A pocket-sized device for isothermal nucleic acid amplification.

Abstract

The pocket-sized device (1) for isothermal nucleic acid amplification comprises an opening (4) for receiving a reaction vessel (15) in which nucleic acid amplification will take place, and an electronic circuit (10). The electronic circuit (10) includes a positive temperature coefficient thermistor (13) as a heating element, a power supply port (9), and optionally a resistor (12).

Description

[Technical field]

[0001] The present invention relates to a pocket-sized device for carrying out nucleic acid amplification at constant elevated temperatures. [Background technology]

[0002] Nucleic acid amplification is based on the ability of some specific enzymes to amplify small amounts of nucleic acid in a sample, generating amplicons that can be subsequently or in real time detected using various methods, such as fluorescent or colorimetric methods. There are a number of different methods for amplifying nucleic acids. Some of these methods, such as polymerase chain reaction (PCR), require the sample to be subjected to repeated cycles of heating and cooling between 55°C and 95°C. Other methods, known as isothermal amplification methods, require the sample to be exposed to a constant elevated temperature, i.e., above room temperature. Many isothermal amplification methods are known in the art, including loop-mediated isothermal amplification (LAMP), recombinase polymerase amplification (RPA), rolling circle amplification (RCA), and isothermal identical multirepeat sequences amplification (isoIMRS). The loop-mediated isothermal amplification (LAMP) method utilizes the complementarity of multiple primers (4 to 6) to different regions of the DNA to be analyzed, together with a DNA polymerase with pronounced strand displacement activity. The use of multiple primers ensures high specificity of the reaction. LAMP is usually performed at a constant temperature between 60 and 65 °C and can amplify up to 10 DNA fragments within 15 to 60 minutes. 9It has exponential properties that allow the generation of DNA copies of a sequence. The isothermal identical multiple repetitive sequence (isoIMRS) amplification assay uses Bst polymerase and a single pair of computationally identified repetitive primers that bind to multiple loci across the genome for exponential DNA amplification at a constant temperature. The recombinase polymerase amplification (RPA) method requires the formation of a complex between the recombinase enzyme and forward and reverse primers. This complex unwinds the DNA double strand and allows exponential amplification of the template. RPA can be performed at 22-45 °C, but the optimal temperature range is 37-42 °C. The rolling circle amplification (RCA) method is based on the use of circular DNA formed by the interaction of the sequence to be analyzed with a single-stranded DNA probe flanked by sequences complementary to the analyte. In the process of complex formation, the 5' and 3' ends of the probe are brought together and then linked to form a circular molecule. The formed circular probe can hybridize with the primer extended by DNA polymerase resulting in the generation of a sequence consisting of multiple copies of the analyte DNA. Typically, RCA is performed at 30-37 °C, and linear amplification at constant temperature takes hours to days, resulting in the synthesis of a large number of analyte copies.

[0003] A common feature among devices for isothermal nucleic acid amplification in the art is the requirement of an electronic circuit including a heating element, a temperature sensor to precisely regulate the temperature, and a processing unit to control the circuit. This is the main reason why performing molecular tests outside of diagnostic laboratories, for example when disposable rapid antibody / antigen tests are used, is still expensive and complicated. To date, several attempts have been developed to create simple, low-cost molecular diagnostic devices, but most of them include complex electronic circuits and microprocessors.

[0004] A. Ganguli et al., Rapid isothermal amplification and portable detection system for SARS-CoV-2, PNAS, 2020, 117, 37, 22727-22735, discloses a microfluidic diagnostic cartridge that combines an instrument with a smartphone to detect fluorescent emissions from a LAMP assay performed in a microfluidic cartridge. The instrument includes four main components corresponding to optical, electrical, and heating components, including a microlens, a printed circuit board (PCB), a long-pass filter, eight LEDs, four short-pass filters, an on-off switch, and an auto-regulating positive temperature coefficient heater.

[0005] NY Jayanath et al., Development of a portable electrochemical loop mediated isothermal amplification (LAMP) device for detection of hepatitis B virus, RSC Adv., 2018,8,34954-34959, discloses the development of a custom portable prototype device for real-time measurement of LAMP reaction using electrochemical method. The system includes a drop cell connector, a heat sink heated to a preset temperature in a water bath, a portable potentiostat, and a microelectrode.

[0006] D. Kaygusuz et al., DaimonDNA: A portable, low-cost, loop-mediated isothermal amplification platform for naked-eye detection of genetically modified organisms in resource-limited settings, Biosensors and Bioelectronics, 2019, 141, 111409, discloses a portable device for colorimetric LAMP amplification and naked-eye result interpretation, which includes the following electronic components: Peltier effect heater, temperature sensor, printed circuit board (PCB), thermocouple, thermocouple digital converter, and power supply (12V DC, 5A). An Arduino Nano microcontroller was also used to control the electronic circuit, which includes MOSFETs and transistors for switching and amplifying the signal that operates the Peltier.

[0007] US Patent Application Publication No. 20200122142(A1) discloses an apparatus for performing bioassays, such as DNA amplification and detecting optical properties of a biological sample under analysis. Utilized components of the apparatus include a heating element, a temperature sensor, a circuit board, a light sensor, a light emitting element, an electronic result display mechanism, and a power supply.

[0008] A thermistor is a type of electrical resistor that has a resistance that is significantly more temperature dependent than a standard resistor. There are two main categories of thermistors: those that exhibit a negative temperature coefficient (NTC), i.e., become better conductors as temperature increases, and those that exhibit a positive temperature coefficient (PTC), i.e., become poorer conductors as temperature increases. Thermistors are widely used as temperature sensors in fire alarms, ovens and refrigerators, digital thermometers, and many autonomous applications, or in inrush current suppression and current limiting devices for the protection of motors and circuits in electrical and electronic equipment. Summary of the Invention [Means for solving the problem]

[0009] The present invention provides a pocket-sized device for isothermal amplification of nucleic acids that combines the accuracy of molecular testing with the simplicity of construction and cost-effectiveness of paper-based antigen tests (rapid tests).

[0010] The device of the present invention comprises an opening configured to receive a reaction vessel in which isothermal nucleic acid amplification is to be performed, and further comprises an electronic circuit including a positive temperature coefficient (PTC) thermistor as a heating element, a power input port, and optionally a resistor. [Brief description of the drawings]

[0011] [Figure 1] FIG. 1 shows an apparatus according to the invention. [Diagram 2] FIG. 1 shows two parts of a device according to the invention. [Diagram 3] FIG. 2 shows parts of a device according to the invention, including a thermistor. [Figure 4] FIG. 2 shows parts of a device according to the invention, including an opening for receiving a reaction vessel. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0012] The present invention provides a pocket-sized device for isothermal nucleic acid amplification, the device comprising an opening configured to receive a reaction vessel in which isothermal nucleic acid amplification will take place, and electronic circuitry for heating the contents of the reaction vessel.

[0013] The electronic circuitry of the device includes a PTC thermistor as a heating element, a power input port, and optionally a resistor.

[0014] The thermistor is positioned to be thermally coupled to the opening such that when the reaction vessel is positioned at the opening, the thermistor is thermally coupled to the reaction vessel.

[0015] As current flows through the thermistor, its temperature rises and its resistance also increases, thus automatically regulating the current passing through it. In this way, the temperature of the thermistor is kept constant. Since there is a relationship between the applied voltage and the temperature of the thermistor, the temperature is predetermined by the voltage applied to the thermistor.

[0016] If the input voltage provided through the power input port is greater than the voltage required for the thermistor to reach the required temperature, the electronic circuit further includes a resistor, which acts as a voltage divider to deliver the desired voltage to the thermistor. Alternatively, the required voltage can be applied directly to the thermistor through the power input port. In this case, the electronic circuit does not need to include a resistor.

[0017] The power input port can have different forms well known in the art, such as a power socket, a power plug, a power outlet, a Molex port, an International Electrotechnical Commission (IEC) connector, a Japanese Solderless Terminal (JST) connector, a coaxial port, a Deutsche Industrie Norm (DIN) connector, a Universal Serial Bus (USB) port, a barrel connector, an Anderson Power Pole, an SAE connector, an XLR connector, an EmPower connector, a Tamiya connector, IP44, or any similar connector capable of delivering power. According to a preferred embodiment, the power input port comprises a USB port, such as a USB port type A, B, C, or a micro USB port, or any similar port, preferably having a small footprint.

[0018] The current applied through the power input port is preferably a direct current (DC). The voltage applied is preferably in the range of 1V to 230V. The current is preferably up to 5A. Alternatively, an alternating current (AC) can also be applied to the device of the present invention. However, according to PTC thermistor manufacturers, due to the structure of the material, PTC thermistors for AC voltages do not act as pure ohmic resistors, but as capacitive resistors due to grain boundary junctions. Therefore, the input voltage to be used in the device of the present invention is preferably a DC voltage. According to a preferred embodiment, the input voltage applied to the device is a DC voltage of 5V and the current is 2-3A.

[0019] According to a preferred embodiment, the device does not comprise any additional electronic circuitry.

[0020] According to another preferred embodiment, the electronic circuitry of the device does not include any additional electrical components.

[0021] The device comprises an aperture configured to receive a reaction vessel in which isothermal nucleic acid amplification will take place. The walls of the aperture are preferably made of a material having a thermal conductivity greater than 1 W(m·K). Examples of such materials include highly filled epoxies, silicones, aluminum oxide (Al 2 O 3 The thermistor may be a metal, a non-metal, or a polymer, such as SiO2, aluminum nitride (AlN), graphene, etc. However, other materials with lower thermal conductivity (e.g., 0.01-0.9 W / (m·K)), such as polypropylene (PP) and acrylonitrile butadiene styrene (ABS), may also be used. The aperture and thermistor are arranged such that the thermistor is thermally coupled to the aperture. Thus, when the reaction vessel is positioned at the aperture, the reaction vessel is thermally coupled to the thermistor. During operation of the apparatus, as the temperature of the thermistor increases, the temperature of the walls of the aperture also increases, thereby heating the reaction vessel and its contents.

[0022] The remainder of the device is preferably made of a material with low thermal conductivity (e.g., 0.01-0.9 W / (m·K)) to keep heat within the opening of the device. Examples of such materials include ABS, acrylic, PP, FR4, glass, poly(4,4'-oxydiphenylene-pyromellitimide) (e.g., Kapton®), nylon, plexiglass, polyethylene (PE), polystyrene (PS), rubber, and polytetrafluoroethylene (e.g., Teflon®).

[0023] Heating of the reaction vessel occurs primarily through conduction, since the reaction vessel is in thermal contact with the thermistor and the walls of the opening, and partially through convection, since the thermistor heats any air trapped in the opening, which in turn heats the reaction vessel.

[0024] The device may comprise two or more openings, for example two openings, each configured to receive a reaction vessel. In such a case, two or more amplification reactions can be carried out simultaneously. In such an embodiment, the heating of all vessels is preferably performed by the same PTC thermistor.

[0025] The reaction vessel can be any vessel commonly used for performing nucleic acid amplification. The volume of the reaction vessel is preferably from 0.1 ml to 5 ml. The reaction vessel is preferably a tube, such as a PCR tube. The reaction vessel is preferably made of a transparent or translucent material, such as polypropylene.

[0026] To perform the analysis, the sample to be analyzed and the required reagents such as primers, enzymes, detection agents, etc. are added to the reaction vessel. The reaction vessel is then inserted into the opening of the device. The power input port is then connected to a power source and kept connected for the time required for the completion of the amplification reaction, typically 15 to 30 minutes. The reaction vessel is then removed from the device and the contents of the reaction vessel are examined, for example visually, to see if the nucleic acid of interest was present in the sample. There are a number of different methods for visually detecting the contents of the reaction vessel, which are well known in the art. For example, visual detection can be based on a color change in the contents of the reaction vessel, by utilizing pH-sensitive dyes or metal-binding indicators or DNA-intercalating dyes, etc. Visual detection can also be performed by visualizing the change in turbidity of the solution after the production of magnesium pyrophosphate. As an alternative to visual detection, the production of DNA amplicons can be detected by other methods such as turbidimetry, fluorescence, or by using a UV light source. A smartphone camera and an appropriate smartphone app can also be used for sensitive detection of DNA amplification products.

[0027] The device of the present invention is used in an isothermal nucleic acid amplification method, i.e., a method in which nucleic acid amplification is carried out at a constant temperature higher than room temperature. The isothermal nucleic acid amplification method is preferably LAMP, RPA, RCA, or Iso-IMRS. More preferably, the isothermal nucleic acid amplification method is LAMP or Iso-IMRS.

[0028] The devices of the invention may be disposable, ie suitable for a single use, or may be non-disposable, ie suitable for more than one use.

[0029] According to a preferred embodiment, the reaction vessel can be secured after insertion into the opening of the device by use of a security means, such as a security ring. In this way, the reaction vessel cannot be inadvertently removed from the device during operation. After completion of the amplification, the security means can be removed from the device and the reaction vessel removed and examined.

[0030] According to another preferred embodiment, the reaction vessel is secured by a security means, such as a non-removable security ring. According to this embodiment, the part of the device containing the opening is removably connected to the part of the device containing the thermistor. The lower part of the reaction vessel preferably protrudes through the lower surface of the part of the device containing the opening. Once the analysis of the sample is completed, the part of the device containing the opening that holds the reaction vessel is removed from the remaining parts of the device and the contents of the reaction vessel are examined to see if the nucleic acid of interest was present in the sample. If the device is not disposable, the part of the device containing the opening is replaced after each test is completed. In this way, the reaction vessel held by this part is properly disposed of.

[0031] The device of the present invention is pocket-sized, i.e., it fits in the palm of an adult user's hand or in a pocket. Moreover, the device is very simple, low-cost and easy to manufacture. Heating the contents of the reaction vessel is performed simply and effectively. Thus, the complex electronic circuitry of the prior art devices is not necessary. Moreover, the operation of the device is very simple, i.e., the user does not need any specialized knowledge or skills to perform nucleic acid amplification. In addition, the device can be used in any situation, not just in a laboratory.

[0032] Figure 1 shows one embodiment of the device according to the invention. The device (1) comprises an upper part (2) which is removably connected to a lower part (3).

[0033] FIG. 2 shows that the top part (2) of the device includes two openings / slots (4) for receiving two reaction vessels (not shown). The walls (7) of the openings (4) surrounding each reaction vessel are made of aluminum, while the remaining part (6) of the top part (2) of the device is made of polypropylene. A ring (5) holds each reaction vessel within the opening (4) of the top part (2). The bottom part (3) of the device includes a circular opening (8) on the side of the thermistor (13) facing the opening (4), which mates with a ring (not shown) of the top part (2) to fix the top part (2) and thus the opening (4) in their position. The bottom part (3) of the device is made of polypropylene. The bottom part (3) further includes a type-C USB port (9) and electronic circuitry (10) on a PCB. The electronic circuit (10) includes conductive traces (11), a resistor (12) and a PTC thermistor (13). The thermistor (13) is thermally coupled to the opening (4) when the upper part (2) is fixed in place.

[0034] FIG. 3 shows a different view of the lower part (3) of the device which includes the features described above with respect to FIG.

[0035] Figure 4 shows a different view of the upper part (2) of the device, including the features described above with respect to Figure 2. Furthermore, Figure 4 shows the ring (14) of the upper part (2) of the device mating with the disk-shaped opening (8) of the lower part (3) of the device. On the right hand side, the figure further shows a reaction vessel (15) inserted in the opening (4).

[0036] Working Example EXAMPLES

[0037] COVID-19 disease The device shown in Figures 1-4 was used for detection of SARS-CoV-2 RNA from saliva, oropharyngeal swabs, or nasopharyngeal swabs without the need for nucleic acid purification. Detection of viral RNA was achieved in less than 20 minutes after incubation at 65°C using a commercially available set of six LAMP primers, an enzyme mix containing reverse transcriptase and Bst DNA polymerase, and a color indicator (phenol red pH indicator or HNB colorimetric dye). The input voltage applied to the device was a DC voltage of 5 V with a current of 2.4 A. The resistors in the device had a resistance of 4.7 Ω. EXAMPLES

[0038] Malaria disease The device shown in Figures 1-4 was used for detection of Plasmodium falciparum DNA after DNA extraction from saliva or from blood samples. Detection of target DNA was achieved in less than 20 minutes after incubation at 65°C using a set of commercial assay primers, either 2 Iso-IMRS or 4-6 LAMP, Bst DNA polymerase enzyme, and a color indicator (phenol red pH indicator or HNB colorimetric dye). The input voltage applied to the device was a DC voltage of 5 V, with a current of 2.4 A. The resistors in the device had a resistance of 4.7 Ω.

Claims

1. A pocket-sized device (1) for isothermal nucleic acid amplification, comprising: an opening (4) configured to receive a reaction vessel (15) containing a sample of nucleic acid to be amplified; an electronic circuit (10), said circuit including a heating element for heating the contents of said reaction vessel, a power input port (9), and optionally a resistor (12); Equipped with A pocket-sized device (1) for thermal nucleic acid amplification, wherein the heating element is a positive temperature coefficient thermistor (13).

2. 2. The pocket-sized device (1) for isothermal nucleic acid amplification according to claim 1, wherein the power input port (9) is selected from a power socket, a power plug, a power outlet, a Molex port, an IEC connector, a JST connector, a coaxial port, a DIN connector, a USB port, a barrel connector, an Anderson PowerPole, an SAE connector, an XLR connector, an EmPower connector, a Tamiya connector, or an IP44.

3. The pocket-sized device (1) for isothermal nucleic acid amplification according to claim 2, wherein the power input port (9) is a USB port.

4. 4. The pocket-sized device (1) for isothermal nucleic acid amplification according to any one of claims 1 to 3, wherein the current applied through the power input port (9) is direct current.

5. 4. The pocket-sized device (1) for isothermal nucleic acid amplification according to any one of claims 1 to 3, wherein the device (1) is made of a material having a thermal conductivity of 0.01 W / (m·K) to 0.9 W / (m·K).

6. 4. The pocket-sized device (1) for isothermal nucleic acid amplification according to any one of claims 1 to 3, wherein the walls (7) of the opening (4) are made of a material having a thermal conductivity of at least 1 W / (m·K).

7. 7. The pocket-sized device (1) for isothermal nucleic acid amplification according to claim 6, wherein the walls (7) of the opening (4) are made of a material selected from metal, non-metal, highly filled epoxy, silicon, aluminum oxide, aluminum nitride, or graphene.

8. Pocket-sized device (1) for isothermal nucleic acid amplification according to any one of claims 1 to 3, wherein the device further comprises means (5) for holding the reaction vessel (15).

9. The pocket-sized device (1) for isothermal nucleic acid amplification according to any one of claims 1 to 3, wherein said isothermal nucleic acid amplification is selected from LAMP, Iso-IMRS, RPA, or RCA.

10. The pocket-sized device (1) for isothermal nucleic acid amplification according to any one of claims 1 to 3, wherein the electronic circuit (10) does not comprise any additional electrical components.

11. Pocket-sized device (1) for isothermal nucleic acid amplification according to any one of claims 1 to 3, wherein the device (1) does not comprise any additional electronic circuitry.

12. Pocket-sized device (1) for isothermal nucleic acid amplification according to any one of claims 1 to 3, wherein the reaction vessel (15) is a tube, preferably a PCR tube.

13. 4. The pocket-sized device (1) for isothermal nucleic acid amplification according to claim 1, wherein the device (1) comprises, at the end of the opening (4) facing the thermistor (13), a ring (14) which mates with a circular opening (8) on the side of the thermistor (13) facing the opening (4) to fix the opening (4) in its fixed position.

14. 4. A pocket-sized device (1) for isothermal nucleic acid amplification according to any one of claims 1 to 3, wherein the part (2) of the device comprising the opening (4) is removably connected to the part (3) of the device comprising the thermistor (13).

15. Pocket-sized device (1) for isothermal nucleic acid amplification according to any one of claims 1 to 3, wherein said device (1) comprises two openings (4).

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