WO2017213587A1 - Déplacement de blocs de chaleur pour l'amplification d'acides nucléiques - Google Patents

Déplacement de blocs de chaleur pour l'amplification d'acides nucléiques Download PDF

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Publication number
WO2017213587A1
WO2017213587A1 PCT/SG2017/050286 SG2017050286W WO2017213587A1 WO 2017213587 A1 WO2017213587 A1 WO 2017213587A1 SG 2017050286 W SG2017050286 W SG 2017050286W WO 2017213587 A1 WO2017213587 A1 WO 2017213587A1
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WO
WIPO (PCT)
Prior art keywords
reactor
heating
heating blocks
blocks
heating block
Prior art date
Application number
PCT/SG2017/050286
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English (en)
Inventor
Haiqing Gong
Wen YAN
Zeng XUDONG
Original Assignee
Star Array Pte Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from SG10201700260XA external-priority patent/SG10201700260XA/en
Application filed by Star Array Pte Ltd filed Critical Star Array Pte Ltd
Publication of WO2017213587A1 publication Critical patent/WO2017213587A1/fr
Priority to US16/215,626 priority Critical patent/US20190111435A1/en

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    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L7/00—Heating or cooling apparatus; Heat insulating devices
    • B01L7/52—Heating or cooling apparatus; Heat insulating devices with provision for submitting samples to a predetermined sequence of different temperatures, e.g. for treating nucleic acid samples
    • B01L7/525—Heating or cooling apparatus; Heat insulating devices with provision for submitting samples to a predetermined sequence of different temperatures, e.g. for treating nucleic acid samples with physical movement of samples between temperature zones
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/508—Containers for the purpose of retaining a material to be analysed, e.g. test tubes rigid containers not provided for above
    • B01L3/5082—Test tubes per se
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L9/00—Supporting devices; Holding devices
    • B01L9/06—Test-tube stands; Test-tube holders
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N35/00—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor
    • G01N2035/00346—Heating or cooling arrangements
    • G01N2035/00356—Holding samples at elevated temperature (incubation)
    • G01N2035/00366—Several different temperatures used
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N35/00—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor
    • G01N2035/00346—Heating or cooling arrangements
    • G01N2035/00356—Holding samples at elevated temperature (incubation)
    • G01N2035/00376—Conductive heating, e.g. heated plates
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N35/00—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor
    • G01N35/02—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor using a plurality of sample containers moved by a conveyor system past one or more treatment or analysis stations
    • G01N35/04—Details of the conveyor system
    • G01N2035/0401—Sample carriers, cuvettes or reaction vessels
    • G01N2035/0403—Sample carriers with closing or sealing means
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N35/00—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N35/00—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor
    • G01N35/02—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor using a plurality of sample containers moved by a conveyor system past one or more treatment or analysis stations
    • G01N35/026—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor using a plurality of sample containers moved by a conveyor system past one or more treatment or analysis stations having blocks or racks of reaction cells or cuvettes

Definitions

  • the present invention relates to the field of amplification of nucleic acids for analyses.
  • Amplification of nucleic acids such as for polymerase chain reaction (PCR), isothermal amplification, and primer extension, is increasingly important to genetic engineering, molecular biology, food safety, environmental monitoring, medicine for analyses and diagnosis.
  • PCR polymerase chain reaction
  • a large number of biological researchers use DNA amplification techniques such as PCR in their work on nucleic acid analyses, due to its high sensitivity and specificity.
  • the DNA amplification such as PCR is conducted in a PCR module where the nucleic acid reaction material undergoes thermal processing such as thermal cycling for DNA amplification.
  • the optical detection may be carried during and/or after the thermal cycling.
  • researchers have been constantly striving to increase the speed of thermal cycling.
  • the time duration of a PCR is typically in the order of an hour, primarily due to a time- consuming PCR thermal cycling process that requires the heating device to be cyclically regulated to attain target temperatures for heating and cooling reactors containing the sample for DNA denaturation, annealing and extension.
  • each device can be set to a specified temperature as required for the thermal cycling.
  • the heating devices commonly used are water baths maintained at specified temperatures. The reactors can then be transported between these devices for the thermal cycling.
  • using the liquid baths have several disadvantages like loss of liquid due to evaporation that needs to be topped up as required, leakages and the kind.
  • the liquid having gone through one or more rounds of thermal cycling may also progressively change its volume and properties thereby affecting the time and temperature calibration of the apparatus, particularly when automated.
  • a faster speed of the PCR may be achieved when the thermal cycling apparatus moves a biochip reactor between two heating blocks whose temperatures are set at the target temperatures as required for nucleic acid amplification reactions.
  • This apparatus works for a PCR biochip only in which the chip has a flat bottom surface to be in contact with the flat heat block.
  • the biochip is far less cost effective and user- friendly than the PCR tubes and wellplates.
  • the conventional PCR tubes and wellplates are also advantageous in their ability to interface with standard liquid handling robots and multi-channel pipettes, allow easy loading and aspiring of liquid in the wells, either manually and robotically.
  • the present invention provides a thermal cycling apparatus with higher efficiency of the thermal processing of reactors such as the conventional PCR tubes, PCR wellplates and biochip.
  • the invention allows an improved architecture of the apparatus with easier maintenance and higher precision for imaging.
  • the term “comprising” and “comprise” and grammatical variants thereof, are intended to represent “open” or “inclusive” language such that they include recited elements but also permit inclusion of additional, unrecited elements.
  • the terminologies 'first heating block', 'second heating block' 'sixth heating block' do not constitute the corresponding number of blocks in a sequence but merely are names for ease of identification with respect to the purpose they serve. These baths may not necessarily represent separate physical entities as some of them may be shareable.
  • the term 'thermal processing' includes: a) thermal cycling, and optionally includes: b) thermal process steps before and/or after thermal cycling.
  • the term 'thermal profile' refers to the temperature-time variation of the reactor(s) during a) alone or during a) with b).
  • Apparatus for thermal processing reaction material containing nucleic acid is provided, the reaction material being contained in reactor(s) and the reactor(s) being in any form such as tube(s) or wellplate(s) or chip(s) or cartridge(s).
  • the apparatus comprises a reactor holder for statically holding the reactor(s); at least two heating blocks each being maintainable at a specified temperature; and transport means for positioning the at least two heating blocks to make a contact with the reactor(s) one at a time for specified duration, the positioning being conductable once or over a plurality of times for thermal processing the reactor(s) between a plurality of user specifiable temperatures.
  • faster speed of the thermal processing is achieved with the multiple heating blocks each set at a specified temperature.
  • the heat blocks advantageously eliminate the disadvantages of using the conventional liquid baths that suffer from leakages, spillages and loss of liquid due to evaporation hence requiring frequent top-up.
  • the liquid vapors may also contaminate the surrounding regions of the apparatus.
  • the liquid having gone through one or more rounds of thermal cycling may also progressively change its properties, thereby affecting the time and temperature calibration of the apparatus.
  • the apparatus with the moving heating blocks enables the reactor(s) to remain static during the thermal processing, therefore also enables the optical module for optical detection of the reaction material in the reactor(s) to remain static during the process. This advantageously avoids potential misalignment that could be caused by any relative movement between the optical module and the reactor(s).
  • the transport means provides linear displacement for the heating blocks, such that the selected heating block may be positioned under the reactor(s), thereafter moved upwards to make contact with the reactor(s) for specified duration and thereafter moved downwards to break the contact and proceed with linear displacement of another heating block.
  • the transport means further provides rotational movements for moving the heating blocks in a circular path, the rotational movement being along a common axis for the at least two heating blocks.
  • the movement of the heating blocks in the circular path minimizes the footprint especially when there are more than two heating blocks.
  • the circular path may be along a vertical plane for a smaller footprint as compared with an embodiment when the circular path is along a horizontal plane.
  • the contact between the reactor(s) with at least one of the heating blocks is made when the reactor(s) gets inserted into cavities in the at least one of the heating blocks, the shape of the cavities being substantially conformal with at least the lower portions of the reactor(s).
  • the heating blocks comprise thermally conductive elastomeric material such as silicone. Since silicone can be deformed, a tight fit of the reactor(s) in the cavities may be obtained with the cavities being slightly smaller than the reactor(s) when made of glass or metal.
  • the heating block comprises porous metal wherein the voids are filled with a thermally conductive liquid. This feature allows a tight fit between the cavities and the reactor(s) by using cavities which are slightly undersized than the glass or metallic reactor(s), such that slight deformation of the cavities occur causing the thermally conductive liquid to fill up any gap in between the cavities and the reactor(s).
  • each of the heating blocks comprises: a first portion that is coupled to the transport means; and a second portion that can be removably coupled to the first portion, the second portion accommodating the cavities.
  • This feature helps the user to easily replace the second portion to suit the available reactor pitch and design of the reactors in the form of tubes or well plates. This feature allows the first portion that is coupled to the transport means to remain undisturbed in the interest of maintaining precision of the positioning the cavities to make the contacts with the reactor(s).
  • each heating block comprises at least one injector means such that in operation, the injector means provides a pressure on the reactor(s) to release the contact, before the reactor(s) separates out of the heating block.
  • the injector means may access the reactor(s) via a through hole in the heating block which provides an effective way to release the contact in a compact arrangement for the injector means.
  • At least one of the heating blocks may be a solid metallic block.
  • the metallic block is easier to fabricate.
  • at least one of the heating blocks is a hollow metallic block that can accommodate heating medium such as liquid or mica. This is advantageous if the liquid or mica makes the block lighter than when the block is a solid metallic block. Lighter blocks are easier to handle by the transport means and facilitate lighter weight of the apparatus. Lighter blocks also allow lesser power consumption by the transport means.
  • the apparatus further comprises fluorescence imaging means that allows light to access the nucleic acid when the reactor(s) is/are in contact with any of the heating block or is/are in air outside the heating block as desired by the user.
  • At least a portion of at least one of the heating blocks is transparent to light for fluorescent imaging of the reaction material while the reactor(s) is/are in the contact with the at least one of the heating blocks.
  • the heating block may be at least partially made of a transparent material that is in contact with a transparent electrical conductive layer for allowing resistive heating.
  • the transparent material may be glass.
  • the transparent electrical conductive material may be ITO (Indium Tin Oxide). The transparency is useful for fluorescent imaging of the reaction material.
  • the apparatus may comprise a temperature sensor means or a time sensor means to determine the specified duration.
  • the temperature sensor means may be located to sense the realtime temperature in a sample reactor containing the reaction material. This closely simulates the reactors undergoing the thermal processing.
  • the apparatus may further comprise a calibration means for user calibrating the time sensor means. This feature is helpful particularly (though not exclusively) when the target temperature is lower than the user specifiable temperature or when the target temperature is higher than the user specifiable temperature in at least one of the heating blocks.
  • the at least two heating blocks comprise: a first heating block where the reactor(s) is/are allowed to attain a predetermined high target temperature T HT , wherein the T HT is in the region 85-99 degree Celsius for denaturation of the nucleic acid; and a second heating block where the reactor(s) is/are allowed to attain a predetermined low target temperature T LT , wherein the T LT is in the region 45-75 degree Celsius for annealing of primers or probes onto nucleic acid or for primer extension for thermal cycling the reactor(s) to attain polymerase chain reaction (PCR) amplification or primer extension.
  • PCR polymerase chain reaction
  • the apparatus further comprises a third heating block where the reactor(s) is/are allowed to attain a predetermined medium target temperature T MT , wherein the T MT is for annealing of primers or probes onto nucleic acid.
  • the apparatus may further comprise a fourth heating block where the reactor(s) is/are allowed to attain a predetermined medium target temperature T MT , wherein the T MT is for extension of primers on nucleic acid.
  • the apparatus may further comprise a fifth heating block where the reactor(s) is/are allowed to attain a temperature T AP to allow an additional process for the reactor(s) before thermal cycling, the additional process being one from the group consisting: reverse transcription - polymerase chain reaction (RT-PCR), hot start process and isothermal amplification reaction.
  • the apparatus may further comprise a sixth heating block that can be progressively heated while conducting melt curve analysis after thermal cycling.
  • the fifth and the sixth blocks allow the thermal cycling to be integrated with the previous and the following steps respectively while advantageously using the inventive concept.
  • At least one of the heating block is further capable of providing temperatures with programmable ramp up or ramp down characteristics for providing user flexibility on the thermal profiles.
  • at least one of the heating block is further capable of providing plurality of the user specifiable temperatures for use before or after the thermal processing such as for reverse transcription -polymerase chain reaction (RT- PCR), isothermal amplification reaction, DNA melting analysis and the kind.
  • the specified duration may be dependent on a target temperature to be achieved by the reactor(s) when in the contact, irrespective of any offset in the temperatures for the blocks so that the thermal processing is not adversely affected.
  • the apparatus further comprises a sealing means for sealing the reactor(s) before the thermal processing to prevent the reaction material from vaporizing during thermal processing.
  • the reactors may be positioned over at least one of the heating block, so that after sealing the reactors need not have to be moved over the heating block during the next step of thermal cycling. This saves time and requires no further operating mechanism thereby further saving on the mass and the footprint of the apparatus.
  • the apparatus further comprises a sample preparation means for preparing the reaction material and loading into the reactor(s) before the thermal processing. Integrating the whole process in a single apparatus helps in automating the processing line and also overcomes the requirement of providing controlled ambience and trained personnel for conducting the process of sample preparation. BRIEF DESCRIPTION OF THE DRAWINGS
  • FIGs. 1(a) to (e) presents elevation cross-sectional views of the apparatus with two heating blocks changing their positions by a linear-rotary motion stage during thermal processing, as according to an embodiment of the invention.
  • FIGs. 2(a) to (f) presents elevation cross-sectional views of the apparatus with three heating blocks changing their positions by a linear-rotary motion stage, as according to an embodiment of the invention.
  • FIGs. 3(a) to (f) presents elevation cross-sectional views of the apparatus with six heating blocks changing their positions by a linear-rotary motion stage, as according to an embodiment of the invention.
  • Figs. 4(a) to (d) presents elevation cross-sectional views of the apparatus with two heating blocks changing their positions by a linear-linear motion stage, as according to an embodiment of the invention.
  • Fig. 5 is a planar view of the apparatus with four heating blocks changing their positions relative to the reactor(s) in a horizontal and circular path, as according to an embodiment of the invention.
  • Fig. 6 is an isometric view of an embodiment of the apparatus including the sealing means and the sample preparation means as according to an embodiment of the invention.
  • Figs. 7 (a) to (c) show elevation cross-sectional views of the reactor when in contact with the cavity, according to an embodiment of the invention.
  • Figs. 8 (a) to (c) in elevation cross-sectional views illustrate an embodiment of the invention using injector pins to release the tight fit between the reactors and the cavities.
  • FIGs. 9 (a) and (b) in a cross-sectional view illustrates an embodiment of the invention wherein the heating block is split into two portions.
  • Figs. 1(a) to (e) illustrate the movement of a first heating block 10 and a second heating block 12 which are maintained at two different temperatures during the thermal cycling of the reaction material within the reactors 5.
  • the reactors 5 thus need to cycle between the two heating blocks 10, 12 several times as predetermined.
  • the heating blocks 10, 12 change their positions by a rotary motion stage 22 and a linear motion stage 24, as according to an embodiment of the invention.
  • the rotary motion stage 22 enables the heating blocks 10 and 12 to rotate along a vertical plane and about a common axis.
  • the reactors 5 presumably containing the reaction material are capped by the cap strip 2.
  • the reactors 5 are held in a static position by the reactor holder 4.
  • the first heating block 10 is placed in the active position where the reactors 5 make contact with the first heating block 10 by each inserting into the cavities 15 of the first heating block 10.
  • the second heating block 12 is shown in a passive position. After a predetermined time as shown at Fig. 1(b), the linear motion stage 24 linearly displaces the first heating block 10 downwards as shown by the dashed block arrow and the rotary motion stage 22 rotates as shown by the curved arrow such that the second heating block 12 is positioned under the reactors 5. At Fig. 1(c), the linear motion stage 24 linearly displaces the second heating block 12 upwards to make contact with the reactors 5 and remain in this active position for another predetermined time. As shown at Fig.
  • the linear motion stage 24 linearly displaces the second heating block downwards as shown by the dashed block arrow and the rotary motion stage 22 is rotated as shown by the curved arrow such that the first heating block 10 is positioned under the reactors 5.
  • the linear motion stage 24 again linearly displaces the first heating block 10 upwards to the active position to make contact with the reactors 5 as at Fig. 1(a). This sequence may be repeated several times as required under the process of thermal processing.
  • the temperatures of the two heating blocks 10, 12 are maintained at predetermined temperatures as required.
  • Figs. 2(a) to (f) illustrate the movement of the first heating block 10, the second heating block 12 and a third heating block 14 which are maintained at three different temperatures during the thermal cycling of the reaction material within the reactors 5.
  • the operations of the motion stages 22, 24 are similar to that described under Fig. 1, but with three heating blocks 10, 12, 14 instead of two 10, 12.
  • the sequence of positioning the heating blocks 10, 12, 14 in the active position may be altered as required for the process of thermal cycling.
  • the sequence shown in the figures is self-explanatory.
  • Figs. 3(a) to (f) illustrate the movement of six heating blocks 10, 12, 14, 16, 18, 20, each being maintained at a different temperature during the thermal processing of the reaction material within the reactors 5.
  • the operations of the motion stages 22, 24 are similar to that described under Figs. 1, but with six heating blocks 10, 12, 14, 16, 18, 20.
  • the sequence of positioning the heating blocks 10, 12, 14, 16, 18, 20 in the active position may be altered as required for the thermal processing.
  • the sequence shown in the figures is self explanatory.
  • the heating blocks 10 may be maintained at temperatures for additional process before thermal cycling like reverse transcription-polymerase chain reaction (RT-PCR), hot start process and isothermal amplification reaction.
  • the heating blocks 12, 14, 16 may be respectively maintained at temperatures for denaturation, annealing and extension.
  • the heating blocks 18 and 20 may be maintained at temperatures for stabilization and imaging.
  • Figs. 4(a) to (d) illustrate the linear movement of two heating blocks 10, 12, being maintained at two different temperatures during the thermal processing of the reaction material within the reactors 5.
  • both the heating blocks 10 and 12 have a common platform 40.
  • the linear motion stage 24 (not shown) provides linear displacement of the heating blocks 10, 12 via the common platform 40.
  • the first heating block 10 is in the active position.
  • the heating blocks 10, 12 are displaced downwards.
  • the heating blocks 10, 12 move towards the left as shown by the dashed block arrow.
  • the heating blocks 10, 12 are linearly displaced upwards to place the second block 12 in the active position. According to other embodiments, more number of heating blocks may be used.
  • Fig. 5 in a plan view illustrates the movement of four heating blocks 10, 12, 14, 16 in a horizontal plane and about a common axis.
  • Each of the heating blocks 10, 12, 14, 16 is maintainable at a different temperature during the thermal processing of the reaction material within the reactors 5.
  • the operation of the motion stages 22, 24 (not shown) is similar to that described under Figs. 1, but with the four heating blocks 10, 12, 14, 16 and rotating in the circular path in the horizontal plane instead of the vertical plane.
  • the sequence of positioning the heating blocks 10, 12, 14, 16 in the active position may be altered as required for the process of thermal processing.
  • Fig. 6 shows the apparatus for amplification of nucleic acid as integrated along with the sealing means 100, the sample preparation means 200 and the fluorescence imaging means or the electrochemical detection means 400.
  • the reactors are shown to be positioned over at least one of the heating block.
  • the reactors 5 in the tube form as shown could be made up of glass or metal or plastic. While making the contact with the heating blocks 10, 12, 14, 16, 18, 20, the plastic deforms to some extent to fit into the cavities 15. Thus the size of the cavities 15 may be slightly smaller than those of the reactors 5 to ensure a tight fit for a good contact with the cavities 15. However since the glass or metallic ones do not deform, slight oversize of the cavities 15 need to be used thereby forming a gap 34 as shown in Fig. 7 (a). The gap 34 needs to be maintained to avoid collision during the relative movement between the cavity 15 and the reactor 5. This gap 36 may be occupied with air or preferably a thermally conductive lubricant liquid (not shown) such as oil, lubricant chemicals, water and the kind.
  • the heating block 10 may be made of porous metal which contains a large number of voids among the metal phase typically obtained by bonding metal particles together under high pressure and temperature or by a metal infiltration process.
  • the voids can store the thermally conductive liquid that is able to form a liquid layer over the surface of the cavity 15 by means of surface tension or by pressure exerted on the liquid to move to the surface of the cavity.
  • the gap 34 may be filled with the thermally conductive liquid seeping out from the void region of the porous metal of the heating block 10, thereby forming a conductive liquid layer to enhance heat transfer between the reactor 5 and the heating block 10.
  • the heating blocks 10 may be made of a thermally conductive elastomeric material such as silicone or rubber. Since the silicone can be deformed easily under a small stress, in order to ensure a tight fit the cavities 15 can be slightly smaller than the reactors 5.
  • Fig. 8 (a) illustrates an embodiment for a case when the reactors 5 are subjected to a tight fit in the cavities 15 for a good thermal contact.
  • the heating block 10 is linearly displaced downwards for breaking the contact, the tight fit resists the detachment of the reactor 5 from the cavity.
  • through-holes 36 are provided in the heating block 10 with injector pins 38 inserted from the bottom side of the heating block 10.
  • the injector pins 38 provide a small upward force along the bottom side of the reactors 5 to loosen the contact. Thereafter the heating block 10 starts moving downwards as shown by the dashed arrow.
  • both the heating block 10 and the pins 38 move downwards for another heating block to take position and make the contact with the reactors 5.
  • the set of injector pins 38 may be provided with each heating block or may be common for all the heating blocks 10, 12, 14, 16, 18, 20, to insert into the through holes 36 when the heating block is in the active position. Such injector pins 38 may be used also for an embodiment where the heating block 10 is stationary and the reactor 5 in the reactor holder 4 moves upwards for breaking the contact.
  • the injector pins 38 may preferably be provided for each reactor 5. However, having fewer ones may also work. The fewer ones may preferably be distributed over the area of the reactors 5 so as to maintain better uniformity of the upward force provided.
  • the heating block 10 is split into a top portion 10a including the cavities 15 and a bottom portion 10b that is coupled to the rotary and linear motion stages 22, 24 as described earlier.
  • the top portion 10a is designed to accommodate the tubular and well-plate reactors 5 with varying sizes and pitches. Hence, depending on the size, shape and pitch of the reactors 5, suitable top portions 10a can be rigidly fixed over the bottom portion 10b by the user.
  • Fig. 9b shows the top portion 10a wherein the size, shape and pitch of the cavities 15 are different from the top potion 10a of Fig. 9a. In this embodiment, the injector pins 38 are shown though it is not a necessary feature.
  • the shape of the cavities 15 may preferably be substantially conformal with the lower portions of the reactors 5 such that tight and uniform contact of the reactor(s) 5 with the heating blocks 10, 12, 14, 16, 18, 20 may be established for speedy heat exchange. This is desirable both for the quality control of the thermal processing and for reducing the cycle time.
  • the pitch of the cavities 15 in the heating blocks 10, 12, 14, 16, 18, 20 need to match the pitch of the reactors 5 whether as individual reactors 5 held by the reactor holder 4 or in the form of well plates. During the thermal processing, the positioning of the heating blocks 10, 12, 14, 16, 18, 20 under the reactors 5 needs to be executed with the required precision.
  • Temperature sensors are provided with each heating block to monitor and regulate the specified temperature.
  • the temperatures of the different heating blocks 10, 12, 14, 16, 18, 20 may co-relate to the target temperatures for the thermal processing such as for DNA denaturation, annealing and extension.
  • the temperature sensor may be mounted inside each heating block or mounted inside a reactor 5 dedicated for temperature sensing which has a similar heat transfer characteristics as the reactor 5 containing the reaction material. Such a reactor 5 with the temperature sensor is inserted into the cavity of the different heating blocks together with the other reactors 5.
  • the apparatus may be programmed such that the duration of the contact is dependent on a target temperature to be achieved by the reactor(s) when in the contact.
  • the temperature of any of the heating blocks 10, 12, 14, 16, 18, 20 may be set to be higher than the target temperature to speed up the rate of heating of the reactor(s). Similarly the temperature of any of the heating blocks 10, 12, 14, 16, 18, 20 may be set to be lower than the target temperature to speed up the rate of cooling of the reactor(s).
  • the user specifiable temperature in one or more of the heating block(s) 10, 12, 14, 16, 18, 20 may be maintained higher than the target temperature such as the denaturation temperature for the reactor 5.
  • the reactor 5 can remain in the heating block(s) 10, 12, 14, 16, 18, 20 for a specified period of time required for denaturation.
  • the user specifiable temperature may be maintained lower than the target temperature such as the annealing or extension temperature.
  • the reactor 5 can remain in the heating block(s) 10, 12, 14, 16, 18, 20 for another specified period of time as required for annealing and extension for the reactor 5.
  • the preceding embodiments show the reactors 5 in the form of tubes and the heating blocks 10, 12, 14, 16, 18, 20 to be provided with cavities 15 to receive the lower side of the reactors 5 when in the active position.
  • Reactors 5 with other shapes including PCR chips and cartridges may also be used.
  • the PCR chip requires multilayer bonding or glue sealing of plates or films which may frequently cause delamination and leaking during the thermal cycling process.
  • the cartridges have similar problems as well.
  • the conventional PCR tubes and wellplates are molded as a single piece plastic without any bonding required. This provides a more reliable vehicle to contain the biological sample and reagent before, during and after the nucleic acid analyses.
  • the PCR chip has a flat surface to be in contact with the heating blocks 10, 12, 14, 16, 18, 20 and such one-sided heating and cooling could generate a thermal gradient across the PCR chip thereby making the temperature inside inaccurate and the temperature control difficult.
  • the reactors 5 in the form of conventional PCR tube or a tube on a PCR wellplate are inserted into the cavities 15 for heating and cooling, and such cavities 15 surround the tube in all directions making the heating and cooling uniform over the domain of the reaction material in the tube. This helps the temperature field to be more uniform inside the reaction material during the thermal cycling.
  • the materials used to construct the reactors 5 may be plastics, elastomer, glass, metal, ceramic and their combinations, in which the plastics include polypropylene and polycarbonate.
  • the glass reactor 5 can be made in a form of a glass capillary of small diameters such as 0.1mm-3mm OD and 0.02mm-2mm ID, and the metal can be aluminum in form of thin film, thin cavity, and capillary.
  • Reactor materials can be made from non-biological active substances with chemical or biological stability. At least a portion of the reactor 15 is preferred to be transparent.
  • the volume of the at least one reactor 5 may be in the range 1 uL to 500 ⁇ Smaller the volume, faster is the heat transfer, higher is the speed of PCR, smaller are the required heating block sizes and more compact is the apparatus.
  • the reaction material in all the reactors 5 in the reactor holder 4 may not be identical. Simultaneous PCR can be advantageously conducted for different materials if the heating block temperatures are suitable. The heating blocks may be shared between different process steps by altering the temperatures.
  • the embodiments described above may be suitable for one reactor 5 or a plurality reactors 5.
  • the reactor 5 may be in the form of tube(s) as shown or as wellplate(s) or chip(s) or cartridge(s) and the like.
  • the reaction material comprises reaction constituents including at least one enzyme, nucleic acid and/or particle containing at least one nucleic acid, primers for PCR, primers for isothermal amplifications, primers for other nucleic acid amplifications and processing, dNTP, Mg 2+ , fluorescent dyes and probes, control DNA, control RNA, control cells, control micro-organisms, and other reagents required for nucleic acid amplification, processing, and analysis.
  • the particle containing nucleic acid mentioned above comprises at least one cell virus, white blood cell and stromal cell, circulating tumor cell, embryo cell.
  • One application may be to use the apparatus to test different kinds of reaction materials against the same set of primer and probes, such as test more than one sample.
  • different kinds of reaction material containing no target primers and/or probes are each loaded into one reactor 5 in a reactor array, with all the reactors 5 being pre-loaded with the same set or the same sets of PCR primers and/or probes.
  • different kinds of reaction materials pre-mixed with respective PCR target primers and/or probes are each loaded into one reactor 5 in a reactor array, with all the reactors 5 being not pre-loaded with the same set of PCR primers and or probes.
  • the reaction materials can include control genes and/or cells and corresponding fluorescent dyes or probes.
  • the different probes emit light of different wavelengths.
  • Another application of the methods and devices are used to test the same reaction material against different sets of primer and probes.
  • One example of such an application is to test one type of sample for more than one purpose.
  • a single reaction material is added into the reactors 5 each loaded with at least one different set PCR primers and or probes.
  • the reaction material can include control genes and/or cells and corresponding fluorescent dyes or probes.
  • the different probes emit light of different wavelengths.
  • the above reaction material is used in polymerase chain reaction, reverse transcription-PCR, end-point PCR, ligase chain reaction, pre-amplification or target enrichment of nucleic acid sequencing or variations of polymerase chain reaction (PCR), isothermal amplification, linear amplification, library preparations for sequencing, bridge amplification used in sequencing.
  • PCR polymerase chain reaction
  • the variation of the polymerase chain reaction mentioned above comprises reverse transcription-PCR, real-time fluorescent quantitative polymerase chain amplification reaction and real-time fluorescent quantitative reverse transcription polymerase chain amplification reaction, inverse polymerase chain amplification reaction, anchored polymerase chain amplification reaction, asymmetric polymerase chain amplification reaction, multiplex PCR, colour complementation polymerase chain amplification reaction, immune polymerase chain amplification reaction, nested polymerase chain amplification reaction, the target enrichment of pre-amplification or nucleic acid sequencing, ELISA-PCR.

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  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Molecular Biology (AREA)
  • Clinical Laboratory Science (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Apparatus Associated With Microorganisms And Enzymes (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

L'invention concerne un appareil pour le traitement thermique d'un matériau de réaction contenant un acide nucléique, par exemple par PCR. L'appareil comprend un support de réacteur (4) destiné à maintenir statiquement une pluralité de réacteurs (5); au moins deux moyens de chauffage (blocs chauffants 10, 12), chacun pouvant être maintenu à une température spécifiable par l'utilisateur; et des moyens de transport (étage de mouvement rotatif 22) pour positionner les moyens de chauffage (10, 12) afin d'établir un contact avec les réacteurs (5) un par un pendant une durée spécifiée. Le positionnement peut être conduit une ou plusieurs fois pour le traitement thermique des réacteurs entre une pluralité de températures. L'utilisation d'une pluralité de moyens de chauffage permet d'obtenir un traitement thermique plus rapide et plus efficace.
PCT/SG2017/050286 2016-06-10 2017-06-06 Déplacement de blocs de chaleur pour l'amplification d'acides nucléiques WO2017213587A1 (fr)

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US201662348155P 2016-06-10 2016-06-10
US62/348,155 2016-06-10
SG10201700260XA SG10201700260XA (en) 2016-06-10 2017-01-12 Rapid thermal cycling for sample analyses and processing
SG10201700260X 2017-01-12
SG10201702663P 2017-03-31
SG10201702663PA SG10201702663PA (en) 2016-06-10 2017-03-31 Moving heat blocks for amplification of nucleic acids

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110142068A (zh) * 2019-06-12 2019-08-20 杭州华得森生物技术有限公司 一种上皮间质混合型循环肿瘤细胞检测试剂盒及方法
CN111220448A (zh) * 2016-06-10 2020-06-02 星阵私人有限公司 用于样品分析与处理的快速热循环
CN116183596A (zh) * 2023-05-04 2023-05-30 常州先趋医疗科技有限公司 多通道lamp自动化检测系统及其工作方法

Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE19646114A1 (de) * 1996-11-08 1998-05-14 Eppendorf Geraetebau Netheler Laborthermostat mit Temperierblöcken
US20040265884A1 (en) * 2003-06-04 2004-12-30 Jochem Knoche Thermocycler
WO2006138586A2 (fr) * 2005-06-16 2006-12-28 Stratagene California Unites de chauffe et procede de chauffe
US20080057544A1 (en) * 2004-04-16 2008-03-06 Spartan Bioscience Inc. System for rapid nucleic acid amplification and detection
US20100279392A1 (en) * 2007-05-23 2010-11-04 Trust Medical Co., Ltd. Container for Liquid Reaction Mixture, Reaction-Promoting Device Using the Same and Method Therefor
US9061285B2 (en) 2010-04-23 2015-06-23 Nanobiosys Inc. PCR device including two heating blocks
WO2016106717A1 (fr) * 2014-12-31 2016-07-07 Coyote Bioscience Co., Ltd. Appareils et procédés permettant de réaliser des réactions chimiques

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE19646114A1 (de) * 1996-11-08 1998-05-14 Eppendorf Geraetebau Netheler Laborthermostat mit Temperierblöcken
US20040265884A1 (en) * 2003-06-04 2004-12-30 Jochem Knoche Thermocycler
US20080057544A1 (en) * 2004-04-16 2008-03-06 Spartan Bioscience Inc. System for rapid nucleic acid amplification and detection
WO2006138586A2 (fr) * 2005-06-16 2006-12-28 Stratagene California Unites de chauffe et procede de chauffe
US20100279392A1 (en) * 2007-05-23 2010-11-04 Trust Medical Co., Ltd. Container for Liquid Reaction Mixture, Reaction-Promoting Device Using the Same and Method Therefor
US9061285B2 (en) 2010-04-23 2015-06-23 Nanobiosys Inc. PCR device including two heating blocks
WO2016106717A1 (fr) * 2014-12-31 2016-07-07 Coyote Bioscience Co., Ltd. Appareils et procédés permettant de réaliser des réactions chimiques

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111220448A (zh) * 2016-06-10 2020-06-02 星阵私人有限公司 用于样品分析与处理的快速热循环
CN110142068A (zh) * 2019-06-12 2019-08-20 杭州华得森生物技术有限公司 一种上皮间质混合型循环肿瘤细胞检测试剂盒及方法
CN110142068B (zh) * 2019-06-12 2024-02-02 杭州华得森生物技术有限公司 一种上皮间质混合型循环肿瘤细胞检测试剂盒及方法
CN116183596A (zh) * 2023-05-04 2023-05-30 常州先趋医疗科技有限公司 多通道lamp自动化检测系统及其工作方法
CN116183596B (zh) * 2023-05-04 2023-07-21 常州先趋医疗科技有限公司 多通道lamp自动化检测系统及其工作方法

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