Devices for rapid amplifying nucleic acid and method of using the same

Abstract

Disclosed herein are a device, an apparatus, and a method for a rapid polymerase chain reaction (PCR) in a liquid sample having a volume of 50-500 µL. The device comprises a reaction vessel, a needle, a receptacle, and a driver, where the driver is configured to drive a liquid sample from the reaction vessel, through the needle and into the receptacle. The apparatus is configured to rapidly heat and rapidly cool the device comprising a bracket member configured to fix the device to the apparatus, a heater block, a motor, and an actuator, where the actuator is configured to actuate the driver to drive a liquid sample from the reaction vessel, through the needle, and into the receptacle.

Description

DEVICES FOR RAPID AMPLIFYING NUCLEIC ACID AND METHOD OF USING THE SAMETECHNICAL FIELD

[0001] This application relates to devices, apparatuses, and methods for rapidly amplifying 50-500 pL DNA by polymerase chain reaction.SEQUENCE LISTING

[0002] This application contains a ST.26 compliant Sequence Listing, which is submitted herewith in XML format via Patent Center, and is hereby incorporated by reference in its entirety. The XML copy, created on August 27, 2024, is named 2024- 08-27 sequence listing 076651 .8035.WQ01 .xml and is 4,590 bytes in size.BACKGROUND

[0003] Nucleic acid detection is an important part of in vitro diagnosis, which relies on polymerase chain reaction (PCR) to perform exponential amplification, making it possible to detect even a single molecule of nucleic acid. Typically, polymerase chain reactions require DNA double strand denaturation at 95QC and annealing and extension of primers and templates at 60sC. The denaturation usually takes about 1 -2s, and the extension of primer takes about 6-10s for 100-150bp of the amplicon length using a “slow” polymerase, and only about 1 -2s when using fast polymerase. The required rounds of amplification are about 40-50, so the total time could be 4-1 Omin without considering other factors. However, the duration required by the existing general-purpose nucleic acid amplifiers for the 40-cycle amplification of the reaction volume of 20-50pl is 30-45min, and most of the time is for temperature heating and cooling between denaturation and annealing / extension. To increase the rate of changing temperature, microfluidic systems are generally adopted to improve the area of the heat transmission between the liquid and the vessel through the channel of less than 1 mm thickness and width. This allows the reaction to be completed within 5-15min, however it confines the volume of reaction liquid to 1 -1 OpL.

[0004] The sensitivity of polymerase amplification systems is mainly limited by the sample volume in the reaction system. Too small reaction volume can lead to loss of sensitivity. This loss often needs to be overcome by adding enrichment steps in thepre-treatment, prolonging the overall analysis time. In addition, although the microfluidic system saves the amount of reagents, its own manufacturing cost is often difficult to bear for practical applications.

[0005] Therefore, there is a need for a rapid PGR amplification system of a large sample volume (for example, greater than 50pL) and rapid temperature variation.BRIEF DESCRIPTION OF THE DRAWINGS

[0006] FIG. 1 shows a side view of an example of a first embodiment of the device of the present technology.

[0007] FIG. 2 shows a front right top perspective view of an example of a first embodiment of the apparatus of the present technology.

[0008] FIG. 3 shows an inset of a front right top perspective view of an example of a first embodiment of the apparatus of the present technology.

[0009] FIG. 4 shows a front view of an example of a first embodiment of the apparatus of the present technology with additional detection device and light source.

[0010] FIG. 5 shows a front right perspective view of an example of a second embodiment of the present technology.

[0011] FIG. 6 shows a front right top perspective view of an example of a second embodiment example of the apparatus of the present technology.

[0012] FIG. 7 shows an example of an amplification plot derived from a commercial method. The X-axis is PGR cycle number, and the Y-axis is fluorescent signal,

[0013] FIG. 8 shows an example of an amplification plot derived from the present technology. The X-axis is PGR cycle number, and the Y-axis is normalized fluorescent signal,

[0014] FIG. 9 shows an example of a temperature plot derived from the present technology. X axis is data point, each data point is roughly 100 millisecond. Y axis is the temperature of the outside surface of the reaction vessel measured by an IR sensor, which cycled from 50-72QC.DETAILED DESCRIPTION

[0015] The present technology includes methods for rapidly amplifying DNA in a sample having a volume of 50pL or more. The present technology also includes a device and an apparatus that can be used to rapidly amplify DNA in a sample having a volume of 50pL or more according to methods of the present technology.

[0016] The word "about" when immediately preceding a numerical value means a range of plus or minus 10 % of that value, e.g., "about 50" means 45 to 55, "about 10" means "9 to 11", etc.A Device for Amplifying DNA

[0017] The present technology includes a device for amplifying DNA by polymerase chain reaction. The device is configured to rapidly decrease the temperature of a relatively large sample (50-500 pL), thereby allowing rapid amplification of DNA in the sample. As such, the device comprises a reaction vessel having a volume capacity of 200pL to 500pL. The device also comprises a needle made of a thermal conducting material configured to rapidly dissipate heat from the sample.

[0018] In some embodiments, the device may be a consumable or disposable device. The device comprises: a reaction vessel made of an inert and thermal conducting material having a volume capacity of 200pL to 500pL; a needle made of an inert and thermal conducting material having a first needle end and a second needle end, wherein the first end is inserted in the reaction vessel and the needle has an outer diameter of 1 mm or less; a receptacle having a first receptacle end and a second receptacle end, wherein the first receptacle end is connected to the second needle end; a driver, wherein the driver is: connected to the second receptacle end of the receptacle, configured to drive a liquid sample from the reaction vessel, through the needle, and into the receptacle, and configured to drive the liquid sample from the receptacle, through the needle, and into the reaction vessel; and wherein the liquid sample is rapidly cooled at a rate of 5-30°C per second when the driver drives the liquid sample from the reaction vessel, through the needle, and into the receptacle.

[0019] In one embodiment, the device is illustrated in FIG. 1 . The reaction vessel 1 is configured to hold a volume of the sample. In some embodiments, the reaction vessel 1 has a volume of at least about 50pL, 10OpL, 150pL, 200pL, or 250pL. In someembodiments, the reaction vessel 1 has a volume capacity of about 200pL to 500pL. The reaction vessel 1 may include a 200pL PCR tube inside the reaction vessel. In some embodiments, the full load of the reaction vessel 1 is 500pL. In some embodiments, the reaction vessel 1 is configured to hold a volume of the sample and a volume of gas or air. The gas or air in the volume may be about 10pL to 200pL, 20pL to 150pL, 30pL to 100pL, 40pL to 80pL or about 50pL.

[0020] The reaction vessel 1 is configured to transfer heat to the sample. As such, the reaction vessel 1 comprises a material that can withstand high temperatures (greater than 95QC) and fast temperature changes. The reaction vessel 1 may also comprise a thermal conducting material that can transfer heat to the sample. The reaction vessel 1 is made of an inert material, such that the reaction vessel 1 does not chemically interfere with DNA amplification. The reaction vessel 1 may comprise a metal or plastic material.

[0021] The needle 2 is configured to hold a volume of the sample. The needle 2 is fluidly connected to the reaction vessel 1 and the receptacle 3. The needle 2 has a first needle end 31 and a second needle end 32. In some embodiments, the needle 2 is fluidly connected to the reaction vessel 1 at the first needle end 31. The needle 2 may be fluidly connected to the receptacle 3 at the second needle end 32.

[0022] The needle 2 is fluidly connect with the reaction vessel 1 such that the sample may be transferred from the reaction vessel 1 to the receptacle 3. In some embodiments, the reaction vessel 1 has a perforation in the reaction vessel top 33. The needle 2 may be inserted through the perforation such that the needle 2 is inserted into the sample in the reaction vessel 1 . In some embodiments, the needle 2 is inserted through the perforation such that the first needle end 31 is in close proximity to the bottom side of the inside of the reaction vessel 1. In some embodiments, as shown in FIG. 5, for example, the needle 2 is fluidly connected to the reaction vessel 1 at the reaction vessel bottom 34. In some embodiments, the needle 2 is attached to the reaction vessel 1 by a temperature resistant adhesive. For example, the temperature resistant adhesive may be a high-temperature epoxy resin.

[0023] The needle 2 preferably is made of a material that can quickly dissipate heat. As such, the needle 2 may comprise a thermal conducting material (e.g., a metal). The needle 2 is thin and has a narrow diameter. In some embodiments, the needle 2may have an outer diameter of about 1 ,5mm, 1 ,4mm, 1 ,3mm, 1 ,2mm, 1 .1 mm, 1 mm, 0.9mm, 0.8mm, 0.7mm, 0.6mm, or 0.5mm or less. In some embodiments, the needle 2 may have an outer diameter of about 1 mm or less. In some embodiments, the needle 2 may have an outer diameter of about 0.5mm-1 .5mm, 0.6mm-1.4mm, 0.7-1 ,3mm, 0.8-1 ,2mm, or 0.9-1 mm. As used herein, the “outer diameter” is the diameter of a cross section of the needle 2, as measured from the outside of the wall of the needle 2 on one side of the needle 2 to the opposite side of the needle 2.

[0024] In some embodiments, the needle 2 may have an inner diameter of about 0.7mm, 0.6mm, 0.5mm, 0.4mm, 0.3mm, 0.2mm, or 0.1 mm or less. In some embodiments, the needle 2 may have an inner diameter of about 0.7mm or less. In some embodiments, the needle 2 may have an inner diameter of about 0.1 mm-0.7mm, 0.2mm-0.6mm, or 0.3-0.5mm. As used herein, the “inner diameter” is the diameter of a cross section of the needle 2, as measured from the inside of the wall of the needle 2 on one side of the needle 2 to the inside of the wall of the opposite side of the needle 2

[0025] In some embodiments, the needle 2 may have a length of at least about 50mm, 60mm, 70mm, 80mm, 90mm, 100mm, or 110mm. In some embodiments, the needle 2 may have a length of about 50-1 10mm, 60-100mm, or 70-90mm. In some embodiments, the needle 2 may have a length of at least about 80mm. As used herein, the “length” of the needle 2 refers to the distance from the needle first end 31 to the needle second end 32. In some embodiments, the needle 2 is configured to hold 10%- 15%, 15%-20%, 20%-25%, 25%-30%, 30%-40%, or 40%-50% of the sample volume. In some embodiments, the needle 2 is configured to hold approximately 20% of the sample volume.

[0026] The receptacle 3 is configured to receive a sample from the needle. The receptacle 3 comprises an inert material that does not interfere with DNA amplification. The receptacle 3 may be configured to dissipate heat. As such, the receptacle 3, may comprise a thermal conducting material. In some embodiments, the receptacle 3 comprises a metal material.

[0027] The receptacle 3 may be configured to hold a fluid volume of at least 50pL. In some embodiments, the receptacle 3 has a volume of at least about 50pL, 100pL, 150pL, 200pL, or 250pL. In some embodiments, the receptacle 3 has a volumecapacity of about 50pL to 1 OOOpL, 1 OOpL to 800pL, 150pL to 600pL, 200pL to 500pL, or 250pL to 400pL. In some embodiments, the receptacle 3 has a volume capacity of about 200pL to 500pL.

[0028] The receptacle 3 may be configured to allow for the detection of a characteristic of the sample. For example, the receptacle 3 may be configured to allow detection of a fluorescent probe using an LED light. As such, the receptacle 3 may comprise a transparent material, such as glass or plastic. The receptacle 3 may comprise any material suitable for detecting a characteristic of a sample in the receptacle 3 using any technique known to one of ordinary skill in the art.

[0029] The receptacle 3 has a first receptacle end 35 and a second receptacle end 36. The receptacle 3 may be configured to fluidly attach at the first receptacle end 35 to the needle 2 at a second needle end 32. The receptacle 3 may be configured to attach a driver 4 at the second receptacle end 36.

[0030] The driver 4 is configured to drive the sample from the reaction vessel 1 , through the needle 2, and into the receptacle 3. The driver 4 may also be configured to drive the sample from the receptacle 3, through the needle 2, and into the reaction vessel 1. The driver may be any element known to one of ordinary skill in the art to drive fluids through a fluid container. In some embodiments, the sample is driven sample from the reaction vessel 1 , through the needle 2, and into the receptacle 3 by gravity. In some embodiments, the sample is driven from the receptacle 3, through the needle 2, and into the reaction vessel 1 by gravity.

[0031] In some embodiments, the driver 4 may be a piston. The piston may be connected to the receptacle 3 at the second receptacle end 36, where the piston is inserted inside the receptacle 3. As such, the driver 4 and the receptacle 3 may comprise a syringe, for example as shown in FIG. 1 . The piston may drive the sample from the reaction vessel 1 , through the needle 2, and into the receptacle 4 by moving the piston away from the first receptacle end 35. The piston may drive the sample from the receptacle 4, through the needle 2, and into the reaction vessel 1 by moving the piston toward the first receptacle end 35. In some embodiments, the driver 4 and the receptacle 3 comprise a 1 mm syringe. The receptacle 3 may connect to the needle 2 separated by a luer connector.

[0032] The driver 4 may be any mechanism for moving liquids, including a pump. The receptacle 3 may be directly connected to a pump, such that the pump drives the sample from the reaction vessel 1 , through the needle 2, and into the receptacle 4. The pump may also drive the sample from the receptacle 4, through the needle 2, and into the reaction vessel 1. In some embodiments, the pump is fluidly connected to the receptacle 3 by a spacer, such as a tube.

[0033] In some embodiments, as shown in FIG. 5, for example, the device 40 may include a framework 22 that attaches to one or more components of the device 40 to enable interaction between the device 40 and an apparatus 50 of the present technology, for example the apparatus 50 shown in FIG. 6. The framework 22 may also facilitate user engagement with the device 40.An Apparatus for Amplifying DNA

[0034] The present technology includes an apparatus to be used with the device described above for amplifying DNA. The apparatus comprises a heater block having a closed configuration and an open configuration, allowing the heater block to rapidly increase the temperature of the reaction vessel of the device without overheating the reaction vessel. In some embodiments, the apparatus has one or more cooling units configured to rapidly reduce the temperature of the device.

[0035] The apparatus comprises: a bracket member releasably attached to a receptacle of the device of claim 7; a heater block having a closed configuration or an open configuration, wherein the heater block is in a closed configuration when the heater block is in contact with the reaction vessel to heat the reaction vessel, and in an open configuration when the heater block is not in contact with the reaction vessel, wherein the heater block is configured to rapidly increase the temperature of the reaction vessel of the device by a rate of 2-6°C per second without overheating the reaction vessel; a motor configured to alternate the heater block between the open configuration and the closed configuration; and an actuator configured to actuate the driver to drive a liquid sample from the reaction vessel, through the needle, and into the receptacle, or to drive the sample from the receptacle, through the needle, and into the reaction vessel.

[0036] In one embodiment, the apparatus is illustrated in FIG. 2. The bracket member 7 is configured to fix the device 40 to the apparatus 50. As such, the device40 may releasably attach to the bracket member 7. In some embodiments, the bracket member 7 may attach to the device 40 at the receptacle 3 of the device 40. The bracket member 7 may be configured to hold the reaction vessel 1 , the needle 2, and the receptacle 3 of the device 40 stationary, while the driver 4 drives the sample from the reaction vessel 1 , through the needle 2 and into the receptacle 3 or from the receptacle 3, through the needle 2, and into the reaction vessel 1.

[0037] The bracket member 7 may also be configured to attach to one or more accessory devices. For example, as shown in FIG. 4, bracket member 7 may be configured to attach to a light source 17, which can be configured to illuminate a probe in the receptacle 3. The bracket member 7 may also be configured to attach to a detection device 18, such as a camera or fluorescence reading device configured to detect, for example, a probe in the receptacle 3. The detection device 18 may be a camera, photomultiplier, or photodiode, for example. The light source 17 may be an illuminator, laser, or LED, for example.

[0038] In some embodiments, the light source 17 is an LED with a peak wavelength of 480nm. The LED may have a power of 1 W. In some embodiments, the light source may be equipped with an excitation filter. The light source may have a central wavelength of 470nm and a bandwidth of 30nm to illuminate the receptacle 3 and excite the fluorescent probes in the sample, for example a fluorescein (FAM) fluorescent dye. In some embodiments, the light source 17 is configured to excite FAM, VIC, ROX and / or Cy5 fluorescent molecules.

[0039] In some embodiments, the detection device 18 is a camera. The camera may be equipped with a receiving light filter. In some embodiments, the detection device 18 is configured to image a 6mm diameter receptacle 3 of a syringe. The detection device 18 may be equipped to obtain a signal of a hydrolyzed FAM fluorescent probe. The central wavelength of the light filter may be 525nm. The bandwidth of the detection device may be 30nm. For example, the camera may be a global shutter camera OV9281 with a shooting parameter of ISO800, and a shutter speed of 12ms. When the driver 4 pulls the sample into the receptacle 3 of the device 40, the camera may take photos. The detection device 18 may be configured to select a specific area to convert it into gray value for recording. The change of gray value reflects the inventory and quantity of the templates in the polymerase chain reactionsystem. In some embodiments, the detection device 18 is configured to excite FAM, VIC, ROX and / or Cy5 fluorescent molecules.

[0040] The heater block 9 transfers heat to the reaction vessel 1 of the device 40. The heater block 9 may comprise a thermal conducting material, such as copper. The heater block 9 may be heated by a heating element. For example, the heating element may be a 50W positive temperature coefficient (PTC) heating rod.

[0041] The heater block 9 has a closed configuration and an open configuration. When the heater block 9 is in a closed configuration, the heater block 9 is in contact with the reaction vessel 1 to heat the reaction vessel 1. In some embodiments, the heater block 9 is in an open configuration when the heater block is not in contact with the reaction vessel. In some embodiments, for example in FIG. 3, the heater block 9 comprises a first heater block and a second heater block, and in the closed configuration, the first heater block substantially contacts the second heater block with the reaction vessel 1 in between. In the open configuration, the first heater block may become separated from the heater block, such that there is space between the reaction vessel 1 and both the first heater block and the second heater block. In some embodiments, the heater block 9 comprises one single heater block, and in its open configuration, the heater block 9 is lowered away from the reaction vessel 1.

[0042] The apparatus 50 has a motor 11 configured to alternate the heater block9 between its open configuration and its closed configuration (see FIG. 2). The motor may be a through-shaft motor, for example.

[0043] The apparatus 50 includes a temperature sensor 10. The temperature sensor 10 may be an infrared sensor. In some embodiments, the temperature sensor10 may be configured to measure or read the temperature of the outside of the reaction vessel 1 (i.e. the outside surface). In some embodiments, the temperature sensor 10 may be configured to measure the temperature of the heater block 9. The heating element may be configured to adjust the temperature of the heater block 9 based on the temperature detected by the temperature sensor 10. For example, if the temperature sensor 10 detects a temperature that is lower than a desired temperature, the heating element may increase the heater block 9 to the desired temperature. In some embodiments, the temperature sensor is a PT1000 temperature sensor. In some embodiments, the temperature sensor 10 is configured to measure the temperature ofthe outside of the reaction vessel 1 , therefore in some embodiments a calibration may be used to correlate the temperature of the outside of the reaction vessel 1 with the internal temperature.

[0044] The apparatus 50 includes an actuator 6 configured to actuate the driver 4 to drive a liquid sample from the reaction vessel 1 , through the needle 2, and into the receptacle 3, or to drive the sample from the receptacle 3, through the needle 2, and into the reaction vessel 1 . In some embodiments, for example in FIG. 2, the driver4 is a piston and the actuator 6 is configured to move the piston relative to the receptacle 3 of the device 40. The actuator 6 may include a piston connector configured to releasably attach to the piston. The actuator 6 may also include a motor5 configured to move the piston. In some embodiments, the driver 4 is a pump and the actuator 6 is configured to activate a pump to drive the sample from the reaction vessel 1 , through the needle 2, and into the receptacle 3, or to drive the sample from the receptacle 3, through the needle 2, and into the reaction vessel 1 .

[0045] In some embodiments, the apparatus 50 includes one or more cooling units. The cooling unit may be a turbofan, magnetic cooler, or peltier cooler, for example.

[0046] For example, the apparatus 50 may include a first cooling unit 12 configured to cool the needle 2 of the device 40. The first cooling unit 12 may be a fan oriented such that the outlet of the fan is positioned near the needle 2. The first cooling unit 12 may include a condenser to focus air flow on the needle 2.

[0047] The apparatus 50 may also include a second cooling unit 8 configured to cool the reaction vessel 1 . The second cooling unit 8 may be a fan oriented such that the outlet of the fan is positioned near the reaction vessel 1. The second cooling unit 12 may include a condenser to focus air flow on the reaction vessel 1. The second cooling unit 8 may be configured to cool the reaction vessel 1 when the heater block 9 is in its open configuration.Methods for rapidly performing PCR on a large volume sample

[0048] The present technology includes a method of amplifying DNA in a sample by polymerase chain reaction (PCR). The method rapidly increases and rapidly decreases a large volume (50-500 pL) of a sample, thereby allowing DNA in the sample to be rapidly amplified.

[0049] The method comprises amplifying DNA in a sample by polymerase chain reaction (PCR), comprising: (a) heating a sample comprising DNA having a volume of 50-500 pL in a reaction vessel for 15 seconds or less to increase a temperature of the sample to 90-98°C; (b) denaturing the DNA to single-strand DNA; (c) cooling the sample for 5 seconds or less to decrease the temperature of the sample to 55-65°C; (d) annealing primers to the single-stranded DNA and extending the new strand of DNA from the primers to synthesize a new strands of DNA; and (e) repeating steps (a)-(d).

[0050] In some embodiments, the sample has a volume of about 50-500pL, or 100-250pL. The sample comprises DNA. The sample may comprise additional biological components, such as protein, nucleic acids, lipids, and carbohydrates. The sample may contain DNA and / or RNA.

[0051] In some embodiments, the method may be used in combination with other molecular biology techniques. The method may be used to amplify DNA that was obtained from RNA by reverse transcriptase. The method may be used to quantitatively measure DNA or RNA using real-time PCR or qPCR. The method may be used for any in vitro or clinical technique, including diagnostic techniques.

[0052] The sample may further comprise a buffer salt, deoxynucleoside triphosphates (dNTPs), two or more primers, and a DNA polymerase. The DNA polymerase may be a natural or synthetic polymerase. The DNA polymerase may be a “fast” DNA polymerase, configured to elongate DNA at a rate of about 6,000 nt per minute.

[0053] The method may include heating the sample comprising DNA in the reaction vessel 1. The DNA may be heated in the reaction vessel 1 to increase the temperature of the sample to about 90-98°C.

[0054] The method may include heating the sample from room temperature or from about 60°C to a temperature of about 90-98°C in about 20, 15, 12, or 10 seconds or less. In some embodiments, the sample may be heated to 90-98°C in about 15 seconds. In some embodiments, the sample is heated to a target temperature by setting the heating block 9 to an overtemperature setting. As used herein, an “overtemperature” setting refers to a temperature that is greater than the target temperature of the sample. As such, the heater block 9 may be set to a temperatureof about 100-150°C, 1 10-140°C, or 120-130°C to rapidly increase the temperature of the sample. In some embodiments, the heater block 9 is set to a temperature of about 1 10-140°C. In some embodiments, the temperature of the sample during heating is increased by a rate of about 2-6°C, 3-5°C, or 4°C per second. In some embodiments, the temperature of the sample is increased at a rate of about 3-5°C per second.

[0055] The method includes denaturing the DNA to single-strand DNA. The DNA may be denatured within 1 to 2 seconds after the temperature of the sample reaches about 90-98°C.

[0056] The method also includes cooling the temperature of the sample. In some embodiments, the temperature of the sample is decreased at a rate of about 5-30°C, 10-25°C or 15-20°C per second. In some embodiments, the temperature is decreased at a rate of more than 15°C per second. In some embodiments, the method may use the present device described above. When the temperature of the sample reaches 90- 98°C in the reaction vessel, the driver 4 then drives the sample from the reaction vessel 1 into the needle 2. In some embodiments, when the temperature of the sample reaches 90-98°C, the heater block 9 alternates to the open configuration. As such, removing the heat source and / or moving the sample through the needle may cause the temperature to drop rapidly.

[0057] In some embodiments, the temperature is cooled to about 55-65°C. In some embodiments, the sample decreased to 55-65°C in less than 10, 9, 8, 7, 6, 5, or 4 seconds. In some embodiments, the temperature decreases to 55-65°C in 5 seconds or less.

[0058] The method includes annealing primers to single-stranded DNA to synthesize new strands of DNA. The sample may contain primers which will anneal to the single stranded DNA. In some embodiments, the annealing step occurs when the temperature reaches 55-65°C.

[0059] The method further includes extending the new DNA strands from the primers. In some embodiments, DNA polymerase in the sample will cause dNTPs from the sample to elongate from the primers according to the DNA template, according to conditions known to one of ordinary skill in the art.

[0060] In some embodiments, the method further includes quantifying the amount of DNA product during or after PCR amplification. In some embodiments,DNA-binding probes will be detected when the sample is in the receptacle 3. For example, probes may be detected and quantified to predict the quantity of DNA in real time, similar to techniques like real-time or quantitative PCR.

[0061] In some embodiments, the DNA amplification technique of the present technology is repeated, for example, repeated at least 20, 25, 30, 35, or 40 times. In some embodiments, the DNA amplification steps of the present technology may be repeated at least 40 times.

[0062] From the foregoing, it will be appreciated that specific embodiments of the invention have been described herein for purposes of illustration, but that various modifications may be made without deviating from the scope of the invention. Accordingly, the invention is not limited except as by the appended claims.A first embodiment of the device and apparatus

[0063] In some embodiments, the device and apparatus cooperate to heat and cool the reaction system of large volume.

[0064] For heating, the reaction vessel 1 of the device 40 can hold at least more than 50pl of liquid and up to 500pl of liquid. The reaction vessel 1 is made of a material that does not interfere with polymerase chain reaction. Since fluorescent reading is not required, aluminum or stainless steel can be used in addition to the commonly used plastics polypropylene or polyethylene to accelerate the temperature of the liquid through greater thermal conductivity. The rapid heating of the device 40 is obtained in part by setting the temperature over the target temperature and rapid detachment from the reaction vessel. For example, if the target liquid temperature is 95 degrees Celsius and the temperature of the heater block s in contact with the liquid vessel is set at 95 degrees Celsius, the rise of the liquid temperature shows an asymptotic line, and the closer it gets to 95 degrees Celsius, the slower it will be, resulting in a long temperature rise period.

[0065] In the present technology, the heater block 9 is set at a temperature of 1 10-140 degrees Celsius, far exceeding the denaturation temperature of 95 degrees Celsius required by the polymerase chain reaction, so that the heating curve of the liquid reaching the denaturation temperature of 95 degrees Celsius is a straight line. In addition, the device 40 is capable of rapid disconnection of the heater block 9 from the reaction vessel, either horizontally or vertically. When the liquid reaches the settemperature, the temperature of the liquid will still rise rapidly due to the overtemperature setting of the heater block. The rising of liquid temperature cannot be quickly stopped by using conventional semiconductor refrigeration reversal. Therefore, a moving mechanism is used to provide this function, while the cooling system of the device is used to reduce the risk of loss of polymerase activity in the reaction system due to liquid temperature overshoot. With the present technology, 250pl_ liquid can rise from 55 to 95 degrees Celsius in 9-1 Os, providing a temperature increase of 4 degrees Celsius per second for the bulk liquid.

[0066] For heat dissipation, the device 4 uses a needle 2 with a large length-to- diameter ratio to increase the area for heat dissipation from the liquid to the environment. At the same time, the needle 2 is made of metal which is not light transmittable and does not interfere with polymerase chain reaction, to increase the speed of heat conduction. The quick detachment of the heat block (alternating the heat block into its open configuration) along with the cooling unit prevents inactivation of polymerase caused by the overshoot of liquid temperature due to the overtemperature setting of the heater block. The needle helps to reduce the liquid temperature from 95 degrees Celsius to below 60 degrees Celsius after passing through the needle twice as well as providing convection from the cooling unit. With the cooperation of the device and apparatus drive module, the movement of the liquid can be completed within 2s, so that the temperature of the liquid of large volume can be reduced by more than 15 degrees Celsius per second.

[0067] For the driving part, the device can drive the liquid to the needle, reaction vessel and receptacle by integrating the piston, the needle and the reaction vessel into an airtight component by means of binding or welding. In air-tight embodiments, the reaction vessel may have a 30-80pl_ space to allow the piston to drive the liquid movement. The device is equipped with a motor fitted with consumable piston to drive the piston movement, and achieve liquid cooling with the movement of heater block, so that the whole cycle time is less than 15s.

[0068] The reagents consist of buffer salts, dNTP, enzymes, primers, probes (which may not be necessary for lateral flow readout), internal control (“IC”) and materials stored in reaction vessels of the device as a frozen-dried ball. During application, appropriately treated samples are injected and the balls are dissolved to perform the polymerase chain reaction. The primers are designed to create anamplicon of 200bp or less to maximize the speed of amplification, and with fast polymerase enzymes that travel at 6,000nt per minute, its amplification can be completed in 1 -2s.

[0069] A fluorescent reading device is set at the device receptacle to read the signal of the device and consumable, which cooperate with multiple LED lights of different wavelengths to read the fluorescence signals generated in the 5-3 circumscribed polymerase chain reaction through avalanche photodiodes (APD), photomultiplier tubes (PMT) or camera; A destructive device is installed below the reaction vessel to introduce the amplification solution into the lateral flow nucleic acid hybridization readout chamber after amplification; Add DNA hybridization chip in the cooling tube to observe the signal after amplification.

[0070] The present technology is described in detail in the following specific embodiments. However, these embodiments are not limited to the present invention, and any transformation in structure, method, or function by ordinary technical personnel in the art according to these embodiments shall be included in the protection scope of the present invention.

[0071] The light source 17 is an LED with a peak wavelength of 480nm, a power of 1 W, an excitation filter at the front end, a central wavelength of 470nm and a bandwidth of 30nm to illuminate the driving part of the consumables and excite the FAM fluorescent molecular in the reaction liquid.

[0072] The detection device 18 is a camera with a receiving light filter located behind the bracket 7 of the apparatus 50 to image a 6mm diameter syringe for obtaining the signal of FAM fluorescent probe hydrolyzed. The central wavelength of the light filter is 525nm, the bandwidth is 30nm. The camera is the global shutter camera OV9281 with a shooting parameter of IS0800, and a shutter speed of 12ms. When the device drive unit pulls the reaction liquid into the drive unit of the device (i.e. the syringe), the camera takes photos, and then selects a specific area to convert it into gray value for recording. The change of gray value reflects the inventory and quantity of the templates in the polymerase chain reaction system.A second embodiment of the device and apparatus

[0073] FIG 5 shows another embodiment of the device. The reaction vessel 1 may have a reaction vessel top 33 with a cover that can be opened by the user to adda sample, for example 250pL of the sample. The tube body (i.e. the tapered portion) of the reaction vessel 20 is where the frozen ball containing buffer salt, dNTP, enzyme, primer and probe is stored. When the user adds sample through the reaction vessel top 33, the frozen ball will dissolve immediately. The needle 2 is a metal tube configured to cool the sample and is connected to the reaction vessel 1 and the receptacle 3 of the liquid drive module. The device may include a framework 22 for all components of the device that enables user operation and interaction between devices. The driver 4 may be a piston that drives of the sample between the reaction vessel 1 , the needle 2, and the receptacle 3.

[0074] FIG. 6 shows another embodiment of the apparatus fitted with the device in FIG. 5. The motor 5 of the actuator 6 in the apparatus 50 enables the driver 4 of the device 40 to move up and down. The light source 17 and the detection device 18 (for example LED light and camera) provide excitation light for FAM, VIC, ROX and Cy5 fluorescent molecules, and contain optical switch optical path inside. The cooling unit 12 of the device is able to cool the heat block 9 and / or the device 40. The heater block 9 can be moved vertically to detach from the device 40.

[0075] The following examples further illustrate the present invention. These examples are intended merely to be illustrative of the present invention and are not to be construed as being limiting.EXAMPLESExample 1 . Method for Amplifying DNA

[0076] The present example compares a faster PCR amplification of DNA in a larger volume sample (250pL) in accordance with the technology (FIG. 8), to amplification by a traditional method with a smaller volume sample (20 pL) (FIG. 7).

[0077] In accordance with the methods described above and using the device described above, a polymerase chain reaction was performed to amplify and detect a monkeypox virus (MPXV) gene in a 250pL sample. The components of the sample for PCR amplification are shown in Table 1 and the conditions were as follows:

[0078] The overtemperature of the heating device was set to 120QC. An IR sensor was set to measure the temperature of the outside surface of the device, and the maximum threshold was set to 72QC (corresponding to the internal temperature of 90- 95QC). In other words, although the heater was set to 120QC, heating would stop whenthe internal temperature of the vessel reached 95QC. Then the sample was allowed to cool by pulling the sample through the needle of the device into the receptacle. The liquid extension time was 2s. Then the cycle repeated after the sample was pushed back through the needle into the vessel. The primers and probes used to amplify and detect MPXV by real-time PCR are shown in Table 2. The temperature for activating hot start polymerase’s activity was 90QC.Table 1 : the solution components of a polymerase chain reaction.Table 2: The forward and reverse primers (F3 and R3), probe (P3) and template sequence (MPXV) of polymerase chain reaction.

[0079] Three samples containing three different concentrations of a linear plasmid with monkeypox fragment as a template (as shown in Table 2) were amplified using a standard volume size (20pL) with a traditional PCR system (the ThermofisherQ5 real-time fluorescent quantitative amplification instrument); the results are shown in FIG. 7. Another three samples containing the same DNA template were also amplified at the same three different concentrations with a volume of 250 pL using the present technology; the results are shown in FIG. 8. The y-axis of FIG. 8 is the normalized relative fluorescence signal and the x-axis is the PCR cycle number. Comparing FIG. 7 and FIG. 8, both techniques were able to amplify the sample in a dose-dependent manner. These results show that the present technology is effective at amplifying DNA in a larger volume sample and in a shorter time, comparing with the traditional method.

[0080] Using the traditional method, amplification of 45 cycles took around 45 minutes. By contrast, the present technology completed 45 cycles of amplification in less than 14 minutes, demonstrating that the present technology amplified DNA to the same degree in a shorter period of time compared to traditional methods.

[0081] The temperature of the outside of the reaction vessel, detected by an I Ft sensor is shown in FIG. 9. The x-axis of FIG. 9 represents datapoints acquired by the system, and each datapoint was collected about every 100ms. Y axis is the temperature of the outside of the reaction vessel, which cycled from 50-72QC. As shown in FIG. 9, the temperature of the outside of a reaction vessel can be rapidly heated to 72°G (corresponding to an internal sample temperature of 90-95QC) in 10 seconds, then rapidly cooled to 50°C (an internal sample temperature about 60°C) in 2 seconds, and then held at 50°C for a 2 second annealing / extension time. Therefore, one PCR cycle could be completed in about 14 seconds. These results demonstrate that the present technology is effective at rapidly increasing the temperature of the sample, and rapidly decreasing the temperature of the sample.Enumerated Embodiments

[0082] Clause 1 . A method of amplifying DNA in a sample by polymerase chain reaction (PCR), comprising: (a) heating a sample comprising DNA having a volume of 50-500 pL in a reaction vessel for 15 seconds or less to increase a temperature of the sample to 90-98°C; (b) denaturing the DNA to single-strand DNA; (c) cooling the sample for 5 seconds or less to decrease the temperature of the sample to 55-65°C; (d) annealing primers to the single-stranded DNA to synthesize a new strands of DNA; and (e) extending the new strand of DNA from the primers.

[0083] Clause 2. The method of clause 1 , where in step (a), the temperature of the sample is increased at a rate 3-5°C per second.

[0084] Clause 3. The method of clause 1 , where in step (c), the temperature of the sample is decreased at a rate of 15-20°C per second.

[0085] Clause 4. The method of clause 1 , after step (d), further comprising a step (e): real-time quantifying an amount of DNA product after PCR amplification.

[0086] Clause 5. The method of clause 1 , wherein the sample has a volume of 100-500 pL.

[0087] Clause 6. The method of clause 1 , wherein the sample further comprises a buffer salt, deoxynucleoside triphosphates (dNTPs), two or more primers, and a DNA polymerase.

[0088] Clause 7. The method of clause 1 , wherein steps (a) - (e) are repeated at least 40 times.

[0089] Clause 8. A device comprising: a reaction vessel having a volume capacity of 200pL to 500pL; a needle having a first needle end and a second needle end, wherein the first end is inserted in the reaction vessel and the needle has an outer diameter of 1 mm or less; a receptacle having a first receptacle end and a second receptacle end, wherein the first receptacle end is connected to the second needle end; and a driver, wherein the driver is: connected to the second receptacle end of the receptacle, configured to drive a liquid sample from the reaction vessel, through the needle, and into the receptacle, and configured to drive the liquid sample from the receptacle, through the needle, and into the reaction vessel.

[0090] Clause 9. The device of clause 8, wherein the reaction vessel is made of an inert material.

[0091] Clause 10. The device of clause 8, wherein the reaction vessel is made of a thermal conducting material.

[0092] Clause 11 . The device of clause 8, wherein the reaction vessel is made of metal.

[0093] Clause 12. The device of clause 8, wherein the needle is at least 80mm in length.

[0094] Clause 13. The device of clause 8, wherein the needle has an inner diameter of 0.7 mm or less.

[0095] Clause 14. The device of clause 8, wherein the reaction vessel has a perforation in a top side of the reaction vessel, and wherein the needle is inserted through the perforation.

[0096] Clause 15. The device of clause 8, wherein the needle is in inserted into the sample in the reaction vessel.

[0097] Clause 16. The device of clause 8, wherein the needle is attached to the reaction vessel by a temperature resistant adhesive.

[0098] Clause 17. The device of clause 16, wherein the temperature resistant adhesive is a high-temperature epoxy resin.

[0099] Clause 18. The device of clause 8, wherein the receptacle and the driver together comprise a syringe.

[0100] Clause 19. The device of clause 18, wherein the syringe is a 1 mm syringe.

[0101] Clause 20. The device of clause 8, wherein the driver is a pump.

[0102] Clause 21 . The device of clause 8, wherein the receptacle is made of an inert material.

[0103] Clause 22. The device of clause 8, wherein the receptacle is made of a thermal conducting material.

[0104] Clause 23. The device of clause 8, wherein the receptacle is made of metal.

[0105] Clause 24. The device of clause 8, wherein the receptacle is made of a transparent material.

[0106] Clause 25. The device of clause 8, wherein the device is disposable.

[0107] Clause 26. An apparatus comprising: a bracket member releasably attached to a receptacle of a device, wherein the device comprises a reaction vessel, a needle, a receptacle, and a driver; a heater block having a closed configuration or an open configuration, wherein the heater block is in a closed configuration when the heater block is in contact with the reaction vessel to heat the reaction vessel, and in an open configuration when the heater block is not in contact with the reaction vessel;a motor configured to alternate the heater block between the open configuration and the closed configuration; and an actuator configured to actuate the driver to drive a liquid sample from the reaction vessel, through the needle, and into the receptacle, or to drive the sample from the receptacle, through the needle, and into the reaction vessel.

[0108] Clause 27. The apparatus of clause 26, wherein the heater block is made of copper.

[0109] Clause 28. The apparatus of clause 26, wherein the motor is a through- shaft motor.

[0110] Clause 29. The apparatus of clause 26, wherein the driver is a piston and the actuator comprises a driving motor and a piston connector, wherein the piston connector is releasably attached to the piston, and the driving motor moves the piston connector with respect to the device.

[0111] Clause 30. The apparatus of clause 26, further comprising a first cooling unit configured to cool the needle.

[0112] Clause 31 . The apparatus of clauses 26 or 30 further comprising a second cooling unit configured to cool the reaction vessel.

[0113] Clause 32. The apparatus of clause 30 or 31 , wherein the first cooling unit and / or the second cooling unit is a turbofan.

[0114] Clause 33. The apparatus of clause 32, wherein the second cooling unit is configured to cool the reaction vessel when the heater block is in its open configuration.

[0115] Clause 34. The apparatus of clause 26, further comprising a heating element configured to provide a heat source for the heater block.

[0116] Clause 35. The apparatus of clause 34, wherein the heating element is a 50W positive temperature coefficient (PTC) heating rod.

[0117] Clause 36. The apparatus of clause 26, further comprising an infrared sensor to read a temperature of the outside of the reaction vessel.

[0118] Clause 37. The apparatus of clause 26, further comprising a temperature sensor for detecting the temperature of the heater block.

[0119] Clause 38. The apparatus of clause 37, wherein the temperature sensor is a PT1000 temperature sensor.

[0120] Clause 39. The apparatus of clause 26, wherein the bracket comprising a fluorescent reading device.

[0121] Clause 40. The apparatus of clause 30 or 31 , wherein the first cooling unit and / or the second cooling unit is a magnet cooler.

[0122] Clause 41 . The apparatus of clause 30 or 31 , wherein the first cooling unit and / or the second cooling unit is a peltier cooler.

Claims

CLAIMSI / We claim:1 . A method of amplifying DNA in a sample by polymerase chain reaction (PCR), comprising:(a) heating a sample comprising DNAs having a volume of 50-500 pL in a reaction vessel for 15 seconds or less to increase a temperature of the sample to 90-98°C;(b) denaturing the DNAs to single-strand DNAs;(c) cooling the sample for 5 seconds or less to decrease the temperature of the sample to 55-65°C;(d) annealing primers to the single-stranded DNAs and extending the primers to synthesize new strands of DNA; and(e) repeating steps (a)-(d).

2. The method of claim 1 , where in step (a), the temperature of the sample is increased at a rate 3-5°C per second, and / or in step (c), the temperature of the sample is decreased at a rate of 15-20°C per second.

3. The method of claim 1 , after step (d), further comprising a step (e): realtime quantifying an amount of DNA product after PCR.

4. The method of claim 1 , wherein the sample has a volume of 100-500 pL.

5. The method of claim 1 , wherein the sample further comprises a buffer salt, deoxynucleoside triphosphates (dNTPs), two or more primers, and a DNA polymerase.

6. The method of claim 1 , wherein step (e) is repeated at least 40 times.

7. A device comprising: a reaction vessel made of an inert and thermal conducting material having a volume capacity of 200pL to 500pL; a needle made of an inert and thermal conducting material having a first needle end and a second needle end, wherein the first end is inserted in the reaction vessel and the needle has an outer diameter of 1 mm or less; a receptacle having a first receptacle end and a second receptacle end, wherein the first receptacle end is connected to the second needle end; a driver, wherein the driver is: connected to the second receptacle end of the receptacle, configured to drive a liquid sample from the reaction vessel, through the needle, and into the receptacle, and configured to drive the liquid sample from the receptacle, through the needle, and into the reaction vessel; and wherein the liquid sample is rapidly cooled at a rate of 5-30°C per second when the driver drives the liquid sample from the reaction vessel, through the needle, and into the receptacle.

8. The device of claim 7, wherein the reaction vessel and / or the receptacle is made of metal.

9. The device of claim 7, wherein the needle is at least 80mm in length and / or an inner diameter of 0.7 mm or less.

10. The device of claim 7, wherein the reaction vessel has a perforation in a top side of the reaction vessel, and wherein the needle is inserted through the perforation.1 1 . The device of claim 7, wherein the needle is in inserted into the sample in the reaction vessel and / or attached to the reaction vessel by a temperature resistant adhesive.

12. The device of claim 7, wherein the receptacle and the driver together comprise a syringe or wherein the driver is a pump.

13. The device of claim 7, wherein the receptacle is made of a transparent material.

14. An apparatus comprising: a bracket member releasably attached to a receptacle of the device of claim 7; a heater block having a closed configuration or an open configuration, wherein the heater block is in a closed configuration when the heater block is in contact with the reaction vessel to heat the reaction vessel, and in an open configuration when the heater block is not in contact with the reaction vessel, wherein the heater block is configured to rapidly increase the temperature of the reaction vessel of the device by a rate of 2-6°C per second without overheating the reaction vessel; a motor configured to alternate the heater block between the open configuration and the closed configuration; and an actuator configured to actuate the driver to drive a liquid sample from the reaction vessel, through the needle, and into the receptacle, or to drive the sample from the receptacle, through the needle, and into the reaction vessel.

15. The apparatus of claim 14, wherein the driver is a piston and the actuator comprises a driving motor and a piston connector, wherein the piston connector is releasably attached to the piston, and the driving motor moves the piston connector with respect to the device.

16. The apparatus of claim 14, further comprising a first cooling unit configured to cool the needle and / or a second cooling unit configured to cool the reaction vessel.

17. The apparatus of claim 16, wherein the second cooling unit is configured to cool the reaction vessel when the heater block is in its open configuration.

18. The apparatus of claim 14, further comprising a heating element configured to provide a heat source for the heater block.

19. The apparatus of claim 14 further comprising an infrared sensor to read a temperature of the reaction vessel or a temperature sensor for detecting the temperature of the heater block20. The apparatus of claim 14, wherein the bracket comprises a fluorescent reading device.

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