Classifications

• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
  • B01L7/00—Heating or cooling apparatus; Heat insulating devices
  • B01L7/52—Heating or cooling apparatus; Heat insulating devices with provision for submitting samples to a predetermined sequence of different temperatures, e.g. for treating nucleic acid samples
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
  • C12Q1/701—Specific hybridization probes
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
  • B01L2300/00—Additional constructional details
  • B01L2300/02—Identification, exchange or storage of information
  • B01L2300/023—Sending and receiving of information, e.g. using bluetooth
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
  • B01L2300/00—Additional constructional details
  • B01L2300/06—Auxiliary integrated devices, integrated components
  • B01L2300/0627—Sensor or part of a sensor is integrated
  • B01L2300/0663—Whole sensors
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
  • B01L2300/00—Additional constructional details
  • B01L2300/18—Means for temperature control
  • B01L2300/1805—Conductive heating, heat from thermostatted solids is conducted to receptacles, e.g. heating plates, blocks
  • B01L2300/1822—Conductive heating, heat from thermostatted solids is conducted to receptacles, e.g. heating plates, blocks using Peltier elements
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
  • B01L2300/00—Additional constructional details
  • B01L2300/18—Means for temperature control
  • B01L2300/1805—Conductive heating, heat from thermostatted solids is conducted to receptacles, e.g. heating plates, blocks
  • B01L2300/1827—Conductive heating, heat from thermostatted solids is conducted to receptacles, e.g. heating plates, blocks using resistive heater

Definitions

• the present invention refers to a method and a portable device for the detection and identification of specific nucleic acids sequences in different types of samples, by means of an optimised reverse transcriptase technique and/or isothermal amplification, usinq specific oliqonucleotide primers of the tarqet reqion(s) to detect, whereby the amplification of the same is conducted in an indirect and non-specific manner, or direct and specific, usinq discriminatory amplification reaqents, which siqnal, colourimetric or fluorescent, is recorded by a portable device where the reaction is carried out, havinq useful application for different types of diaqnostics in different clinical, pharmaceutical, veterinary, food, environmental, biotechnoloqical, or biosafety areas.
• PCR polymerase chain reaction
• the diagnostics by means of the normal or real time PCR suffers from limitations which prohibits it from being applied to Point-of-Care diagnostics (PoC). Namely, they require specialised and costly staff and equipment, take several hours to provide a result and are easily susceptible to being inhibited by contaminants that are frequently present in the samples to be analysed (such as, for example, haemoglobin, salts, chelators, alcohols, among others) this being the main reason for the failure in amplification even in the presence of sufficient copies of target nucleic acid to amplify, leading to false negatives.
• the real-time PCR technology adds the limitation that a multiplex reaction is limited to the detection capacity of the apparatus, and further, that a high technical and knowledge capacity are needed to develop a new test, which is not available in a commercial kit.
• LAMP Loop-mediated isothermal amplification
• LAMP is a DNA amplification technique.
• the RT-LAMP combines LAMP with a reverse transcription step to allow RNA detection. This technique is isothermal, being executed at a constant temperature and does not require a thermal cycler.
• the target sequence is amplified at a constant temperature of 60-65 °C using several sets of oligonucleotide primers and a polymerase with high strand-displacement, as well as the replication capacity.
• the amplified product can be detected by photometry, measuring the turbidity caused by the precipitate in solution, which is produced as a secondary product of the amplification, or by colour change of pH sensitive dye, which varies as a consequence of the polymerase activity during the amplification. This allows detection of the amplification to the naked eye with a simple apparatus.
• the reaction can be detected in real time measuring the turbidity or by fluorescence, using for such intercalating fluorophores such as SYTOTM 9.
• fluorophores such as SYBRTM green can be used to create a visible colour change without the need for expensive detection equipment.
• SYBRTM green can be used to create a visible colour change without the need for expensive detection equipment.
• the fluorophore molecules intercalate with the DNA, there is a correlation with the amplification of the same, therefore the LAMP can be quantitative.
• LAMP is a relatively new DNA amplification technique that due to its simplicity and low cost can bring several benefits. LAMP can be used in field studies and PoC tests. The LAMP technique has shown more robust results regarding the inhibitors of the normal PCR reactions in blood samples, which allows the application of the technique in cases where the optimum extraction of DNA and RNA is not possible.
• LAMP is less versatile than PCR since, although it is primarily useful as a detection tool, it does not allow all the other molecular biology techniques of PCR. Since LAMP uses 4 to 6 target regions for the oligonucleotide primers it is also extremely difficult to design oligonucleotide primers for the technique.
• One of the consequences of such a cocktail of oligonucleotide primers is the increase in non-specific amplifications, and consequent false positives, which by using a DNA strand- displacement polymerase without exonuclease activity does not allow the use of Taqman probes, for example, nor do the indirect methods allow said false positives to be discriminated.
• HDA uses a helicase enzyme to generate single-stranded templates for hybridisation of oligonucleotide primers and subsequent extension of the oligonucleotide primer by a polymerase.
• the HDA offers several advantages over other isothermal DNA amplification methods, having a simple reaction scheme and being an isothermal reaction, which can be carried out at a single and constant temperature during the entire process. These properties offer a great potential for the development of simple portable devices for DNA diagnostic to be used in loco and in PoC.
• HDA high-density polymerase chain reaction
• PCR reactions carried out in a thermal cycler that can contain plates of samples from several wells allow the amplification and detection of the intended target DNA a from a maximum of 96 samples at a time.
• the cost of purchasing reagents for HDA is also relatively expensive in comparison with the reagents for the PCR reagents (Vincent M et al (2004) EMBO, 5(8):795-800.
• RCA Rolling Circle Amplification
• RCA was developed as a simplified version of the Rolling Circle Replication, an isothermal DNA amplification technique.
• the RCA mechanism is widely used in molecular biology and biomedical nanotechnology, particularly in the field of biosensing (as a method for signal amplification).
• RCA was used successfully to detect the existence of viral and bacterial DNA from clinical samples, which is extremely beneficial for the rapid diagnostic of infectious diseases. It was also used as a method for on-chip signal amplification, for nucleic acid microarrays (both for DNA as for RNA).
• the RCA technique can also be applied to the construction of DNA nanostructures and DNA hydrogels.
• the RCA products can also be used as templates for periodic assembly of nano species or proteins, synthesis of metallic nanowires and formation of nano-islands.
• RCA is a highly versatile DNA amplification tool, widely used in many fields where the limitations of sensitivity and/or specificity, preparation of laborious samples and/or signal amplification procedures had previously prevented the use of other tools.
• SDA Strand Displacement Amplification
• MDA Multiple Displacement Amplification
• SDA and MDA are DNA amplification techniques without resorting to PCR. These methods can rapidly amplify minimum DNA sample quantifies sufficient for a genomic analysis.
• the reaction starts by annealing random hexamer oligonucleotide primers to the template: the DNA synthesis is carried out by a high-fidelity enzyme at a constant temperature.
• SDA is an isothermal in vitro technique for amplification of nucleic acids, based on the Hind-I capacity of cutting the non- modified strand of a form of hemi-phosphorothioate from the recognised location thereof and the capacity of the exonuclease-deficient klenow (exo-klenow) to extend the 3' end and dislocate the DNA strand downstream.
• the exponential amplification results from the coupling of the sense and anti-sense reactions wherein the displaced strands of a sense reaction serve as target for an anti-sense reaction and vice- versa.
• ADO Allelic dropout
• RPA Recombinase Polymerase Amplification
• RPA is an isothermal alternative to the PCR reaction.
• a reverse transcriptase enzyme By adding a reverse transcriptase enzyme to an RPA reaction, we can detect both RNA and DNA, without the need for a separate step to produce cDNA.
• RPA uses much simpler equipment than the PCR.
• TMA Transcription Mediated Amplification
• NASBA Nucleic Acid Sequenced Based Amplification
• Both TMA and NASBA are isothermal amplification reactions that imitate the replication of the retroviral RNA. Both are specific for target RNA sequences and have achieved popularity since they demonstrated a wide range of applications to detect pathogen agents in clinical, environmental, and food samples. Both have commercially available kits.
• the NASBA and the TMA have the advantage of not requiring a thermal cycler protocol. Both techniques use RNA polymerase to produce RNA from a promoter created in the region of oligonucleotide primers and a reverse transcriptase, to produce DNA from RNA templates. For the TMA, the reverse transcriptase itself degrades the initial RNA template as it synthesizes the complementary DNA thereof.
• RNA amplification technique was improved, with the introduction of a third enzymatic activity, the RNase H, to destroy the RNA from the CDNA without the step of heat denaturation.
• the oligonucleotide forward primer binds with any target RNA present in a sample.
• the reverse transcriptase enzymes and RNase H together with the reverse oligonucleotide primer, produce a dsDNA with the target sequence and a T7 promoter.
• the polymerase of RNA dependent on DNA T7 uses this dsDNA to produce many RNA complementary strands to the original target RNA.
• each recently synthesised RNA can be copied in a cyclic phase, resulting in an exponential amplification of the RNA that is complementary to the target.
• the thermal cycling step was eliminated, generating an isothermal amplification method named self-sustained sequence replication.
• the final products of NASBA and TMA can be detected using gel electrophoresis and colourimetric test.
• the new coronavirus named "SARS-CoV-2" was detected for the first time in China in December 2019 and, since 11 March 2020, has been classified as a pandemic by the WHO, causing a respiratory disease called "COVID-19".
• the coronaviruses are a large family of common viruses in people and in many different species of animals, including camels, cattle, cats, and bats.
• the animal coronaviruses rarely can infect human beings and subsequently transmit themselves among people such as the MERS-CoV, SARS-CoV and now this new virus SARS-CoV-2. While we are still learning how it disseminates and the severity of the disease that it causes, this new COVID-19 disease has rapidly spread to more than 100 locations throughout the world.
• the diagnostic of the COVID-19 disease, caused by the SARS-CoV-2 virus is performed mainly by the molecular pathway, and the chosen diagnostic means for this type of diagnostic is a real-time PCR, there being a series of commercial kits available (for example, cobas SARS-CoV-2 (Roche), TaqPath COVID-19 Combo Kit (Thermo Fisher), lcopy COVID-19 QPCR Kit (1DROP Inc), Abbott Real-time SARS-COV-2 (Abbott)).
• the portable device that was developed allows the rapid detection ( ⁇ 30 minutes) of the presence of the virus (SARS-Cov-2) in several types of samples, whereby it may be used by any person (layperson in this field) and as it is portable, allowing this test to be carried out virtually anywhere. All of this eliminates the risk of contagion between the potentially infected person and the health professionals who before were involved in the process of collection and analysis of the samples. Notwithstanding, the tracking of positive results by the health entities is also able to be performed in real time, since the diagnostic is made by means of a mobile device which communicates with the portable device and sends all the information to a remote server in the cloud.
• the portable device comprises a battery, and the truly small dimension thereof (in other words, it fits in a coat pocket), makes it truly portable for PoC.
• the present invention refers to an integrated system and method for the detection and identification of specific sequences of nucleic acids in different types of samples with the presence of a portable device (1) controlled by a mobile application (9) which records the data acquired by the portable device (1), analyses and stores it in a remote server in a cloud (10) and method for the detection and identification of specific nucleic acid sequences in different types of samples.
• the mobile application (9) also records the data acquired by the portable device (1), analyses and stores it in a remote server in a cloud (10) wherein the latter, in turn, analyses the global set of all the data in order to allow the global analysis of, for example, the evolution of epidemics.
• FIG. 1 Scheme of the components of the portable device (1) controlled by a mobile application (9) and connected directly or indirectly to a remote server in a cloud (10).
• Figure 2 Example of a test with a negative sample carried out and monitored on the portable device (1), for the Gene N, using a specific oligonucleotide probe marked with fluorophore FAM. The intensity of the fluorescence is maintained unaltered throughout the entire test, demonstrating that the reverse transcription and isothermal amplification did not occur, due to the absence of the target viral RNA.
• Figure 3 Example of a test with a positive sample carried out and monitored on the portable device (1), for the Gene N, using a specific oligonucleotide probe marked with fluorophore FAM. An exponential increase in the intensity of the fluorescence is verified from ⁇ 1100seconds, a consequence of the reverse transcription and isothermal amplification, disputed by the presence of the target viral
• the present invention refers to an integrated system for the detection and identification of specific sequences of nucleic acids in suspected coronavirus samples with the presence of a portable device (1) controlled by a mobile application (9) which records the data acquired by the portable device (1), analyses and stores it in a remote server in a cloud (10).
• the detection and identification of the specific sequences of nucleic acids in suspected coronavirus samples is carried out by means of an optimised reverse transcriptase and/or isothermal amplification technique, usinq specific oliqonucleotide primers of the tarqet reqion(s) to detect, whereby the amplification of the same is carried out in an indirect and non-specific manner, or direct and specific, usinq discriminatory amplification reaqents, which siqnal, colourimetric or by fluorescence, is recorded by a portable device (1) where the reaction is executed, beinq controlled by a mobile application (9) and connected directly or indirectly to a remote server in a cloud (10), and havinq useful application for different types of diaqnostic in different clinical, pharmaceutical, veterinary, food, environmental, biotechnoloqical , and biosafety areas.
• an optimised reverse transcriptase and/or isothermal amplification technique usinq specific oliqonucleotide primer
• the present invention further refers to a method for the detection and identification of the referred specific nucleic acid sequences in suspected coronavirus samples.
• the present invention is useful for the detection and identification of relevant nucleic sequences for different types of diagnostic in different pharmaceutical, veterinary, food, environmental, biotechnological, or biosafety areas, being particularly useful in the rapid diagnostic, with low cost and a point of interest for the SARS-CoV-2 virus.
• the nucleic acid sequences to be detected with the said method are sequences of deoxyribonucleic acid (DNA) or ribonucleic acid (RNA).
• sequences of nucleic acids are of human, animal and/or pathogen origin, including bacteria, fungi, virus, or other microorganisms.
• sequences of nucleic acids belong to a virus, according to the Baltimore classification, Group I - double- stranded DNA viruses (ex. Adenovirus, herpes virus, Poxvirus), Group II - single-stranded DNA viruses (ex. Parvoviruses), Group III - double-stranded RNA viruses (Ex. Reovirus), Group IV - positive sense single-stranded RNA viruses (ex. Coronavirus, Picornavirus, Togavirus), Group V - negative or anti-sense single-stranded RNA viruses (ex.
• Group I - double- stranded DNA viruses ex. Adenovirus, herpes virus, Poxvirus
• Group II - single-stranded DNA viruses ex. Parvoviruses
• Group III - double-stranded RNA viruses Ex. Reovirus
• Group IV - positive sense single-stranded RNA viruses ex. Coronavirus, Picornavirus, Togavirus
• sequences of nucleic acids belong to a virus of the Coronaviridae family, and, particularly, to the severe acute respiratory syndrome coronavirus 2 (SARS- Cov-2).
• SARS- Cov-2 severe acute respiratory syndrome coronavirus 2
• the reverse transcription can be carried out using a polymerase DNA enzyme with reverse transcriptase activity, such as HIV-1, M-MLV, AMV, among others.
• a polymerase DNA enzyme with reverse transcriptase activity such as HIV-1, M-MLV, AMV, among others.
• the polymerase DNA enzyme with reverse transcriptase activity can, or not, be coupled reversibly to an aptamer, which inhibits its activity at temperatures below 40 °C.
• the isothermal amplification can be executed using an isothermal amplification method, such as: Loop-mediated isothermal amplification (LAMP), Helicase-Dependent Amplification (HDA), Rolling Circle Amplification (RCA), Strand Displacement Amplification (SDA), Multiple Displacement Amplification (MDA), Recombinase Polymerase Amplification (RPA), Transcription Mediated Amplification (TMA), Nucleic Acid Sequenced Based Amplification (NASBA), among others.
• LAMP Loop-mediated isothermal amplification
• HDA Helicase-Dependent Amplification
• RCA Rolling Circle Amplification
• SDA Strand Displacement Amplification
• MDA Multiple Displacement Amplification
• RPA Recombinase Polymerase Amplification
• TMA Transcription Mediated Amplification
• NASBA Nucleic Acid Sequenced Based Amplification
• thermostable polymerase DNA enzyme with strand displacement activity such as the Bst enzyme, among others.
• thermostable polymerase DNA enzyme with strand displacement activity can, or not, be reversibly coupled to an aptamer, which inhibits the activities thereof at temperatures lower than 40 °C.
• thermostable polymerase DNA enzyme with strand displacement activity can also have reverse transcriptase activity, allowing carrying out the reverse transcription and isothermal amplification with just one enzyme, such as the Bst enzyme, among others.
• the reaction mixture of the reverse transcription amplification and/or isothermal amplification can be comprised of a combination of oligonucleotide primers, dNTPs, thermostable polymerase DNA with strand displacement activity and/or polymerase DNA enzyme with reverse transcriptase activity, buffer optimised for the activity of the polymerase DNA enzymes, and reagent for discrimination in real time of the amplified fragments.
• the reverse transcription and/or isothermal amplification is carried out at a temperature from 37 °C to 70 °C, preferably at 65 °C, during an interval of more than 10 minutes, preferably between 30 to 60 minutes.
• the real-time discrimination of the amplified fragments can be made in an indirect or direct form using one or more reagents.
• the indirect real-time discrimination of the amplified fragments is made using a pH colourimetric indicator agent, such as phenol red, among others, or a DNA intercalating fluorescent reagent, such as SYBR green, among others.
• a pH colourimetric indicator agent such as phenol red, among others
• a DNA intercalating fluorescent reagent such as SYBR green
• the direct real-time discrimination of the amplified fragments is made using one or more oligonucleotide probes with complementary specific sequence to one or more region (s) of the relatively conserved amplified fragment(s).
• the oligonucleotide probes are conjugated with a fluorophore, such as, for example, FAM, HEX, ROX or Cy5, or similar, and/or a quencher, such as, for example, DABCYL, BHQ-1, BHQ-2, BHQ-3, or similar, or a combination of both.
• a fluorophore such as, for example, FAM, HEX, ROX or Cy5, or similar
• a quencher such as, for example, DABCYL, BHQ-1, BHQ-2, BHQ-3, or similar, or a combination of both.
• the oligonucleotide probes are RNA, DNA, LNA or PNA probes.
• the reverse transcription reaction and/or isothermal amplification results from the addition of a sample of extracted and purified RNA or DNA of samples presumably containing target nucleic acids, such as oral swabs, nasopharynx or nasal samples, blood, urine, saliva, sputum, faeces, environmental or food samples, or from surface contact, among others, or with the direct addition without extraction and purification of the RNA or DNA of these same samples to the reaction amplification mixture.
• target nucleic acids such as oral swabs, nasopharynx or nasal samples, blood, urine, saliva, sputum, faeces, environmental or food samples, or from surface contact, among others, or with the direct addition without extraction and purification of the RNA or DNA of these same samples to the reaction amplification mixture.
• the collection of samples to be analysed can be performed using a swab, syringe, collection tube or any other device designed for collection of samples.
• the method is characterised by the presence and consequent reverse transcription and/or isothermal amplification of a target sequence resulting in an alteration in colourimetric or fluorescence intensity, detected by the spectrophotometric sensor(s) (7) of the portable device (1).
• samples potentially containing viral particles of SARS-CoV-2 or the exposed viral RNA itself can be carried out in several manners, depending on the type of sample: a) Samples from the human or animal upper respiratory tract, including:
• swab Exudate from the nasopharynx, collected by swab and placed in tube containing up to 3mL of medium for transport of viruses, or saline solution, or another solution liable to lyse the viral particles and/or preserve the viral RNA intact.
• the swab must be inserted in one of the nostrils parallel to the palate until a slight resistance is felt. Leave the swab for a few seconds for absorption of the secretions. Gently remove with a rotating movement and repeat the collection in the other nostril.
• Oropharyngeal exudate collected by swab and placed in tube containing up to 3mL of medium for transport of viruses, or saline solution, or another solution liable to lyse the viral particles and/or preserve the viral RNA intact.
• the swab must be inserted in the oral cavity and rubbed against the pharyngeal wall and the oropharyngeal pillars.
• Broncho alveolar lavage collected by sterile container for liquid samples.
• a synthetic fibre swab with a plastic stick must be used, or any other that does not inhibit the reverse transcription reaction and/or isothermal amplification or which can potentially degrade the viral RNA sample.
• the samples can be used for extraction and purification of viral RNA, using traditional extraction and purification methods for nucleic acids or commercial kits for the purpose.
• a fraction of the sample can be placed in an aqueous solution containing a buffer and one or more RNases inhibitor(s), whereby the lysing of the viral particles for release of the viral RNA for subsequent analysis can occur by means of osmotic, mechanical and/or temperature lysis during the isothermal amplification process.
• the reverse transcription and amplification reactions of the cDNA resulting from the reverse transcription are carried out in just one step. Furthermore, the amplification is made in an isothermal manner to be compatible with the reverse transcription reaction. As such, different isothermal amplification techniques can be used:
• HDA Helicase-Dependent Amplification
• TMA Transcription Mediated Amplification
• NASBA Nucleic Acid Sequenced Based Amplification
• the discrimination of the real-time amplification can be carried out in two ways:
• the amplification is carried out in an unspecific manner, whether by monitoring a variable that is associated with the target template amplification (for example, variation of the pH of the reaction, turbidity, etc.), or by means of the colourimetric or intercalating fluorescent reagents to double-stranded DNA produced throughout the reaction (when in presence of a positive template).
• a variable that is associated with the target template amplification for example, variation of the pH of the reaction, turbidity, etc.
• this indirect way is not desirable since it may report false positive results, which can occur if there is non-specific amplification of the template or even of the oligonucleotide primer dimers.
• the amplification is carried out in a specific manner, using specific oligonucleotide probes which specifically hybridise in a region of the amplified zone, producing an alteration to the signal that is proportional to the amplified DNA.
• specific oligonucleotide probes which specifically hybridise in a region of the amplified zone, producing an alteration to the signal that is proportional to the amplified DNA.
• Specific conjugated oligonucleotide probes with self-quenched fluorophore Specific conjugated oligonucleotide probes with a fluorophore at one of the ends and a quencher at the opposite end, whether in hairpin or linear form (for the cases where the polymerase DNA has exonuclease activity); Combination of a specific conjugated oligonucleotide probe with a fluorophore and a specific conjugated nucleotide probe with a quencher, which sequences are contiguous to approximate the fluorophore to the quencher in case both hybridize specifically with the amplified DNA.
• the reverse transcription and/or isothermal amplification can be made with the intention of amplifying and detecting only one region or several simultaneously, using a multiplex approach to this end.
• one of the regions can serve as internal control of the sample, for example, by using as the target organism maintenance genes to be tested (ex. human, animal, bacterial, viral, among others), such as for example the actin gene, GAPDH or ubiquitin.
• the target organism maintenance genes to be tested ex. human, animal, bacterial, viral, among others
• the sample to be tested does not originate from an organism (such as for example, surface or environmental samples)
• a control target sequence such as, for example, actin genes, GAPDH or ubiquitin, or another synthetic target.
• the present invention refers to an integrated system for the detection and identification of specific sequences of nucleic acids in suspected coronavirus samples with the presence of a portable device (1) controlled by a mobile application (9) which records the data acquired by the portable device (1), analyses and stores it in a remote server in a cloud (10).
• the present invention further refers to a method for the detection and identification of the referred specific sequences of nucleic acids in samples that are suspected to contain coronavirus which cover the portable device (1), the mobile application (9) and the remote server in a cloud (10), which work together as a system.
• the present invention comprises a portable device (1) which is complementary to the method, the purpose of which is to control the test and record the data which allow an analysis for the determination of a last result to be established.
• the device (1) comprises one or more of the following components: a) A microcontroller (MCU) (5) with integrated Wi-Fi and/or Bluetooth, such as for example, an ESP32, the purpose of which is to control the different components and administrate all the information generated by some of the components which generate data that is useful to the test, namely, by the temperature sensor (6) and spectrophotometric sensor (s) (7), all managed by a firmware.
• MCU microcontroller
• ESP32 spectrophotometric sensor
• the firmware controls the different components of the device (1), particularly: it maintains an isothermal temperature during the course of the test; it controls the lighting sources and records the spectral variations during the course of the test; it communicates bi-directionally with the mobile application by means of Bluetooth or Wi-Fi, or with a remote server in a cloud by Wi-Fi.
• One or more spectrophotometric sensor(s) (7) which allow the emission of photons to be quantified differentially in different wavelengths of the UV, visible and/or infrared spectrum, preferably with a resolution less than or equal to 40nm.
• the detection is performed by subtraction of the photons, usually from white light, generated by the lighting system at 180° from the spectrophotometric sensor, and which are absorbed by the sample present in the reaction container.
• the detection is performed after excitation of the fluorophores using the lighting sources at 90° from the spectrophotometric sensor (7), and consequent detection by the spectrophotometric sensor (7) of the emission of photons generated by the excited fluorophore (s).
• These sensors (7) can be of the photovoltaic, photodiode, photoresistor, or phototransistor type, and may be coupled to light filters, for example a band-pass filter, to better define the spectral detection zone.
• All the photons detected by the spectrophotometric sensor (s) (7) are communicated to the MCU (5) by electronic means.
• One or more lighting sources (3) the purpose of which is to help the spectrophotometric sensor (s) (7) read the colourimetric or fluorescence changes, by means of the absorption of the light thereof emitted by the reagent (s) used in the real-time discrimination of the amplified fragments.
• These lighting sources (3) can be of the incandescent type, light-emitting diode (LED), laser (light amplification by stimulated emission of radiation) or any other source for emitting photons, and may be coupled to light filters, for example band-pass filter, to better define the spectral zone of the emission, emitting photons with a wavelength that is compatible with the selective excitation of the fluorophores of the probes, or in a broad visible spectrum.
• a heat source (8) the purpose of which is to heat the reaction container (2) to an optimum temperature for the DNA polymerase enzymes to work.
• This source (8) can be constituted by one or more resistor (s), Peltier module or any other heat source that is capable of reaching 100 °C and a minimum of 4 °C above room temperature.
• a temperature sensor (6) the purpose of which is to measure the temperature generated by the heat source (8) so that, with the help of the MCU (5), it is possible to "thermostatise” the heating of the reaction container (2) at the optimum temperature for the DNA polymerase enzymes to work, further guaranteeing that the inactivation temperature thereof is not reached inadvertently.
• the temperature sensor (6) is of the thermistor type (NTC or PTC), thermoresistance (RTD), thermocouple (ex.
• Battery (4) the purpose of which is to charge and operate the portable device (1) in an autonomous manner for a period of time, which allows for the execution of at least one test.
• the battery (4) is charged by DC charging, which can also charge the device (1) alternatively to the battery (4).
• the rechargeable battery (4) can be constituted by ion- aluminium, -lithium, -potassium or -magnesium, carbon, of flow (vanadium or zinc), lead-acid or polymer based, among others.
• the portable device (1) is controlled by a mobile application (9) connected to a remote server in a cloud (10) so that there is transmission of data and commands between the portable device (1), the mobile application (9) and the remote server in a cloud (10).
• the portable device (1) can also connect itself directly to the remote server in a cloud (10) for data transmission.
• the reaction mixture is incubated in the portable device (1) at a constant temperature between 37 °C and 70 °C for 30 minutes, and this time can be extended up to 90 minutes, or even 120 minutes, according to the isothermal amplification technique used.
• the monitoring of the amplification of the sample is carried out indirectly using fluorescent intercalating reagents (ex. SYBRTM green) and/or directly using the specific oligonucleotide probes marked with fluorophore (ex. FAM, HEX, ROX or Cy5, or similar), using the respective source of lighting (3) as excitation source of the fluorophore and its emission recorded by the spectrophotometric sensor (7) in different wavelengths of the visible/infrared UV spectrum (315 to 1400nm, in 40nm intervals at most).
• the same sensor (7) may also monitor alterations to the colour of the reaction mixture, as a form of determining indirectly whether amplification occurred or not.
• the entire process is started and controlled using a mobile application (9), which analyses the data of the spectrophotometric sensor (7) throughout the time to determine the final result.
• the present invention further comprises a mobile application (9) which connects with the portable device (1) by means of Bluetooth or, optionally by means of Wi-Fi.
• the mobile application (9) for control of the portable device (1) is compatible with Android or iOS, or any other operating system for a mobile device.
• the mobile application (9) carries out the processing of the data generated by the portable device (1) and communication of same to the remote server in a cloud (10).
• the application (9) allows for making the record of the test by using the camera of the mobile device to record the barcode associated to the reagent tube which contains the reaction mixture, to be able to record the reference of the test that it will process.
• the mobile application (9) communicates with the remote server in a cloud (10) in order to collect the parameters of the test (time, temperature, data to be analysed, cut-off for analysis, etc.) and transfers these parameters to the portable device (1) to be able to start the test.
• the mobile application (9) also allows for recording the user ID, the sample ID and to attribute a sole ID to the test (which is attributed by the remote server in a cloud (10)).
• the application (9) also records the location of the test to allow a geographic follow-up of epidemics/pandemics, while safeguarding the anonymity of the personal data of the user.
• the test transfers to the application (9) the data collected by the spectrophotometric sensor (7) during the course of the test.
• This data will be analysed by the mobile application (9), which will consider positive, a sample of which the variation in fluorescence intensity in the respective wavelengths of emission of the fluorophore used is higher than 10% of the initial signal of fluorescence, and when this occurs in a time less than 1600 seconds (this cut-off may vary between 700 and 3000, according to the test in question).
• the monitoring can also be made in multiplex, using more than one specific oligonucleotide probe marked with different fluorophores (ex. FAM, HEX, ROX or Cy5, or similar) which can be monitored in distinct wavelengths (ex. 520nm, 556nm, 602nm, 670nm, among others).
• the present invention further comprises a remote server in a cloud (10) which stores and processes the data from all the tests in a centralised manner as well as the parameters used (reaction time, reaction temperature, data to be recorded, intervals of data to be recorded, parameters for analysis of the results, among others) for each of the tests that can be carried out on the portable device (1).
• the server (10) further attributes, by means of the mobile application (9), one sole ID for each one of the tests, associating this ID with the results and other information (sample ID, location of test, user, among others).
• the server (10) can also include an artificial intelligence algorithm for a dynamic analysis of all the data united therein, as well as timely alerting the respective health entities. All the data in the server (10) is encrypted to safeguard the cyber-security of all the data stored and being transmitted between server and mobile application (9) and/or portable device (1).
• the sample to be analysed is usually collected with a swab and placed in a tube containing up to 3mL of medium for transport of viruses, or saline solution, or another solution capable of lysing the viral particles and/or preserving the viral DNA intact.
• a fraction between 1 and 40 microlitres of this aqueous medium is added to the reaction mixture previously prepared and provided to the final user.
• the collected samples can be processed for the extraction and purification of the viral RNA, using traditional extraction and purification methods for nucleic acids or commercial kits for this purpose, resuspending the purified nucleic acids in distilled water (Type-I) free of ribonucleases, being subsequently used a fraction for analysis (between 1 and 40 microlitres) of these purified resuspended nucleic acids.
• distilled water Type-I
• oligonucleotide primers for the reverse transcription and isothermal amplification by the LAMP method, from different genomic regions relatively preserved of SARS-CoV-2. Namely, for the region: Table 1. RNA templates relative to regions of the genome of the coronavirus severe acute respiratory syndrome 2 (SARS- CoV-2).
• a set of oligonucleotide probes conjugated internally with a fluorophore was defined, which is self-quenched in the isolated form thereof, and which sequences hybridize in specific preserved zones of different genomic regions of the SARS-CoV-2, located in the fragment amplified by LAMP, whereby they work by including as oligonucleotide primers of direct or reverse loop, according to region.
• the fluorophore ceases to self-quench, increasing the emission of fluorescence thereof. Namely, these are the probes for each region: Table 3. Probes for specific discrimination of the reverse transcription and/or isothermal amplification of regions of the genome of the coronavirus severe acute respiratory syndrome 2 (SARS-CoV-2).
• the mixture of the LAMP reaction was optimised for the method for detection and identification of SARS-Cov-2 by means of the present invention, containing: 1.6mM of each nucleotide primer FIP and BIP; 0.2mM of each nucleotide primer F3 and B3; 0.4mM of each nucleotide primer LF or LB and of each specific oligonucleotide probe LB or LF, according to the region to be analysed; 1.4mM of each dNTP; 320 U/ml Bst DNA Polymerase; 500 U/mL Reverse Transcriptase; 20 mM Tris-HCl pH 8.8 @25 °C; 8mM (NH 4 ) 2 S0 4 ; lOmM MgS0 4 ; 50 mM KC1; and 0.1% Tween® 20. Table 4. Combination of oligonucleotide primers for the reverse transcription and/or isothermal amplification of regions of the genome of the coronavirus severe
• the final volume of the reaction mixture used is of 20 m ⁇ , considering a sample volume of 8m]3. However, the final volume of the reaction mixture can be proportionally adjusted between 5 to 100m]0.
• 20m]3 of mineral oil or liquid wax are added.
• PCR tubes of 0.2mL are used as a reaction container, and tubes from 0.1 mL to 0.5mL may also be used.
• the reaction mixture was incubated in the portable device (1) at a constant temperature of 65 °C for 30 minutes. At the end of that time, the data from the spectrophotometric sensor (7) was sent to the mobile application (9) and analysed.

Applications Claiming Priority (2)

Publications (1)

Family

ID=75787159

Family Applications (1)

Country Status (8)

Families Citing this family (3)

Family Cites Families (11)

• 2021
  • 2021-04-28 JP JP2022566717A patent/JP2023524117A/ja active Pending
  • 2021-04-28 BR BR112022021086A patent/BR112022021086A2/pt not_active Application Discontinuation
  • 2021-04-28 AU AU2021265579A patent/AU2021265579A1/en active Pending
  • 2021-04-28 CA CA3175648A patent/CA3175648A1/en active Pending
  • 2021-04-28 WO PCT/IB2021/053530 patent/WO2021220192A2/en unknown
  • 2021-04-28 EP EP21723396.4A patent/EP4142945A2/de not_active Withdrawn
  • 2021-04-28 CN CN202180031710.9A patent/CN115836133A/zh active Pending
• 2022
  • 2022-10-12 IL IL297281A patent/IL297281A/en unknown

Also Published As

Similar Documents

Legal Events