening (7) is located in line with its reaction zone (12) and the covers in part or in whole so as to allow the elution of the above-mentioned nucleic acids,

[0119] the arrangement of the absorbent material (15) being such that, during rotation, the window (9) cannot come to the right and into contact with it, and

[0120] • return, by an inverse rotation, to the above-mentioned first position, so that, for this detection unit (4), its window (9) is located at the right of its reaction zone (12), so as to allow the amplification and the detection of the above-mentioned at least one nucleic acid sequence of interest likely to be present in the biological sample,

[0121] the arrangement of the absorbent material (15) being such that, during reverse rotation, the window (9) cannot come to the right and into contact with it.

[0122] In particular, this embodiment may comprise:

[0123] • the diagnostic device (1) as described above in which the reaction zone (12) comprises two pellets, one (13a or 13b) serving as a test pellet and the other (13b or 13a) serving as a negative control pellet; and / or • the diagnostic device (1) as described above in which the reaction zone (12) comprises two pellets, one (13a or 13b) serving as a test pellet and the other (13b or 13a) serving as a positive control pellet; and / or • the diagnostic device (1) as described above in which the reaction zone (12) comprises three pellets, one serving as a test pellet and the other two serving as negative control and positive control pellets.

[0124] By "test pellet" is meant a biologically compatible pellet on which all the reagents allowing the amplification and detection of at least one nucleic acid sequence of interest likely to be present in the biological sample to be tested are lyophilized in situ. In this way, if a positive signal is obtained at the end of the test then the sample contains said at least one nucleic acid sequence of interest and the diagnosis is positive (subject to the results obtained on the control pellet(s)). In other words, in the current health context (i.e. COVID-19 pandemic), if a positive signal is obtained on this test pellet, the patient has a SARS-CoV-2 infection and the appropriate measures (medical treatment, confinement, etc.) can be taken.

[0125] By "negative control pellet" is meant a biologically compatible pellet on which none of the reagents allowing the amplification and detection of at least one nucleic acid sequence of interest likely to be present in the biological sample to be tested is lyophilized. Also meant is a biologically compatible pellet on which one of the elements essential for the amplification or detection of at least one nucleic acid sequence of interest likely to be present in the biological sample to be tested is not lyophilized. In this way, This pellet at the end of the test cannot positively reveal the presence of at least one nucleic acid sequence of interest. If this happens, the test is invalidated and must be repeated.

[0126] By “positive control pellet” is meant a biologically compatible pellet on which is lyophilized in situ:

[0127] • all of the reagents allowing the amplification and detection of at least one nucleic acid sequence of interest likely to be present in the biological sample to be tested or of another nucleic acid sequence; and • said at least one nucleic acid sequence of interest or said other nucleic acid sequence.

[0128] In this way, this pellet at the end of the test must positively reveal the presence of a nucleic acid sequence. If this does not happen, the test is invalidated and must be repeated. Advantageously, it should be noted that the nucleic acid sequence revealed by means of the positive control pellet and the in situ lyophilized reagents can be dissociated (different) from said at least one nucleic acid sequence of interest likely to be present in the biological sample to be tested.

[0129] Among these embodiments, the invention therefore relates to the diagnostic device (1) as described above in which the reaction zone (12) comprises:

[0130] • a test pellet (13a or 13b) on which all of the reagents allowing the amplification and detection of at least one nucleic acid sequence of interest likely to be present in the biological sample to be tested are lyophilized in situ; and • a negative control pellet (13b or 13a) on which: • none of the reagents allowing the amplification and detection of at least one nucleic acid sequence of interest likely to be present in the biological sample to be tested is lyophilized; or • one of the elements essential to the amplification or detection of at least one nucleic acid sequence of interest likely to be present in the biological sample to be tested is not lyophilized.

[0131] Among these embodiments, the invention also relates to the diagnostic device (1) as described above in which the reaction zone (12) comprises:

[0132] • a test pellet (13a or 13b) on which all of the reagents allowing the amplification and detection of at least one nucleic acid sequence of interest likely to be present in the biological sample to be tested are lyophilized in situ; and • a positive control pellet (13b or 13a) on which the following is freeze-dried in situ: • all the reagents allowing the amplification and detection of at at least one nucleic acid sequence of interest likely to be present in the biological sample to be tested or another nucleic acid sequence; and • said at least one nucleic acid sequence of interest or said other nucleic acid sequence.

[0133] Among these embodiments, the invention also relates to the diagnostic device (1) as described above in which the reaction zone (12) comprises:

[0134] • a test pellet on which all of the reagents allowing the amplification and detection of at least one nucleic acid sequence of interest likely to be present in the biological sample to be tested are lyophilized in situ; • a negative control pellet (13b or 13a) on which: • none of the reagents allowing the amplification and detection of at least one nucleic acid sequence of interest likely to be present in the biological sample to be tested is lyophilized, or • one of the elements essential to the amplification or detection of at least one nucleic acid sequence of interest likely to be present in the biological sample to be tested is not lyophilized; and • a positive control pellet (13b or 13a) on which is lyophilized in situ: • all the reagents allowing the amplification and detection of at least one nucleic acid sequence of interest likely to be present in the biological sample to be tested or another nucleic acid sequence; and • said at least one nucleic acid sequence of interest or said other nucleic acid sequence.

[0135] Advantageously, this latter particular embodiment allows better reading of the results thanks to the color contrasts that can be observed between the three pellets. This also makes it possible to further secure the interpretation of the results. Alternatively, this embodiment comprising three pellets can be described as being the diagnostic device (1) as described above in which at least one, preferably at least two, biologically compatible amplification pellets (13a, 13b) contain lyophilized reagents capable of allowing the amplification and detection of at least one nucleic acid sequence of interest likely to be present in the above-mentioned biological sample, and at least one of which also contains a lyophilized nucleic acid of interest capable of being amplified by the above-mentioned lyophilized reagents.

[0136] As mentioned previously, the term “lyophilized reagents capable of enabling the amplification and detection of at least one nucleic acid sequence of interest” is understood to mean likely to be present in the aforementioned biological sample” the molecular tools (primers possibly modified at the 5' and / or 3' ends) capable of hybridizing to a nucleic acid sequence of interest (DNA and / or RNA), of amplifying it (enzymes, dNTPs) and of visually (fluorescence, colorimetry, etc.) revealing its presence. Among these means, we find, in a non-exhaustive manner, molecular tools allowing the implementation of isothermal nucleic acid (DNA and / or RNA) amplification technologies (e.g. RCA, LAMP, RPA, LAMP-QUASR, etc.) which do not require thermocycling. Note that RNA amplification requires an upstream step of reverse transcription (RT) of the RNA into DNA by means of specific enzymes (e.g. viral reverse transcriptases, etc.), which step is also carried out at the level of the pellets (13a and 13b) and whose molecular tools can also be lyophilized.For the purposes of the invention, "amplification" therefore means the step of reverse transcription of a target RNA (i.e. carrying said at least one nucleic acid sequence of interest) into DNA and the step of amplification of this DNA by a polymerase. Consequently, if said at least one nucleic acid sequence of interest likely to be present in the sample to be tested comes from an RNA sequence (e.g. viral genomic RNA whose presence is to be detected), it is essential that the device includes the means allowing its reverse transcription and amplification, then its detection (e.g. RT-LAMP, RT-LAMP-QUASR, etc.).

[0137] According to another embodiment, the invention therefore relates to the diagnostic device (1) as described above comprising at least one reaction zone (12) comprising at least one, preferably at least two, biologically compatible, adjacent and independent amplification pellets (13a, 13b), at least one containing lyophilized reagents capable of allowing the amplification and detection of at least one nucleic acid sequence of interest likely to be present in the above-mentioned biological sample, said lyophilized reagents being at least:

[0138] • a polymerase; • dNTPs; • a set of at least six primers comprising primer F3, primer B3, primer FIP, primer BIP, primer LoopF ​​and primer LoopB; and • an intercalating agent.

[0139] According to another embodiment, the invention relates to the diagnostic device (1) as described above comprising at least one reaction zone (12) comprising at least one, preferably at least two, biologically compatible, adjacent and independent amplification pellets (13a, 13b), at least one containing lyophilized reagents capable of allowing the amplification and the detection of at least one nucleic acid sequence of interest likely to be present in the aforementioned biological sample, said lyophilized reagents being at least:

[0140] • a reverse transcriptase; • a polymerase; • dNTPs; • a set of at least six primers comprising primer F3, primer B3, primer FIP, primer BIP, primer LoopF ​​and primer LoopB; and • an intercalating agent.

[0141] According to another embodiment, the invention also relates to the diagnostic device (1) as described above comprising at least one reaction zone (12) comprising at least one, preferably at least two, biologically compatible, adjacent and independent amplification pellets (13a, 13b), at least one containing lyophilized reagents capable of allowing the amplification and detection of at least one nucleic acid sequence of interest likely to be present in the above-mentioned biological sample, said lyophilized reagents being at least:

[0142] • a polymerase; • dNTPs; • a set of at least six primers including primer F3, primer B3, FIP primer, BIP primer, LoopF ​​primer and LoopB primer,

[0143] at least one of which, with the exception of primer F3 and primer B3, is modified in 5' by the addition of a fluorophore; and

[0144] • at least one oligonucleotide complementary to the FIP primer, the BIP primer, the LoopF ​​primer or the LoopB primer,

[0145] said oligonucleotide being capable of reversibly coupling to the primer of which it is the complement and said oligonucleotide being modified at 3' by a quencher.

[0146] According to a particular embodiment, the invention relates to the diagnostic device (1) as described above comprising at least one reaction zone (12) comprising at least one, preferably at least two, biologically compatible, adjacent and independent amplification pellets (13a, 13b), at least one containing lyophilized reagents capable of allowing the amplification and detection of at least one nucleic acid sequence of interest likely to be present in the above-mentioned biological sample, said lyophilized reagents being at least:

[0147] • a polymerase; • dNTPs; • a set of at least six primers including primer F3, primer B3, FIP primer, BIP primer, LoopF ​​primer and LoopB primer,

[0148] whose primers FIP, BIP, LoopF ​​and LoopB are modified in 5' by the addition of a fluorophore; and

[0149] • four oligonucleotides complementary to the primers FIP, BIP, LoopF ​​and LoopB,

[0150] said oligonucleotides being capable of reversibly coupling to the primers of which they are complementary and said oligonucleotides being modified in 3' by a quencher.

[0151] According to another embodiment, the invention relates to the diagnostic device (1) as described above comprising at least one reaction zone (12) comprising at least one, preferably at least two, biologically compatible, adjacent and independent amplification pellets (13a, 13b), at least one containing lyophilized reagents capable of allowing the amplification and detection of at least one nucleic acid sequence of interest likely to be present in the above-mentioned biological sample, said lyophilized reagents being at least:

[0152] • a reverse transcriptase; • a polymerase; • dNTPs; • a set of at least six primers including primer F3, primer B3, primer FIP, primer BIP, primer LoopF ​​and primer LoopB,

[0153] at least one of which, with the exception of primer F3 and primer B3, is modified in 5' by the addition of a fluorophore; and

[0154] • at least one oligonucleotide complementary to the FIP primer, the BIP primer, the LoopF ​​primer or the LoopB primer,

[0155] said oligonucleotide being capable of reversibly coupling to the primer of which it is the complement and said oligonucleotide being modified in 3' by a quencher.

[0156] According to a particular embodiment, the invention relates to the diagnostic device (1) as described above comprising at least one reaction zone (12) comprising at least one, preferably at least two, biologically compatible, adjacent and independent amplification pellets (13a, 13b), at least one containing lyophilized reagents capable of allowing the amplification and detection of at least one nucleic acid sequence of interest likely to be present in the above-mentioned biological sample, said lyophilized reagents being at least:

[0157] • a reverse transcriptase; • a polymerase; • dNTPs; • a set of at least six primers comprising primer F3, primer B3, primer FIP, primer BIP, primer LoopF ​​and primer LoopB,

[0158] of which primers FIP, BIP, LoopF ​​and LoopB are modified in 5' by the addition of a fluorophore; and

[0159] • four oligonucleotides complementary to the primers FIP, BIP, LoopF ​​and LoopB,

[0160] said oligonucleotides being capable of reversibly coupling to the primers of which they are complementary and said oligonucleotides being modified in 3' by a quencher.

[0161] Among the lyophilized reagents, MgSO4, Betaine and Trehalose can be added and lyophilized.

[0162] By "reverse transcriptase", also called reverse transcriptase, is meant an enzyme capable of converting RNA into DNA. This may be AMV reverse transcriptase or any other reverse transcriptase purified from a retro virus or retro transposon, or any other reverse transcriptase having undergone optimization by directed evolution or otherwise to improve its performance. In particular, the invention relates to the diagnostic device (1) as described above in which said reverse transcriptase is AMV reverse transcriptase.

[0163] By "polymerase" is meant an enzyme capable of replicating DNA into DNA. This may be the Bst polymerase enzyme, the GspSSD polymerase enzyme or any other polymerase enzyme used in nucleic acid amplification (PCR or isothermal amplification type). In particular, the invention relates to the diagnostic device (1) as described above in which said polymerase is chosen from: the Bst polymerase enzyme and the GspSSD polymerase enzyme.

[0164] By "dNTPs" is meant the mixture of the four deoxyribonucleotides: dATP (deoxy adenine tri-phosphate), dCTP (deoxy cytosine tri-phosphate), dGTP (deoxy guanine tri-phosphate) and dTTP (deoxy thymine tri-phosphate).

[0165] By "a set of at least six primers" is meant nucleic acid sequences allowing the isothermal amplification of at least one nucleic acid sequence of interest likely to be present in the biological sample to be tested. Furthermore, by "at least one of which ... is modified in 5' by the addition of a fluorophore" is meant that one, two, three or four primers are modified in 5' by the addition of a fluorophore with the exception of primers F3 and B3 which are never modified. These primers modified in 5' by the addition of a fluorophore can also be called probes since they allow the detection of the signal. Preferably, the invention uses four of them, modified in 5', namely: primer FIP, primer BIP, primer LoopF ​​and primer LoopB. In particular, these at least six primers can be:

[0166] • the primers of sequences SEQ ID NOs: 1 to 6 allowing the detection of a nucleic acid sequence of interest of Dengue virus serotype 2 (DENV2); or • primers with sequences SEQ ID NOs: 7 to 12 allowing the detection of a nucleic acid sequence of interest of the SARS-CoV-2 virus responsible for COVID-19 (Lin Yu et al., Rapid colorimetry detection of COVID-19 co-ronavirus using a reverse transcriptional loop-mediated isothermal amplification (RT-LAMP) diagnostic platform: iLACO; DOI: 10.1101 / 2020.02.20.20025874).

[0167] By "oligonucleotide" is meant a sequence of about ten nucleotides. For example, an oligonucleotide of 5 to 20 nucleotides, i.e. 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 nucleotides. By "complementary oligonucleotide" is meant an oligonucleotide that can bind specifically to a nucleic acid sequence. In this case, these are oligonucleotides complementary to the primers FIP, BIP, LoopF ​​and LoopB, at least one, or two, or three and preferably all four of which are modified in 3' by a quencher. The latter is a quencher capable of quenching the fluorophore grafted in 5' of one or more primers FIB, BIP, LoopF ​​and / or LoopB. In particular, this at least one and preferably four oligonucleotides are chosen from the sequences SEQ ID NOs: 13 to 17 including:

[0168] • the sequence SEQ ID NO: 13 is complementary to the BIP primer of sequence SEQ ID NO: 4 used in the diagnosis of an infection with Dengue virus serotype 2; and • the sequences SEQ ID NOs: 14 to 17 are respectively the complements of the sequences SEQ ID NOs: 9 to 12 used in the diagnosis of an infection with the SARS-CoV-2 virus.

[0169] By "intercalating agent" is meant a molecule capable of reversibly intercalating into DNA and becoming fluorescent when it is in the double helix. This may be SYBRGreen, a SYTO-type fluorophore (SYTO9, SYTO82, etc.), EvaGreen, ethidium bromide or any other DNA intercalating agent used in real-time nucleic acid amplification monitoring. In particular, the invention relates to the diagnostic device (1) as described above in which said intercalating agent is chosen from: SYBRGreen, a SYTO-type fluorophore (SYTO9, SYTO82, etc.), EvaGreen and ethidium bromide.

[0170] By "fluorophore", also called fluorochrome, is meant a chemical or protein substance capable of emitting fluorescence light after excitation. In particular, the invention relates to the diagnostic device (1) as described above in which said fluorophore is chosen from: Alexa Fluor type fluorophores (Alexa 350, Alexa 488, Alexa 555, Alexa 647, etc.), Cyanine type fluorophores (CY3, CY3.5, CY5, etc.), FAM type fluorophores (fluorescein amidite), Fluorescein isothiocyanate (FITC), HEX-type fluorophores, Texas Red fluorophores and ATTO-type fluorophores. Preferably, FAM-type fluorophores and Texas Red fluorophores are used.

[0171] By "quencher", also called deactivator, is meant a chemical or protein substance capable of deactivating (quenching) the excited state of a fluorophore. In particular, the invention relates to the diagnostic device (1) as described above in which said quencher is chosen from: DabCyl, BHQ-1, BHQ-2, BHQ-3, Cy5Q, Cy7Q, Iowa Black FQ, Iowa Black RQ, ITRDye QC-1, TQSY35, QSY7, QXL520, QXL570, QXL610 and QXL680. Preferably, the quenchers lowa Black FQ and lowa Black RQ are used.

[0172] It should be noted, however, that when the invention uses isothermal amplification technology and detection via a fluorophore / quencher system (eg (RT)-LAMP-QUASR), it is essential to choose the right pair. Table 1 below (Peng, X. et al., 2009, A nonfluorescent, broad-range quencher dye for Fôrster resonance energy transfer assays. Analytical biochemistry, 388(2), 220-228) allows this choice to be made. For example, if the fluorophore used is Texas Red then it is preferable to use Iowa Black RQ.

[0173] [Tables 1] UU OOU, this QO "IS ca Fluorophore | 472 | If? | Oregon (îwt) 4^X. 51B | Huotescm $20 | RhoOWineGreervX $31 d $53 d Cy3 $64 O | gOEW* 5W56S SôS ili | Ate» Hw 54« 571 1 TWA sas [ ROX 608 | fexaFkw $94 61$ j $Ï7 | 8OOiFYSWô5à-X $53 1 Alex » Fluor «47 670 — — ■ ili ■ | $707 Z j ________W [Alex» Fluorine 750 $77 | avg WCW W mi [ avg 800________$09

[0174] Table 1. List of spectrally compatible fluorophores and quenchers (gray box = compatible)

[0175] As described above, the diagnostic device (1) of the invention comprises a single detection unit (4) and makes it possible to test only a single biological sample. Advantageously, it can be configured to accommodate different detection units distributed over the entire upper (3) and lower (5) discs. This configuration thus offers the user a very practical multi-sample device due to its versatility since, depending on its configuration, it can be used:

[0176] • either from a sample, to carry out different diagnoses (eg Zika virus, SARS-CoV-2, HIV, Salmonella, etc.); • either from several samples, to carry out a single type of diagnosis (eg Zika virus or SARS-CoV-2 or HIV or Salmonella, etc.); • either from several samples, to carry out different diagnoses (eg Zika virus, SARS-CoV-2, HIV, Salmonella, etc.).

[0177] According to another embodiment, the invention therefore relates to the device of

[0178]

[0179]

[0180]

[0181]

[0182]

[0183]

[0184]

[0185]

[0186] diagnostic (1) as described above comprising at least two detection units which are distributed on the upper (3) and lower (5) discs so that their respective receiving openings are located on the same radius of the upper disc. According to another embodiment, the invention therefore relates to the diagnostic device (1) as described above comprising at least two detection units which are distributed on the upper (3) and lower (5) discs so that their respective receiving openings are located on the same arc of a circle of the upper disc. According to another embodiment, the invention therefore relates to the diagnostic device (1) as described above comprising: • at least two detection units which are distributed on the upper (3) and lower (5) discs so that their respective receiving openings are located on the same radius of the upper disc, and • at least two detection units which are distributed on the upper (3) and lower (5) discs so that their respective receiving openings are located on the same arc of a circle of the upper disc. According to another embodiment, the invention therefore relates to the diagnostic device (1) as described above comprising: • at least two detection units which are distributed on the upper (3) and lower (5) discs so that their respective receiving openings are located on the same radius of the upper disc, or • at least two detection units which are distributed on the upper (3) and lower (5) discs so that their respective receiving openings are located on the same arc of a circle of the upper disc. Advantageously and according to this multi-sample configuration, the windows (9) arranged on the upper disc (3) of the detection units arranged on the same radius can be grouped together and form only one. The same applies to any recesses (6) arranged on the same radius on the lower disc (5) which can form only one. According to another embodiment, the invention therefore relates to the diagnostic device (1) as described above in which: • the windows (9) arranged on the same radius of the upper disc (3) are grouped together and form only one, and / or • any recesses (6) arranged on the same radius of the lower disc (5) are grouped together and form only one. Alternatively and with regard to this multi-sample configuration, the invention can be defined as being a portable diagnostic device (1) in the form of a flat closed cylindrical box comprising: • a lower disc (5); and • an upper disc (3),

[0187] the two disks being:

[0188] • mounted on top of each other and movable relative to each other so that the upper disc (3) is able to perform a rotational movement on the lower disc (5) in both possible directions of rotation; and • segmented into x diagnostic blocks (2), x being an integer greater than or equal to 1 and each diagnostic block comprising y detection units (4) of samples to be tested, each detection unit comprising:

[0189] - a receptacle comprising an absorbent material (15) arranged either on the upper surface of the lower disc (5), or integrated into the mass of said upper surface of the lower disc (5),

[0190] and said absorbent material (15) being capable of containing a volume of at least 1 mL;

[0191] - a reaction zone (12) arranged in the lower disc, said reaction zone (12) comprising at least one, preferably at least two, biologically compatible, adjacent and independent pellets (13a and 13b) on the same circle of the lower disc (5) or the same radius of the lower disc (5),

[0192] and at least one of which optionally comprises on its surface lyophilized reagents allowing the amplification and detection of at least one nucleic acid sequence of interest likely to be present in a sample to be tested;

[0193] - possibly a recess (6) arranged in the lower disc (5), said recess (6) being provided with a waterproof material (10),

[0194] said recess (6) provided with a waterproof material (10) being at right angles to the reaction zone (12) and integral with it, and in particular allowing light to reach said at least one, preferably at least two, biologically compatible pellets (13a and 13b), and

[0195] said waterproof material (10) being either positioned on the recess (6) in a removable manner, or integrated into the mass of the lower disc (5);

[0196] - a receiving opening (7) arranged in the upper disc (3), said opening being: • positioned on a circle of the upper disc, which circle is parallel to the circle of the lower disc (3) on which the aforementioned reaction zone (12) is positioned; • equipped with a biologically compatible membrane (8) allowing the extraction and retention of nucleic acids from the sample to be tested in the presence of appropriate reagents; and • able to be positioned in line with the aforementioned reaction zone and cover it in part or in whole;

[0197] and

[0198] - a window (9) arranged in the upper disc (3), said window (9) being provided with an optical filter (11),

[0199] said window (9) provided with an optical filter (11) being able to be positioned in line with the reaction zone (12) of the detection unit (4), and

[0200] said optical filter (11) being either positioned on the window (9) in a removable manner, or integrated into the mass of the upper disc (3),

[0201] y being an integer greater than or equal to 1 and when y is greater than or equal to 2, the different elements of the lower disc (5) and the upper disc (3) of the detection units (4) are respectively adjacent and independent along a respective radius of the lower disc (5) and the upper disc (3),

[0202] and each diagnostic block (2) being configured so that the upper disc (3) can, by rotation through an angle of at least 30°, pass:

[0203] • of a first position in which: - the receiving opening (7) equipped with a biologically compatible membrane (8) of a detection unit (4) is located at the right of the absorbent material (15) of said detection unit (4) to allow the extraction and retention of the nucleic acids of said sample to be tested; and - the window (9) provided with an optical filter (11) of said detection unit (4) is located at the right of the reaction zone (12) of said detection unit (4), • to a second position in which: - the receiving opening (7) equipped with a biologically compatible membrane (8) of said detection unit (4) is located at the right of the reaction zone (12) of said detection unit (4) and covers it in part or in full to allow the elution of the nucleic acids of said sample to be tested in the presence of the appropriate reagents, said nucleic acids by gravity being found on said at least one, preferably at least two, biologically compatible pellets (13a and 13b) of said detection unit (4); and - the window (9) provided with an optical filter (11) of said detection unit (4) is offset by an angle of at least 30°, • then to position itself on said first position so that the window (9) provided with an optical filter (11) of said detection unit (4) is again located at the right of the reaction zone (12) of said detection unit (4) to form an amplification and detection chamber substantially hermetic and substantially watertight, said chamber allowing the amplification and detection of at least one nucleic acid sequence of interest likely to be present in the sample to be tested.

[0204] In the same way, the passage from the above-mentioned second position to the above-mentioned first position can therefore be done either by a rotation opposite to that allowing the passage from the above-mentioned first position to the above-mentioned second position, or by a rotation in the same direction as the latter. Advantageously, the invention relates to the portable diagnostic device (1) in the form of a flat closed cylindrical box as described above comprising:

[0205] • a lower disc (5); and • an upper disc (3),

[0206] the two discs being:

[0207] • mounted on top of each other and movable relative to each other so that the upper disc (3) is able to perform a rotational movement on the lower disc (5) in both possible directions of rotation; and • segmented into x diagnostic blocks (2), x being an integer greater than or equal to 1 and each diagnostic block comprising y detection units (4) of samples to be tested, each detection unit comprising:

[0208] - a receptacle comprising an absorbent material (15) arranged either on the upper surface of the lower disc (5), or integrated into the mass of said upper surface of the lower disc (5),

[0209] and said absorbent material (15) being capable of containing a volume of at least 1 mL;

[0210] - a reaction zone (12) arranged in the lower disc, said reaction zone (12) comprising at least one, preferably at least two, biologically compatible, adjacent and independent pellets (13a and 13b) on the same circle of the lower disc (5) or the same radius of the lower disc (5),

[0211] and at least one of which optionally comprises on its surface lyophilized reagents allowing the amplification and detection of at least one nucleic acid sequence of interest likely to be present in a sample to be tested;

[0212] - possibly a recess (6) arranged in the lower disc (5), said recess (6) being provided with a waterproof material (10),

[0213] said recess (6) provided with a waterproof material (10) being at right angles to the reaction zone (12) and integral with it, and in particular allowing light to reach said at least one, preferably at least two, biologically compatible pellets (13a and 13b), and

[0214] said waterproof material (10) being either positioned on the recess (6) so as to removable, either integrated into the mass of the lower disc (5);

[0215] - a receiving opening (7) arranged in the upper disc (3), said opening being: • positioned on a circle of the upper disc, which circle is parallel to the circle of the lower disc (3) on which the aforementioned reaction zone (12) is positioned; • equipped with a biologically compatible membrane (8) allowing the extraction and retention of nucleic acids from the sample to be tested in the presence of appropriate reagents; and • capable of being positioned in line with the aforementioned reaction zone and of covering it in part or in full;

[0216] and

[0217] - a window (9) arranged in the upper disc (3), said window (9) being provided with an optical filter (11),

[0218] said window (9) provided with an optical filter (11) being able to be positioned in line with the reaction zone (12) of the detection unit (4), and

[0219] said optical filter (11) being either positioned on the window (9) in a removable manner, or integrated into the mass of the upper disc (3),

[0220] y being an integer greater than or equal to 1 and when y is greater than or equal to 2, the different elements of the lower disc (5) and the upper disc (3) of the detection units (4) are respectively adjacent and independent along a respective radius of the lower disc (5) and the upper disc (3),

[0221] and each diagnostic block (2) being configured so that the upper disc (3) can, by rotation through an angle of at least 30°, pass:

[0222] • of a first position in which: - the receiving opening (7) equipped with a biologically compatible membrane (8) of a detection unit (4) is located at the right of the absorbent material (15) of said detection unit (4) to allow the extraction and retention of the nucleic acids of said sample to be tested; and - the window (9) provided with an optical filter (11) of said detection unit (4) is located at the right of the reaction zone (12) of said detection unit (4), • to a second position in which: - the receiving opening (7) equipped with a biologically compatible membrane (8) of said detection unit (4) is located at the right of the reaction zone (12) of said detection unit (4) and covers it in part or in full to allow the elution of the acids nucleic acids of said sample to be tested in the presence of appropriate reagents, said nucleic acids by gravity being found on said at least one, preferably at least two, biologically compatible pellets (13a and 13b) of said detection unit (4); and - the window (9) provided with an optical filter (11) of said detection unit (4) is offset by an angle of at least 30°, • then to return by reverse rotation to said first position so that the window (9) provided with an optical filter (11) of said detection unit (4) is again located at the right of the reaction zone (12) of said detection unit (4) to form a substantially hermetic and substantially sealed amplification and detection chamber, said chamber allowing the amplification and detection of at least one nucleic acid sequence of interest likely to be present in the sample to be tested,

[0223] and each diagnostic block (2) also being configured so that the window(s) (9a and 9b) provided with an optical filter (11) of one or more detection units (4) of a diagnostic block (2) cannot be at the right of the receptacle(s) comprising an absorbent material (15) of said diagnostic block (2) or of another diagnostic block (2') whatever the position of the upper disc (3) relative to the lower disc (5).

[0224] Since all embodiments are compatible with each other and can be combined with each other, they apply to this alternative definition of the invention. The reverse is also true. In addition, all definitions provided apply to all embodiments described.

[0225] By "x being an integer greater than or equal to 1", it is meant that x can be equal to 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. It is also meant that x can be from 1 to 10, from 1 to 5, from 2 to 10, from 4 to 8. By "y being an integer greater than or equal to 1", it is meant that y can be equal to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20. It is also meant that y can be from 1 to 20, from 1 to 6, from 1 to 12, from 1 to 5, from 1 to 10, from 5 to 15 or 10 to 20.

[0226] By "an angle of at least 30°" is meant that the rotation of the upper disc (3) on the lower disc (5) can be 30°, 35°, 40°, 45°, 50°, 55°, 60°, 65°, 70°, 75°, 80°, 85°, 90°, 95°, 100°, 105°, 110°, 115°, 120°, 125°, 130°, 135°, 140°, 145°, 150°, 155°, 160°, 165°, 170°, 175°, 180°, 185°, 195°, 200°, 205°, 210°, 215°, 220°, 225°, 230°, 235°, 240°, 245°, 250°, 255°, 260°, 265°, 270°, 275°, 280°, 285°, 295°, 300°, 305°, 310°, 315° or 320° or even 360° allowing the upper disc (3) to rotate freely (i.e. make a complete turn) on the lower disc (5) and vice versa. This also means an angle between 30° and 320°. Of course, it should be noted that the possible rotation angle depends on the number of diagnostic blocks (2) that one has on the diagnostic device (1) of the invention, so that all of the diagnostic blocks operate, preferably, simultaneously.

[0227] In particular, according to this alternative the invention relates to the diagnostic device (1) as described above in which x and y are chosen from the pairs:

[0228] • x=lety=l (corresponding to the configuration of a biological sample); • x = 1 and y is from 1 to 6; • x = 2 and y is from 1 to 6; • x = 3 and y is from 1 to 6.

[0229] According to this alternative, the invention advantageously relates to the diagnostic device (1) as described above in which each of the x diagnostic blocks (2) comprises y detection units (4) of which the set of y windows (9) provided with an optical filter (11) arranged on the upper disc (3) forms only one.

[0230] According to this alternative, the invention advantageously relates to the diagnostic device (1) as described above in which each of the x diagnostic blocks (2) comprises y detection units (4) of which all of the y possible recesses (6) provided with a sealing material (10) arranged on the lower disc (5) form only one.

[0231] According to this alternative, the invention also relates to the diagnostic device (1) as described above in which each of the x diagnostic blocks (2) comprises y detection units (4) of which the set of y windows (9) provided with an optical filter (11) arranged on the upper disc (3) forms only one and

[0232] of which the set of possible y obviously (6) provided with a waterproof material (10) arranged on the lower disc (5) forms only one.

[0233] According to another embodiment, the invention relates to the diagnostic device (1) as described above further comprising means, in particular:

[0234] • at least one clamp capable of gripping said device and applying pressure to block and clamp the upper disc on the lower disc, said pressure ensuring the hermeticity and sealing of the device, and

[0235] equipped with a heating system capable of being positioned in line with the reaction zone(s) of the device, avoiding the creation of a temperature gradient on the reaction zone(s) to be heated.

[0236] A second aspect of the invention relates to the use of the portable diagnostic device (1) in the form of a cylindrical housing as described above for detecting in vitro at least one nucleic acid sequence of interest likely to be present in a biological sample to be tested. This at least one nucleic acid sequence (DNA and / or RNA) of interest may be:

[0237] • a nucleic acid sequence present in the genome of a pathogen (virus, bacteria, microorganism, etc.), or • a nucleic acid sequence present in the genome (DNA) or the transcriptome (RNA) of a mammal (e.g., humans) whose presence would be linked to a predisposition to a pathology,

[0238] the use according to the invention allows the diagnosis of infections due to one or more pathogens and also the diagnosis of genetic diseases. This usefulness and versatility of the diagnostic device (1) of the invention are of course conditioned by the reagents (molecular tools) possibly lyophilized on the surface of one or more so-called "test" pellets. For example, if it is lyophilized (or added extemporaneously), the set of primers allowing the detection of at least one nucleic acid sequence of the SARS-CoV-2 coronavirus, the use described makes it possible to diagnose an infection with this virus. Similarly, if it is lyophilized (or added extemporaneously), the set of primers allowing the detection of at least one nucleic acid sequence of the Dengue virus serotype 1, the use described makes it possible to diagnose an infection with this virus. Etc.

[0239] Advantageously, the multi-sample configuration makes it possible to diagnose for one or more biological samples to be tested several types of infections. For example, in the configuration where x = 2 and y = 3, a diagnostic block would be used to test in vitro a first biological sample and detect in vitro whether it suffers from an infection with the influenza virus, the COVID-19 virus (SARS-CoV-2) and / or the Staphylococcus aureus bacteria, and

[0240] the second diagnostic block would be used to test in vitro a second biological sample and detect in vitro whether it suffers from an infection with the influenza virus, the COVID-19 virus (SARS-CoV-2) and / or the Staphylococcus aureus bacteria. This example can be generalized to x diagnostic block for x biological samples, each of the x blocks comprising y detection units making it possible to test in vitro the presence (or not) of y viral, bacterial or other microorganism (eg fungal) infections. In the same way, the diagnostic device (1) of the invention can be used to test in vitro y genetic diseases.

[0241] According to another embodiment, the invention therefore relates to the use as described above for detecting in vitro a viral infection (e.g. HIV, Dengue virus, Zika virus, Sigma 3 virus, SARS-CoV-2, etc.), a bacterial infection (e.g. Salmonella, Pseudomonas, Staphylococcus, Escherichia, Streptococcus, Helicobacter, etc.), an infection linked to a microorganism (e.g. fungus) and / or a genetic disease (e.g. cystic fibrosis, neurofibromatosis type 1, hemophilia, trisomy 21, myopathy, etc.). In particular, the invention relates to the use as described above for detecting in vitro a viral infection. The invention also relates to the use as described above for detecting in vitro a viral infection and in particular for detecting in vitro a SARS-CoV-2 viral infection. Preferably, The invention relates to the use as described above for detecting in vitro a SARS-Cov-2 viral infection. As mentioned previously, this embodiment of the invention is conditioned by the use, for example, of primers of sequences SEQ ID NO: 7 to 12 on the test pellet present on the reaction zone (12).

[0242] Another aspect of the invention relates to the method for in vitro detection of at least one nucleic acid sequence of interest likely to be present in a biological sample to be tested, said method being implemented using the portable diagnostic device (1) in the form of a cylindrical housing as described above.

[0243] In particular, the invention relates to the method for in vitro detection of at least one nucleic acid sequence of interest likely to be present in a biological sample to be tested, said method being implemented using the portable diagnostic device (1) in the form of a cylindrical housing as described above and said method comprising at least the following steps:

[0244] a. extracting the nucleic acids from the biological sample to be tested onto a biologically compatible membrane (8) of a detection unit (4); b. rotating the upper disc (3) to position said biologically compatible membrane (8) in line with the reaction zone (12) of this detection unit and covering it in part or in full; c. eluting the nucleic acids to pass them from said biologically compatible membrane (8) to the at least one, preferably at least two, biologically compatible pellets (13a, 13b) of this detection unit (4); d. turn the upper disc (3) to return to the initial position; and e. amplify and then detect whether amplification of at least one nucleic acid sequence of interest likely to be present in a biological sample has taken place.

[0245] Depending on the configuration of the diagnostic device (1) of the invention, the above-mentioned step d. can be carried out either by a rotation in the opposite direction to that of step b., or by a rotation in the same direction as that of step b. Advantageously, the invention relates to the method for in vitro detection of at least one nucleic acid sequence of interest likely to be present in a biological sample to be tested, said method being implemented using the portable diagnostic device (1) in the form of a cylindrical housing as described above and said method comprising at least the following steps:

[0246] a. extracting the nucleic acids from the biological sample to be tested onto a biologically compatible membrane (8) of a detection unit (4); b. turn the upper disc (3) to position said membrane (8) biolo logically compatible with the reaction zone (12) of this detection unit and cover it in part or in full; c. eluting the nucleic acids to pass them from said biologically compatible membrane (8) to the at least one, preferably at least two, biologically compatible pellets (13a, 13b) of this detection unit (4); d. turn the upper disc (3) in the opposite direction to return to the initial position; and e. amplify and then detect whether amplification of at least one nucleic acid sequence of interest likely to be present in a biological sample has taken place.

[0247] Since the above method involves the use of the diagnostic device (1) of the invention, everything that applies to the uses (definitions, etc.) also applies to the methods.

[0248] According to another embodiment, the invention relates to the method as described above, said method comprising at least the following steps:

[0249] a. depositing the biological sample to be tested on a biologically compatible membrane (8) of a detection unit (4); b. extract the nucleic acids from the biological sample to be tested at this biologically compatible membrane (8); c. washing said biologically compatible membrane (8) of this detection unit (4); d. rotating the upper disc (3) to position said biologically compatible membrane (8) in line with the reaction zone (12) of this detection unit and covering it in part or in full; e. drying said biologically compatible membrane (8) of this detection unit (4) f. eluting the nucleic acids to pass them from said biologically compatible membrane (8) to the at least one, preferably at least two, biologically compatible pellets (13a, 13b) of this detection unit (4); g. turn the upper disc (3) in the same direction as in step b. or in the opposite direction to step b. to return to the initial position; h. heating the reaction zone (12) to 65°C for 40 min to amplify said at least one nucleic acid sequence of interest likely to be present in the biological sample; and i. detect whether an amplification of at least one nucleic acid sequence of interest likely to be present in a biological sample has taken place.

[0250] Whether the invention relates to the uses as described above or to the methods as described above, the observation of a positive signal (e.g. emission of light by fluorescence, appearance of a coloration) at the test pellet means that said at least one nucleic acid sequence (DNA and / or RNA) has been amplified and is present in the biological sample. For example, if it were desired to test in vitro for a SARS-CoV-2 infection in a patient, the observation of this positive signal means that the patient is infected with the SARS-CoV-2 coronavirus and is suffering from COVID-19. Conversely and for example, if it were desired to test in vitro for a SARS-CoV-2 infection in a patient, the non-observation of a positive signal means that the patient is not infected with the SARS-CoV-2 coronavirus and is not suffering from COVID-19.However, to ensure the veracity of this in vitro test, it is also appropriate to consider the results of a positive control and / or a positive control, which can be carried out outside the diagnostic device (1) of the invention. Preferably, these controls are integrated into the diagnostic device (1) of the invention via the positive control pellet which must always reveal a positive signal and / or via the negative control pellet which must always reveal a negative signal (i.e. absence of fluorescence, absence of color). If this is not the case at the end of the test, it is invalidated and must be repeated. The presence of a second pellet on the reaction zone is therefore preferred for the reliability and safety of the in vitro diagnostic test which can be implemented by the diagnostic device (1) of the invention. Furthermore, it should be noted that the observation of the signals on each of the pellets obtained by colorimetry, fluorometry, etc., can be carried out using the human eye, a camera or a camera traditionally used in laboratories or hospitals. It can even be a smartphone camera. In addition, and depending on the detection technology chosen, it is possible to carry out kinetic monitoring of the test (e.g. (RT)-LAMP) or “endpoint” monitoring (e.g. (RT)-LAMP-QUASR).

[0251] Another aspect of the invention relates to the method of manufacturing (eg industrial) the portable diagnostic device (1) in the form of a cylindrical housing as described above. In particular, the invention relates to the method of manufacturing the portable diagnostic device (1) in the form of a cylindrical housing as described above, said method comprising at least the following steps:

[0252] a. manufacture (eg 3D printing or injection into a mold) the upper (3) and lower (5) discs; b. providing at least one biologically compatible membrane (8), at least one, preferably at least two, biologically compatible pellets (13a and 13b), at least one absorbent material (15) and at least one optical filter (11); and c. assemble the upper (3) and lower (5) discs.

[0253] According to another embodiment, the invention relates to the manufacturing method as described above, said method comprising at least the following steps:

[0254] a. manufacture (eg 3D printing or injection into a mold) the upper (3) and lower (5) discs; b. providing at least one biologically compatible membrane (8), at least one, preferably at least two, biologically compatible pellets (13a and 13b), at least one absorbent material (15), at least one optical filter (11) and optionally at least one waterproof material (10); and c. assemble the upper (3) and lower (5) discs.

[0255] According to another embodiment, the invention relates to the manufacturing method as described above, said method comprising at least the following steps:

[0256] a. manufacture (eg 3D printing) the upper (3) and lower (5) discs; b. wash the upper (3) and lower (5) discs with RNAfree solution; c. providing at least one biologically compatible membrane (8), at least one, preferably at least two, biologically compatible pellets (13a and 13b), at least one absorbent material (15), at least one optical filter (11) and optionally at least one waterproof material (10); and d. assemble the upper (3) and lower (5) discs.

[0257] According to another embodiment, the invention relates to the manufacturing method as described above, said method comprising at least the following steps:

[0258] a. manufacture (eg 3D printing) the upper (3) and lower (5) discs; b. wash the upper (3) and lower (5) discs with RNAfree solution; c. providing at least one biologically compatible membrane (8), at least one, preferably at least two, biologically compatible pellets (13a and 13b), at least one absorbent material (15), at least one optical filter (11) and optionally at least one waterproof material (10); d. assemble the upper (3) and lower (5) discs; and e. freeze-drying on at least one pellet (13a or 13b) reagents capable of enabling the amplification and detection of at least one nucleic acid sequence of interest likely to be present in the aforementioned biological sample.

[0259] Alternatively, the invention relates to the manufacturing method as described above, said method comprising at least the following steps:

[0260] a. manufacture (eg 3D printing) the upper (3) and lower (5) discs; b. freeze-dry on at least one pellet (13a or 13b) reagents capable of allow the amplification and detection of at least one nucleic acid sequence of interest likely to be present in the aforementioned biological sample. c. wash the upper (3) and lower (5) discs with RNAfree solution; d. providing at least one biologically compatible membrane (8), at least one, preferably at least two, biologically compatible pellets (13a and 13b), at least one absorbent material (15), at least one optical filter (11) and optionally at least one waterproof material (10); and e. assemble the upper (3) and lower (5) discs. EXAMPLES

[0261] The following examples illustrate the invention, without limiting its scope.

[0262] EXAMPLE 1 - MANUFACTURE OF THE DIAGNOSTIC DEVICE PORTABLE IN THE SHAPE OF A CYLINDRICAL CASE OF THE INVENTION

[0263] Creating disks

[0264] (1) Using the Fusion 360 Autodesk license software, 2 circular parts were produced culars with a diameter of 65 mm and the other of 58 mm hereinafter called respectively upper disc and lower disc.

[0265] On the upper disc the dimensions of diagram 1 shown in Figure 9 have been reproduced.

[0266] On the lower disc the dimensions of diagram 2 shown in Figure 10 have been reproduced.

[0267] The dimensions of the diagrams are in millimeters (mm).

[0268] (2) The 3D parts of the 2 discs were exported in STL format and implemented in the Makerboot software.

[0269] (3) Printing with the Replicator 2X printer was configured with the pa following print parameters:

[0270] • Nozzle temperature: 210°C • Plateau temperature: 50°C • Layer thickness: 100 pm • Writing speed: 110 mm / s • Filling: 45% • Filling technique: Hexagonal

[0271] (4) 3gs format print trajectories have been exported and implemented into the printer and printing was started.

[0272] (5) After 3 hours of printing, the 2 discs were recovered by detaching them from the tray printing.

[0273] Assembly of the different elements on the discs

[0274] (1) After cleaning the upper disc with RNAfree solution:

[0275] • double-sided tape was placed on the contours of the 2 openings (3 mm wide), the taped face being the inner face of the upper disc where there is the guide groove; • the biologically compatible membrane allowing the extraction and retention of nucleic acids, called the capture membrane, was placed at the receiving opening in the form of an oblong hole, said capture membrane having been previously encapsulated in PCR tape perforated on each side; and • the assembly was held in place by pressing on the contours of the oblong hole to ensure the best possible contact between the tape and the upper disc.

[0276] (2) Operation (1) was repeated with the previously cut optical filter and assembled with a heat-shrinkable PCR sheet for the face in contact with the upper disc.

[0277] (3) After cleaning the lower disc with RNAfree solution:

[0278] • the absorbent material, called a capillary pad, was placed in the receptacle (or cavity) in a semicircle; and • the 2 amplification and detection pellets were placed in the hollow circular housings provided for this purpose in the reaction zone, which recess in particular allows light to reach said pellets.

[0279] Assembling the discs

[0280] The two upper and lower discs were assembled, taking care that the stop lug (or lug) was properly in the guide groove and that the hole in the center of the upper disc was aligned with the central stud of the lower disc.

[0281] The device thus prepared can either be stored and used later, or used directly.

[0282] In situ freeze-drying of reagents

[0283] In addition to the manufacture of the diagnostic device of the invention, a step of lyophilization of the molecular tools allowing the amplification and detection of at least one nucleic acid sequence likely to be present in the sample to be tested can be added. For this, dNTPs, enzymes, primers, etc. are mixed then adsorbed on the pellets. Then, the reaction pellets are incubated at - 20°C for 20 minutes, then at - 80°C for 20 minutes, then lyophilized in a lyophilizer at a pressure of 3 mbar overnight (8 hours). The pellets thus lyophilized can then be placed in the housings provided for this purpose in the device of the invention.

[0284] EXAMPLE 2 - USES OF THE DIAGNOSTIC DEVICE OF THE INVENTION FOR DIAGNOSING DENGUE VIRUS INFECTION AND SARS-COV-2 CORONAVIRUS INFECTION

[0285] Materials & Methods Freeze-drying of compounds

[0286] The following compounds were lyophilized in each pellet (18 μL):

[0287] • 0.85 mM of each deoxyribonucleotide triphosphate (dATP, dTTP, dGTP, dCTP) • 5 mM MgSO4 • 0.5 mM Betaine • 0.2 pM of each primer F3 and B3 • 0.8 pM of each FIP and BIP primer • 0.4 pM of each LoopF ​​and LoopB primer • a quencher concentration one and a half times higher than the concentration of the probe primer (1.2 pM if the probe primer is FIP or BIP, 0.6 pM if the probe primer is LoopF ​​or LoopB) • 7.2 units of GspSSD2.0 polymerase enzyme • 0.9 units of AMV-RT reverse transcriptase enzyme • 5% v / v of Trehalose, allowing the stability of the lyophilisate.

[0288] For the detection of the DENV2 virus, the primers of sequences SEQ ID NOs: 1 to 6 were used, which amplify a target sequence of the NS4B gene, and the BIP primer (SEQ ID NO: 4) served as a probe since it was modified in 5' by the addition of a FAM fluorophore. The oligonucleotide quencher of sequence SEQ ID NO: 13 was used and modified in 3' by the addition of Iowa Black FQ.

[0289] For the detection of the SARS-CoV-2 coronavirus, the primers with sequences SEQ ID NOs: 7 to 12 were used, which amplify a target sequence of the ORFlab gene (Lin Yu et al., Rapid colorimetry detection of COVID-19 coronavirus using a reverse transcriptional loop-mediated isothermal amplification (RT-LAMP) diagnostic plat-form: ilACO; DOI: 10.1101 / 2020.02.20.20025874), and the LoopF ​​primer (SEQ ID NO: 11) served as a probe since it was modified in the 5' end by the addition of a Texas Red fluorophore. The oligonucleotide quencher with sequence SEQ ID NO: 16 was used and modified in the 3' end by the addition of Iowa Black RQ.

[0290] These primers (etc.) were lyophilized (see above) in a test pellet alongside an internal positive control pellet in which the template DNA / RNA to initiate the reaction was also lyophilized.

[0291] Once freeze-dried, the pellets were placed in the housings provided for this purpose. Use of the device

[0292] (1) Extraction of nucleic acids from the sample

[0293] • Mix 50 pL of sample with the lysis buffer “AVL buffer” (Qiagen®) or any other lysis buffer whose composition is close to the published lysis buffer: Boom et al., JPME (1990). Rapid and simple method for purification of nucleic acids. Journal of clinical microbiology, 28(3), 495-503 Vortex for 15 seconds Incubate at room temperature for 10 minutes. Add 200 pL of pure ethanol Vortex for 15 seconds Place the lysate on the capture membrane. Rinse the capture membrane with one or more rinse buffers: 200 pL of Buffer AW1 (Qiagen®), then 200 pL of Buffer AW2 (Qiagen®) or any other rinsing buffer whose composition is close to the published rinsing buffer: Boom et al., JPME (1990). Rapid and simple method for purification of nucleic acids. Journal of clinical microbiology, 28(3), 495-503 Allow the capture membrane to dry for 15 minutes.

[0294] (2) Elution in the reaction pellets and heating step

[0295] Turn the upper disc to face the capture membrane and pellets, and rehydrate the capture membrane with 43 pL of “Buffer” reaction buffer diluted to IX (Qiagen®) allowing the elution of nucleic acids Turn the upper disc either in the same direction or in the opposite direction so as to cover the pads with the optical filter, here, a “PCR tape” type adhesive. Incubate the device at 65°C for 30 to 45 minutes, e.g. in an oven or on heating elements.

[0296] For the purposes of this example, the samples used were a sample infected with the DENV2 virus and a sample not infected with the DENV2 virus. Also used were a SARS-CoV-2 RNA extract and a negative control without this RNA extract. Data acquisition

[0297] The acquisition of end-point fluorescence via the probes used and the associated quenchers was carried out:

[0298] • for the DENV2 virus (fluorophore = FAM) with an excitation length of 945 nm and an emission wavelength of 520 nm; and for the SARS-CoV-2 coronavirus (fluorophore = Texas Red) with an excitation wavelength of 586 nm and an emission wavelength of 603 nm.

[0299] Photographs were also taken with a smartphone (Iphone®) or a

[0300] Nikon® commercial camera. Results

[0301] Figure 11 shows the results obtained, which confirmed that the device is capable, safely and reliably, of detecting the DENV2 virus and the SARS-CoV-2 coronavirus only in samples where their respective genetic material is present.

Claims

1. Claims Portable diagnostic device (1) in the form of a cylindrical case comprising two superimposed coaxial discs (3, 5) mounted to rotate relative to each other and defining a closed volume, namely: • an upper disc (3) comprising: - at least one receiving opening (7) for a biological sample to be tested which is provided with a biologically compatible membrane (8) capable of allowing the extraction and retention of nucleic acids from this biological sample in the presence of appropriate reagents, and - at least one window (9) which is either equipped with an optical filter (11) integrated into the mass of the upper disc, or capable of receiving an optical filter (11) positionable on said window in a removable manner, this receiving opening (7) and this window (9) being angularly spaced apart by an angle at the center of at least 30°, • a lower disc (5) comprising: - an absorbent material (15) capable of containing a volume of liquid, and - at least one reaction zone (12) comprising at least one, preferably at least two, biologically compatible, adjacent, independent pellets (13a, 13b) capable of enabling the amplification and detection of at least one nucleic acid sequence of interest likely to be present in the above-mentioned biological sample in the presence of appropriate reagents, the receiving opening (7), the window (9), the absorbent material (15) and the reaction zone (12) forming a detection unit (4) such that, by rotation, the upper disc can successively • move from a first position in which, for this unit detection unit (4), its receiving opening (7) is located in line with its absorbent material (15), so as to allow the extraction and retention of the above-mentioned nucleic acids, and its window (9) is located in line with its reaction zone (12), • at a second position in which, for this detection unit (4), its receiving opening (7) is located in line with its reaction zone (12) and covers it in part or in full so as to allow the elution of the above-mentioned nucleic acids, and • position itself, by rotation, on the above-mentioned first position, so that, for this detection unit (4), its window (9) is located in line with its reaction zone (12), so as to allow the amplification and detection of the above-mentioned at least one nucleic acid sequence of interest likely to be present in the biological sample.

2. Diagnostic device (1) according to claim 1 wherein the passage from the above-mentioned second position to the above-mentioned first position is done by a rotation opposite to that allowing the passage from the above-mentioned first position to the above-mentioned second position.

3. A diagnostic device (1) according to claim 1 or 2 wherein the absorbent material (15) is disposed in a receptacle which is positioned either on the upper surface of the lower disc or integrated into the mass of the latter, and said absorbent material (15) is preferably capable of containing a volume of at least 1 mL.

4. Diagnostic device (1) according to one of the preceding claims in which the absorbent material (15) is in a material chosen from: an absorbent pad made of cellulose, fiberglass or cotton wool and an absorbent pad usable for an immunochromatographic test.

5. Diagnostic device (1) according to one of the preceding claims in which the membrane (8) biologically compatible and capable of allowing the extraction and retention of nucleic acids is made of a material chosen from: cellulose, silica and glass fiber.

6. Diagnostic device (1) according to any one of the preceding claims wherein the at least one, preferably at least two, biologically compatible amplification pellets (13a, 13b) are in a material chosen from: fiberglass (containing or not an adjuvant binder) and cellulose.

7. Diagnostic device (1) according to any one of the preceding claims in which at least one reaction zone (12) comprises at least one, preferably at least two, biologically compatible, adjacent and independent amplification pellets (13a, 13b), at least one containing lyophilized reagents capable of allowing the amplification and detection of at least one nucleic acid sequence of interest likely to be present in the above-mentioned biological sample.

8. Diagnostic device (1) according to any one of the preceding claims in which at least one, preferably at least two, biologically compatible amplification pellets (13a, 13b) contain lyophilized reagents capable of allowing the amplification and detection of at least one nucleic acid sequence of interest likely to be present in the above-mentioned biological sample, and at least one of which also contains a lyophilized nucleic acid of interest capable of being amplified by the above-mentioned lyophilized reagents.

9. A diagnostic device (1) according to any preceding claim, the diameter to height ratio of the device being from 5 to 30.

10. Diagnostic device (1) according to any one of the preceding claims in which said rotations are guided by a system of groove(s) and lug(s).

11. Diagnostic device (1) according to any one of the preceding claims comprising: • at least two detection units (4) which are distributed on the upper (3) and lower (5) discs so that their respective receiving openings are located on the same radius of the upper disc, or • at least two detection units (4) which are distributed on the upper (3) and lower (5) discs so that their respective receiving openings are located on the same arc of a circle of the upper disc.

12. A diagnostic device (1) according to any one of the claims previous ones further comprising means, in particular: • at least one clamp capable of gripping said device and applying pressure making it possible to block and clamp the upper disc on the lower disc, said pressure ensuring the hermeticity and sealing of the device, and equipped with a heating system capable of being positioned in line with the reaction zone(s) of the device avoiding the creation of a temperature gradient on the reaction zone(s) to be heated.

13. Use of the portable diagnostic device (1) in the form of a cylindrical housing according to any one of claims 1 to 12 for detecting in vitro at least one nucleic acid sequence of interest likely to be present in a biological sample to be tested.

14. Use according to claim 13 for detecting in vitro a viral infection and in particular for detecting in vitro a viral infection with SARS-CoV-2.

15. Method for in vitro detection of at least one nucleic acid sequence of interest likely to be present in a biological sample to be tested, said method being implemented using the portable diagnostic device (1) in the form of a cylindrical housing according to any one of claims 1 to 12 and said method comprising at least the following steps: a. extracting the nucleic acids from the biological sample to be tested onto a biologically compatible membrane (8) of a detection unit (4); b. rotating the upper disc (3) to position said biologically compatible membrane (8) in line with the reaction zone (12) of this detection unit and covering it in part or in full; c. eluting the nucleic acids to pass them from said biologically compatible membrane (8) to the at least one, preferably at least two, biologically compatible pellets (13a, 13b) of this detection unit (4); d.rotating the upper disc (3) to return to the initial position; and e. amplifying and then detecting whether an amplification of at least one nucleic acid sequence of interest likely to be present in a biological sample has taken place.

16. Method of manufacturing the portable diagnostic device (1) in the form cylindrical housing according to any one of claims 1 to 12, said method comprising at least the following steps: a. manufacture the upper (3) and lower (5) discs; b. providing at least one biologically compatible membrane (8), at least one, preferably at least two, biologically compatible pellets (13a and 13b), at least one absorbent material (15) and at least one optical filter (11); And c. assemble the upper (3) and lower (5) discs.

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