WO2021201801A2 - Dispositif portable, kit et domaines d'utilisation pour diagnostic basés sur l'amplification isotherme d'acides nucléiques - Google Patents

Dispositif portable, kit et domaines d'utilisation pour diagnostic basés sur l'amplification isotherme d'acides nucléiques Download PDF

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WO2021201801A2
WO2021201801A2 PCT/TR2021/050264 TR2021050264W WO2021201801A2 WO 2021201801 A2 WO2021201801 A2 WO 2021201801A2 TR 2021050264 W TR2021050264 W TR 2021050264W WO 2021201801 A2 WO2021201801 A2 WO 2021201801A2
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reaction
module
bag
tubes
results
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WO2021201801A3 (fr
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Hüseyin Avni ÖKTEM
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Nanobi̇z Teknoloji̇ Anoni̇m Şi̇rketi̇
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  • the invention relates to devices, apparatus, test kits that can be used in the diagnosis of various biological agents with nucleic acid-based methods in settings other than equipped laboratories, and to their usage areas.
  • ELISA polymerase chain reaction
  • RT-PCR reverse transcription-polymerase chain reaction
  • NA nucleic acid
  • nucleic acid-based techniques In order to obtain nucleic acid from the sample containing the target agent for the application of nucleic acid-based techniques, different requirements must be met such as pre-enrichment, nucleic acid isolation, preparation of different reagents for the tubes to be used in the reaction, reagents that need to be stored at -20 degrees such as enzymes, primers, precise pipetting at the microliter level, preferably a sterile environment to prevent possible contamination. Due to these requirements, a well-equipped molecular biology laboratory is required to apply NA- based techniques and expert personnel are required to make the above-mentioned preparations.
  • NA-based analyzes Another important issue in NA-based analyzes is the problems that may occur during the transfer of samples collected from the field to the laboratory for analysis (such as sample deterioration, contamination) and the result may be delayed due to elapsed time. Accordingly, different problems can be experienced in different sectors. For example, in a patient, treatment can be started late, as in the COVID19 pandemic, contamination and disease can become widespread and the damages can reach enormous dimensions since isolation and treatment are started late in veterinary practices. In the food sector, significant losses can occur due to delays in shipment or withdrawal of shipped goods.
  • This invention defines devices, apparatus, kits and their areas of use that make it possible to perform NA-based analyzes on the bedside or pen side in the field.
  • Two basic versions of the device are defined in the invention. One of them is designed in a format that the screen, keyboard and electronics can be carried in a single bag.
  • the other device version being described contains all of the above-mentioned components in a case that is small enough to be carried by hand.
  • the device can also perform NA-based tests using the kits defined in this invention, automatically analyze the test results and display the results on the screen, and perform all functions required to transmit them to third parties by SMS, e-mail and voice calls in accordance with the Internet of Things (IoT) concept.
  • the device of the invention can also exchange data by communicating with smart devices such as mobile phones and tablets.
  • kits used in the devices are designed to contain all the reagents required to perform isothermal amplification-based nucleic acid tests for diagnosis of the target biological agent and the chemicals necessary for the release of nucleic acids from biological agents, all these components are contained in a single tube and are presented in reactive gel or dry format.
  • kits format when samples containing biological agents (such as nose/throat swabs, blood/serum samples taken from the patient) have sufficient agent concentration, they are applied directly to reaction tubes without any pre -preparation, pre-enrichment and/or nucleic acid purification, and the reaction is carried out by placing the tubes in the device defined in the invention, the result is automatically obtained, displayed on the screen of the devices, and can be transmitted to a central software and third parties via SMS, e-mail and voice calls, including the GPS data of the location where the test is performed and sample information when desired.
  • a portable meteorology station can be connected to the system and the meteorological data at the point where the analysis is made can be transmitted to the user or third parties defined in the system as stated above.
  • Figure 3 View of the portable device in bag format from different angles (A. Top view, B. Front view, C. Screen view, D. Side view)
  • Figure 6. View of the hand-held device from different angles (A. Top view, B. Front view, C. Perspective view, D. Side view)
  • Figure 8. View of the reaction cartridge from different angles (A. Front view, B. Back view, C. Perspective view, D. Side view)
  • FIG. 1-8 The elements of the device forming the invention are shown in Figure- 1-8.
  • Two basic versions of the device are defined in the invention. One of them is designed in a format that the screen, keyboard and electronics can be carried in a single bag.
  • the other device version being described contains all of the above-mentioned components in a case that is large enough to be carried by hand.
  • Figure 1 shows the diagnostic device in portable bag (1) format and its parts.
  • Figure 2 shows the inside view of the diagnostic device in bag format.
  • Figure 3 shows the diagnostic device in bag format view from different perspectives.
  • Figure 4 shows the diagnostic device in hand-held format.
  • Figure 5 shows the diagnostic device in hand-held format view from different perspectives.
  • Figure 6 shows the inside view of the diagnostic device in hand-held format.
  • Figure 7 shows the cartridge used in the diagnostic device and its parts.
  • Figure 8 shows the cartridge used in the diagnostic device and its parts viewed from different perspectives.
  • the inventive test system which includes two separate applications in a bag (LAMP4U ® - BagLab) and hand-held (LAMP4U ® -NanoLab) format, can perform highly sensitive molecular methods based on nucleic acid amplification, such as polymerase chain reaction (PCR) and loop-mediated isothermal amplification (LAMP) reactions without the need for any other device and/or laboratory infrastructure.
  • PCR polymerase chain reaction
  • LAMP loop-mediated isothermal amplification
  • the biological agent diagnostic device of the invention includes;
  • At least one reaction module in which cartridges are placed, which performs the functions of heating and cooling the reaction tubes, detecting the reaction results, and
  • the term "hand-held case” defines the carrying bag (1) in the first embodiment of the invention and the device body in the second embodiment.
  • data input module where the user can input information defines the keyboard (2) and the touch screen (14) in embodiments of the invention.
  • data display module where the user can view information defines the screen and the computer (7) in the first embodiment of the invention and the touch screen (14) in the second embodiment of the invention.
  • the device in Bag format (LAMP4U ® -BagLab) is contained in a portable bag (1) that is impact resistant and waterproof (IP67).
  • the dimensions of the portable bag (1) are 40-45 cm x 30-35 cm x 17-23 cm in width-length-height, respectively. Bag dimensions can also be smaller, such as 30-55 cm x 25-30 cm x 5-10 cm, provided that their functions remain the same. Bag dimensions are not limited to these.
  • the portable device in bag format includes a keyboard (2) where the user can input information, at least one reaction module (3), in which the cartridges used for diagnosis are placed and that performs the functions of heating, cooling, illuminating the reaction tubes, determining reaction results, turn on-off button (4), socket for energy connection (5), data cable (6) providing communication of screen and computer (7) with the electronic units (13), USB sockets (8) for connecting different devices such as touch screen and computer (7), meteorology station (10), barcode reader (9) to identify cartridges to the system, bag multi- electronic unit (13) including heating and cooling of at least one reaction module (3), image and signal capture, signal processing, communication, GPS data determination, communication units, connection panels (11) for connecting touch screen-computer-electronic units to the bag body (1) and bag cover (12) and screws for mounting.
  • a keyboard (2) where the user can input information
  • at least one reaction module (3) in which the cartridges used for diagnosis are placed and that performs the functions of heating, cooling, illuminating the reaction tubes, determining reaction results, turn on-off
  • the hand-held format system in the hand-held format system (LAMP4U ® -NanoLab), it includes the touch screen (14), the screen upper cover (15), the screen lower cover (16), the reaction module cover (17), the body upper part (18), the body lower part (19), at least one reaction module (3), which performs the functions of heating, cooling, illuminating the reaction tubes, determining the reaction results, the hand-held apparatus electronic unit (20) including heating, cooling of at least one reaction module (3), image and signal capture, signal processing, communication, GPS data determination and communication units, the rechargeable battery (21) and screws for mounting.
  • the hand-held apparatus electronic unit (20) including heating, cooling of at least one reaction module (3), image and signal capture, signal processing, communication, GPS data determination and communication units, the rechargeable battery (21) and screws for mounting.
  • the device in both embodiments can wirelessly communicate with smart devices such as mobile phones or tablets via radio frequency (RF) or internet connection and can be controlled in accordance with the IoT concept.
  • RF radio frequency
  • Reaction cartridges ( Figure 7) contain the cartridge body (23), disposable reaction tubes (24), cartridge cover (25), barcode or QR code positioned on the cartridge.
  • the cartridges include reaction tubes (24) containing chemicals required for the release of acids into the environment in the biological agents and all reaction components required for the diagnosis of biological agents by isothermal nucleic acid amplification method such as primer sets, enzyme, salt, detergent, buffer, dNTP molecules, ions, betaine, EDTA, signal-generating colored or fluorescent molecules and buffer chemicals in dry or gel format and a barcode printed label.
  • the operating principles of diagnostic systems have been created to be extremely simple, by means of the software they contain and simple user interfaces. An individual reaction tube has been created for each biological agent to be diagnosed.
  • reaction tubes contain all components that can amplify the target biological agent-specific gene region or gene regions by isothermal nucleic acid (DNA or RNA) amplification and make it possible to measure the amplified nucleic acids by changes in color change, fluorescence or magnetic properties.
  • isothermal nucleic acid DNA or RNA
  • Reaction tubes were prepared to contain all components in dried or gel format for single use. By means of these features, reaction tubes make it possible to get results by adding the biological agent directly to the reaction tube without the need for any nucleic acid purification from the biological agent to be diagnosed. In addition, in the case of nucleic acid purification from a biological agent, the purified nucleic acids can be added to the reaction tube and a diagnosis can be made.
  • the tubes are placed in the carrier cartridge or the samples are added to the reaction tubes while the tubes are in the carrier cartridge.
  • the label on the cartridge contains information on the preparation date of the reaction tubes, the expiry date, which biological agent or agents is/are to be diagnosed. This information is also processed into the barcode and/or QR code on the label. Experimental parameters to be used in the diagnosis of the target biological agent are also processed on the barcodes. In this way, before the cartridge is placed in the devices, barcode is scanned with a barcode reader or smart device, and after the cartridge is placed in the reaction module (3), the analysis can be started by commanding via the touch screens. Reactions can also be started with the information inputted on the device screens without reading the barcode.
  • the analysis results are determined by the sensing units in the reaction module (3) and processed by the components in the electronic unit, and the result is displayed to the user after the reaction.
  • the results obtained with these processes are simultaneously transmitted to users registered in the system via e-mail, SMS and voice calls via the communication module in the electronic unit.
  • RNA or DNA nucleic acids
  • PCR and LAMP Molecular test systems based on nucleic acid amplification such as PCR and LAMP are methods used to determine biological agents (such as COVID19) with high sensitivity and accuracy.
  • a well-equipped infrastructure and expert personnel are required for the application of these methods.
  • nucleic acids RNA or DNA
  • PCR nucleic acid purification kit
  • various laboratory devices such as centrifuge, vortex, micropipette and spectrophotometer.
  • the reagents must be carefully pipetted in special sterile cabinets (biosafety cabinets) in order to prevent contamination from the environment during the preparation of the reaction tubes.
  • the reaction is started by placing the tubes in devices called thermal cycle devices.
  • the reaction may take 80 to 120 minutes to result. At the end of this period, it is concluded that the reaction is positive or negative and necessary reporting procedures are carried out. As can be understood from here, this process takes approximately 3-4 hours and cannot be performed outside of the laboratory.
  • the simple-to-use apparatus and disposable kits defined in the invention it is possible to perform molecular tests in the field and at the bedside. Since the ready-to-use kits contain all the components necessary for the release of nucleic acids from the biological agents in the sample and for the amplification reaction to be performed, it is sufficient to directly add the samples taken from the patient to the reaction tubes in order to perform the test. Then, the reaction tubes are placed in the cartridge, and the cartridge is placed in the reaction module (3) in the portable devices, and the reactions can be started using the touch screens (7, 14) of the devices. The reactions are completed in 45-60 minutes and the results are displayed to the user on the screen. In addition, the results can be transmitted to the addresses registered in the system as SMS, voice calls and e-mails when requested.
  • devices and kits have the potential to be used for different purposes in different sectors such as medicine, environment, agriculture, animal husbandry, but not limited to the examples below.
  • LAMP4U ⁇ devices and kits can be used outside the laboratory, it makes it possible to perform molecular tests, especially at sample collection and test stations to be established in the field, first-level health institutions such as family health centers, non-fully-fledged hospitals and even at the bedside at home. In this way, people with virus infection can be diagnosed quickly by applying molecular tests and based on this diagnosis, appropriate triage can be made and necessary precautions can be taken and the spread of the pandemic can be prevented.
  • apparatus and kits have the potential to be used in land border crossings, ports, airports, military units, refugee camps, road control points, field hospitals, and temporarily established health units.
  • LAMP4U ⁇ devices and kits can be widely used in the agriculture sector. For example, with samples taken from greenhouses and fields, the system will be able to quickly detect biological agents (such as mold, bacteria, fungi, viruses) that can cause disease and by taking the necessary precautions, a large-scale disease and therefore product loss can be prevented. It can also be used to determine whether there is a genetically modified organism (GMO) in different plant samples and/or plant products.
  • GMO genetically modified organism
  • a pathogen detected in different samples collected from poultry houses and barns where animals are densely sheltered will enable to take precautions with early warning.
  • An important application in this regard is the fast and accurate detection of calf diarrhea.
  • the system of the invention identifies the biological agents such as spore, bacteria, vims, fungus (mold), yeast, algae, pollen, parasite, parasite egg in samples taken directly or processed after collection or in samples such as tissue, blood, serum, saliva, urine, sweat, milk, honey, egg, body fluid, semen, sperm, vaginal fluid, stool, hair, feather, in soil, fresh water, sea water, environmental samples taken from the air; processed or unprocessed red meat, white meat, water products, such as fish and shrimp; in food products such as vegetables, fruits, grains, legumes, honey, molasses, jam; in plant parts such as leaves, roots, stems and flowers; flying- walking insects, snakes, lice, fleas and insects such as ticks; in poultry, ovine and bovine animals; in domestic animals (pets) such as horses, donkeys, cats- dogs-birds; in humans by a method based on nucleic acid amplification (such as loop- mediated isothermal amplification). Examples of
  • EXAMPLE 1 Identification of E.coli 0157:H7 bacteria by LAMP method without DNA isolation.
  • EXAMPLE 2 Identification of Salmonella bacteria by LAMP method without DNA isolation.
  • this result was sent to the phone number registered in the system via e-mail and SMS.
  • This example shows that the signal (color change) generated as a result of the reaction is determined by the sensing unit, processed by the electronic processor and control unit within the electronic unit, the results are displayed and can be transmitted to the registered persons by SMS and E-mail by the software.
  • EXAMPLE 3 Diagnosis of COVID-19 (SARS-CoV-2) Virus reference material by Reverse transcriptase (RT)-LAMP method without RNA isolation.
  • EXAMPLE 4 Diagnosis of COVID-19 (SARS-CoV-2) Virus in Clinical Samples with or without RNA isolation by Reverse transcriptase (RT) -LAMP method.
  • RNA purification was performed with silica column or magnetic sphere-based kits of different brands on clinical samples contained in VTM or VNAT. 4 microliters of the purified RNA samples were added to reaction tubes, and the reaction was performed in the devices described in the present invention. Color change was observed in the tubes to which RNA was added.

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Abstract

La présente invention concerne un appareil/dispositif portable, un procédé et leurs domaines d'utilisation selon le concept de l'Internet des objets, qui peuvent être utilisés pour identifier des agents biologiques tels que des bactéries, des spores bactériennes, des champignons (moisissure), des levures, des virus, des algues et du pollen, des parasites, des œufs de parasite, des espèces de viande et des organismes génétiquement modifiés à l'aide de techniques basées sur les acides nucléiques à l'extérieur du laboratoire.
PCT/TR2021/050264 2020-03-30 2021-03-25 Dispositif portable, kit et domaines d'utilisation pour diagnostic basés sur l'amplification isotherme d'acides nucléiques Ceased WO2021201801A2 (fr)

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
TR2020/04939 2020-03-30
TR202004939 2020-03-30
TR2021/003965 2021-03-01
TR202103965 2021-03-01

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WO2021201801A2 true WO2021201801A2 (fr) 2021-10-07
WO2021201801A3 WO2021201801A3 (fr) 2021-11-04

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
NL2030742B1 (en) * 2022-01-27 2022-11-30 Dong Jiayin Specimen display device for high school biology teaching and use method
RU216488U1 (ru) * 2022-12-01 2023-02-07 Общество с ограниченной ответственностью "Троицкий инженерный центр" Станция для одноразового картриджа для изотермической амплификации

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CA2692477A1 (fr) * 2007-07-03 2009-01-08 Smiths Detection Inc. Systeme et procede de detection portables
GB0720264D0 (en) * 2007-10-17 2007-11-28 Smiths Detection Watford Ltd Sample preparation devices and analyzers
KR20160090927A (ko) * 2015-01-22 2016-08-02 (주)미코바이오메드 휴대용 실시간 dna 분석 장치

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
NL2030742B1 (en) * 2022-01-27 2022-11-30 Dong Jiayin Specimen display device for high school biology teaching and use method
RU216488U1 (ru) * 2022-12-01 2023-02-07 Общество с ограниченной ответственностью "Троицкий инженерный центр" Станция для одноразового картриджа для изотермической амплификации

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