WO2017121238A1 - 热循环反应组件及具有其的实时检测装置 - Google Patents

热循环反应组件及具有其的实时检测装置 Download PDF

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Publication number
WO2017121238A1
WO2017121238A1 PCT/CN2016/112367 CN2016112367W WO2017121238A1 WO 2017121238 A1 WO2017121238 A1 WO 2017121238A1 CN 2016112367 W CN2016112367 W CN 2016112367W WO 2017121238 A1 WO2017121238 A1 WO 2017121238A1
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Prior art keywords
assembly
capillary
cylindrical heating
thermal cycle
disposed
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PCT/CN2016/112367
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English (en)
French (fr)
Inventor
邱海维
虞之龙
胡琬璐
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北京酷搏科技有限公司
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Publication of WO2017121238A1 publication Critical patent/WO2017121238A1/zh

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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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M1/00—Apparatus for enzymology or microbiology
    • C12M1/36—Apparatus for enzymology or microbiology including condition or time responsive control, e.g. automatically controlled fermentors
    • C12M1/38—Temperature-responsive control
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • 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
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/08—Geometry, shape and general structure
    • B01L2300/0832—Geometry, shape and general structure cylindrical, tube shaped
    • B01L2300/0838—Capillaries
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/18—Means for temperature control
    • B01L2300/1805—Conductive heating, heat from thermostatted solids is conducted to receptacles, e.g. heating plates, blocks

Definitions

  • PCR Polymerase Chain Reaction
  • QPCR Real-time Quantitative PCR Detecting System
  • the QPCR device includes a thermal cycler for the PCR reaction and a fluorometer for detecting the fluorescence of the reaction product.
  • the thermal cycler consists of a thermal block, a temperature control circuit and a heat sink.
  • the thermal block is used to conduct heat to the reaction tube containing the sample.
  • the fluorometer includes a light source that emits excitation light, an optical component for selecting and conducting a fluorescent signal, and a light sensor that measures the intensity of the fluorescent signal.
  • the fluorescence value of the sample is measured by the light sensor under excitation of the light source, thereby showing the real-time progress of the PCR.
  • the prior art thermal cycler specifically includes an annular heating block 1 and a capillary tube 2 having a hollow portion in the middle, and the inside of the heating block 1 is divided into two layers so that The temperature outside the heating block 1 is different from the temperature inside the heating block 1, and the capillary 2 passes through the hollow portion of the heating block 1 and is wound around the heating block 1, and is wound a plurality of turns to conduct different temperatures to the capillary light, thereby enabling It is subjected to thermal cycle heating.
  • the residue of the former sample in the capillary 2 may contaminate the new sample, resulting in inaccurate reaction. Therefore, the capillary needs to be replaced before the new sample is detected.
  • the capillary 2 when the capillary 2 is replaced, it is necessary to sequentially take out each of the capillaries 2 and re-wrap the new capillaries 2, making it extremely inconvenient to replace the capillaries 2.
  • the present invention provides a thermal cycle reaction assembly and a real-time detection device therewith to solve the problem of inconvenience in replacing the capillary tube in the prior art.
  • a thermal cycle reaction assembly comprising a plurality of cylindrical heating assemblies, a plurality of cylindrical heating assemblies spaced apart, at least two cylindrical heating assemblies having different heating temperatures;
  • the prefabricated assembly, the capillary prefabricated assembly is disposed on a plurality of cylindrical heating assemblies.
  • the capillary prefabrication assembly comprises a first stage capillary tube, the first stage capillary tube is used for winding on the plurality of column heating assemblies; the second stage capillary tube is used for winding on one of the column heating elements, first Segment capillary The second segment of the capillary is in communication with each other, and the first segment of the capillary tube and the second segment of the capillary tube are respectively wound on the plurality of columnar heating assemblies with a plurality of layers, and the adjacent two layers of the capillary tubes are fixed to each other.
  • the cylindrical heating assembly includes a housing, the capillary prefabricated assembly is wound on the outer wall of the housing; the heating portion is disposed inside the housing; the temperature controller is electrically connected to the heating portion, and the temperature controller is used to control the cylindrical heating assembly temperature.
  • the thermal cycle reaction assembly further includes a propulsion mechanism disposed at the inlet end of the capillary prefabrication assembly.
  • the thermal cycle reaction assembly further includes a collection device disposed at the outlet end of the capillary preform assembly.
  • the thermal cycle reaction assembly further includes a mount assembly, and the plurality of post heating assemblies are fixedly disposed on the mount assembly.
  • the fixing base assembly includes a fixing seat having a plurality of spaced apart first fixing holes, one ends of the plurality of cylindrical heating assemblies are disposed in the plurality of first fixing holes one by one;
  • the limiting seat has a plurality of The second fixing holes are spaced apart from each other, and the spacing distance of the second fixing holes is greater than the spacing distance of the first fixing holes, and the other ends of the plurality of cylindrical heating assemblies are disposed in the plurality of second fixing holes one by one.
  • the cylindrical heating assembly includes a first cylindrical heating assembly and a second cylindrical heating assembly, the first cylindrical heating assembly and the second cylindrical heating assembly are spaced apart, the heating temperature of the first cylindrical heating assembly is second The heating temperature of the cylindrical heating assembly is different, and the capillary prefabrication assembly is disposed on the first cylindrical heating assembly and the second cylindrical heating assembly.
  • a real-time detecting device comprising a thermal cycle reaction assembly, the thermal cycle reaction assembly being the thermal cycle reaction assembly provided above, and a light-emitting portion for emitting excitation light to the capillary prefabricated assembly a fluorescence detecting component for detecting fluorescence generated by irradiation of the sample in the capillary prefabricated component through the light emitting portion.
  • the light emitting portion includes a light source for emitting excitation light to the capillary prefabrication assembly; an excitation band pass filter disposed on the optical path of the excitation light, and an excitation band pass filter for adjusting the wavelength of the excitation light.
  • the fluorescence detecting component comprises a fluorescent converging lens for concentrating the fluorescence generated by the sample in the capillary prefabricated component; the detecting sensor is disposed on the optical path of the fluorescent condensed lens condensing; the band pass filter is disposed on the fluorescent concentrating lens and detecting Between the sensors, a bandpass filter is used for wavelength selection, and a detection sensor is used to detect fluorescence after wavelength selection by the bandpass filter.
  • the fluorescence detecting component further comprises a bracket, and the plurality of sets of band pass filters are disposed on the bracket; the motor and the motor are drivingly connected with the bracket, and the motor drives the bracket to rotate to switch the plurality of sets of band pass filters.
  • the capillary prefabricated assembly is disposed on the plurality of cylindrical heating assemblies, and when the capillary prefabricated assembly is replaced, the capillary prefabricated assembly is entirely removed from the cylindrical heating assembly, and the new capillary is removed.
  • Pre The whole assembly of the assembly is arranged on the cylindrical heating assembly to complete the replacement, so that the worker can replace the capillary prefabricated assembly and improve the detection efficiency.
  • FIG. 1 is a schematic structural view of a thermal cycler provided by the prior art
  • FIG. 4 is a schematic structural view of a capillary prefabrication assembly provided in Embodiment 2;
  • Figure 5 shows a cross-sectional view of the cylindrical heating assembly of Figure 2 and the base
  • Figure 6 is a schematic view showing the temperature distribution of the cylindrical heating assembly for heating the capillary prefabricated assembly
  • FIG. 7 is a schematic structural diagram of a real-time detecting apparatus provided in Embodiment 3.
  • FIG. 8 is a schematic structural diagram of a real-time detecting apparatus provided in Embodiment 4.
  • the first embodiment is a thermal cycle reaction assembly including a plurality of cylindrical heating assemblies 10 and a capillary prefabrication assembly 20.
  • a plurality of cylindrical heating assemblies 10 are spaced apart, and the heating temperatures of the cylindrical heating assemblies 10 are different.
  • Capillary preform assembly 20 is disposed on a plurality of cylindrical heating assemblies 10.
  • the number and temperature of the cylindrical heating assembly 10 can be specifically set according to the test. In the PCR reaction, the temperature in the denaturation stage is between 92 ° C and 98 ° C, and in the primer binding and DNA extension stages, the required temperature is Between 50 ° C and 75 ° C.
  • two cylindrical heating assemblies 10 are provided, wherein the temperature of one cylindrical heating assembly 10 is set at 95 ° C, and the temperature of the other cylindrical heating assembly 10 is set at 60 ° C, in order to facilitate the solution. It is stated that the temperature of the large-diameter cylindrical heating assembly 10 is set at 60 ° C, and the temperature of the small-diameter cylindrical heating assembly 10 is set at 95 ° C.
  • the heating assembly 10 denatures the sample and anneals and extends the sample through the large diameter cylindrical heating assembly 10.
  • Figure 6 shows the temperature distribution of the sample in the capillary preform assembly 20 during the actual heating process.
  • the temperature at a is 60 ° C
  • the temperature at b is 60 ° C to 95 ° C
  • the temperature at c is 95 ° C
  • the temperature at d is 95. °C to 60 °C.
  • the capillary prefabrication assembly 20 is disposed on the plurality of cylindrical heating assemblies 10, and the capillary prefabricated assembly 20 can be completely removed from the cylindrical heating assembly 10 when the capillary prefabricated assembly 20 is replaced. Then, the new capillary prefabrication assembly 20 is integrally disposed on the cylindrical heating assembly 10 to complete the replacement, so that the worker can replace the capillary prefabrication assembly 20, thereby improving the detection efficiency.
  • the winding mode facilitates forming the capillary preform assembly 20 into an integral annular structure such that when the capillary preform assembly 20 is repositioned on the cylindrical heating assembly 10, the capillary preform assembly 20 is integrally nested on the cylindrical heating assembly 10. Just go up.
  • the capillary prefabricated assembly 20 can be bonded into a unitary structure by an adhesive, or the outer wall of the capillary can be melted or softened by heating to adhere to each other. Hehe.
  • the capillary prefabricated assembly 20 can be turned into an integral annular structure, which facilitates overall removal or installation, thereby further saving installation time and improving installation efficiency.
  • the cylindrical heating assembly 10 is provided in a cylindrical shape to facilitate the mounting of the capillary prefabricated assembly 20 and is easy to manufacture.
  • the cylindrical heating assembly 10 may be provided as other cylindrical structures such as a prismatic shape and an elliptical cross section as needed.
  • the diameters of the two cylindrical heating assemblies 10 are 10 mm and 20 mm, respectively, and the center distance between the two cylindrical heating assemblies 10 is 40 mm, but the diameters and spacings of the two cylindrical heating assemblies 10 are not limited thereto, and the sizes and intervals thereof.
  • the diameter of the cylindrical heating assembly 10 is typically set between 2 mm and 50 mm.
  • the length of the cylindrical heating assembly 10 is set to be between 20 mm and 200 mm.
  • the capillary preform assembly 20 of FIG. 3 includes a first length of capillary 21 and a second length of capillary 22.
  • the first stage capillary 21 sequentially bypasses the plurality of cylindrical heating assemblies 10 and is wound around the plurality of cylindrical heating assemblies 10, and the second length of the capillary tubes 22 is wound around one of the cylindrical heating assemblies 10.
  • the first-stage capillary tube 21 and the second-stage capillary tube 22 are wound on the plurality of column-shaped heating assemblies 10, respectively, and the adjacent upper and lower layers of the capillary tubes are fixed to each other by the above-described bonding method.
  • the second stage capillary 22 is wound around the small diameter cylindrical heating assembly 10. Since in the heat-activated DNA polymerase, an antibody, an inhibitor or other substance capable of blocking the activity of the polymerase at a normal temperature is usually added. Therefore, before the start of the PCR reaction, the inhibition of the DNA polymerase is usually released by heating, so that the PCR reaction can proceed normally.
  • the second stage capillary 22 is wound 10 times on the small diameter cylindrical heating assembly 10. And the heating temperature is generally at 95 ° C, and the passage time is from 1 to 10 minutes.
  • the inhibition of the DNA polymerase can be released by winding the second length of the capillary 22 on the columnar heating assembly 10 having a higher temperature without separate heating means. Heating the sample facilitates the progress of the PCR reaction and reduces the number of reaction devices required.
  • the material of the capillary prefabrication assembly 20 is polytetrafluoroethylene, but not limited to polytetrafluoroethylene, and may be polyvinylidene fluoride, perfluoroethylene propylene polymer, polypropylene, polycarbonate, polydimethylsiloxane. Alkane, fused silica, silicate glass, borate glass or composites, copolymers thereof, and the like. It is also possible that the inner wall of the capillary made of one of the above materials is covered with another material or more. In the present embodiment, the capillary preform assembly 20 has an outer diameter of 0.6 mm and an inner diameter of 0.3 mm, but is not limited thereto.
  • the outer diameter of the capillary preform assembly 20 can be set between 0.2 mm and 2.0 mm, and the inner diameter of the capillary preform assembly 20 can be set between 0.05 mm and 1.8 mm.
  • a cross-sectional view of a cylindrical heating assembly 10 is provided, wherein the cylindrical heating assembly 10 includes a housing 11, a heating portion 12, and a temperature controller 13.
  • the capillary prefabrication assembly 20 is wound around the outer wall of the housing 11, the heating portion 12 is disposed inside the housing 11, and the temperature controller 13 is electrically connected to the heating portion 12, and the heating of the cylindrical heating assembly 10 can be controlled by the temperature controller 13.
  • the heating portion 12 may be a heating core, and may be composed of a resistance wire, or may be provided with a heating sheet, an electric heating tube, a heating wire, a circuit board, a Peltier unit, or the like.
  • the heating portion 12 may heat the casing 11 by generating heat radiation by electric current, or may heat the casing 11 by means of heat convection, such as heating water or other liquid, gas, or phase change heat conduction. Further, the heat generated by the heating unit 12 is detected and adjusted in real time by the temperature controller 13, so that the heat supplied to the capillary prefabrication unit 20 can be accurately controlled to ensure the normal progress of the PCR reaction.
  • the thermal cycle reaction assembly further includes a propulsion mechanism 30 disposed at the inlet end of the capillary prefabrication assembly 20, and the sample liquid is pushed by the propulsion mechanism 30 to move within the capillary prefabrication assembly 20.
  • the advancement mechanism 30 is disposed at the port of the second stage capillary 22, and the advancement mechanism 30 can be configured as a syringe pump.
  • the propulsion mechanism 30 may be a peristaltic pump, an air pressure pump, or a liquid generated by compressing a gas or an electrolyte body to generate a gas, or a method of using a gas to heat volume expansion to realize a sample liquid in a capillary prefabricated assembly.
  • the pushing flow rate of the propulsion mechanism 30 is about 0.4 uL/s, and after 600 s, it can be filled to the 36th turn of the first stage capillary 21 to complete the PCR reaction.
  • the pushing speed and the advancing time of the reaction sample are not limited to this.
  • the pushing flow rate of the propulsion mechanism 30 can vary from 0.01 uL/s to 10 uL/s, and the advancement time of a single sample to be tested is usually between 300 s and 1200 s. between.
  • the thermal cycle reaction assembly further includes a collection device 40 disposed at the outlet end of the capillary prefabrication assembly 20.
  • the collecting device 40 is disposed at the port of the first stage capillary 21, and the reaction liquid after the reaction is collected by the collecting device 40.
  • the collecting device 40 can be arranged as a flexible balloon, or can be set in other containers of constant or variable volume, such as a syringe, a plastic bottle or the like.
  • the collecting device 40 may be filled with air, a water absorbing material (such as a sponge), a substance reactive with water (such as quick-drying agent such as quicklime, gypsum, cobalt chloride), a DNA adsorbing material (such as porous silicon oxide), or the like.
  • a water absorbing material such as a sponge
  • a substance reactive with water such as quick-drying agent such as quicklime, gypsum, cobalt chloride
  • a DNA adsorbing material such as porous silicon oxide
  • the thermal cycle reaction assembly further includes a fixing base assembly, wherein the plurality of cylindrical heating assemblies 10 are fixedly disposed on the fixing base assembly. To avoid sloshing of the cylindrical heating assembly 10 during the test, the test was carried out.
  • the fixing base assembly includes a fixing base 51 and a limiting seat 52.
  • the fixing base 51 has a plurality of spaced apart first fixing holes, and one ends of the plurality of cylindrical heating assemblies 10 are disposed one by one. In a plurality of first fixing holes.
  • a plurality of second fixing holes are also disposed on the limiting seat 52, and the other ends of the plurality of cylindrical heating assemblies 10 are disposed in the plurality of second fixing holes one by one.
  • the spacing distance of the second fixing holes is greater than the spacing distance of the first fixing holes, so as to increase the column shape
  • one end of the plurality of cylindrical heating assemblies 10 is first fixed on the fixing base 51, and the capillary prefabricated assembly 20 is placed on the plurality of cylindrical heating assemblies 10, and then the limiting seat 52 is placed. Fixedly disposed at the other end of the plurality of cylindrical heating assemblies 10, since the spacing distance of the second fixing holes is greater than the spacing distance of the first fixing holes, the plurality of cylindrical heating assemblies 10 are moved to both sides, thereby being able to open the capillary prefabrication The assembly 20 secures the capillary preform assembly 20 to the plurality of cylindrical heating assemblies 10.
  • the capillary prefabrication assembly 20 may not be filled.
  • another inert liquid that is immiscible with the sample can be selected as the medium carried by the sample to assist in pushing the sample forward in the capillary.
  • silicone oil may be used as a medium for carrying the sample, but it is not limited to the use of silicone oil, and may be a fluoroalkane, a fluoroether, a liquid paraffin or the like.
  • a sample of 1 uL is mixed with 400 uL of silicone oil, and the sample is passed through the carrier medium, thus ensuring the successful completion of the PCR reaction.
  • a third embodiment of the present invention is a real-time detecting device, which includes a thermal cycle reaction component 100, a light emitting portion 60, and a fluorescence detecting component 70.
  • the thermal cycle reaction assembly 100 is the thermal cycle reaction assembly 100 provided by the above embodiment.
  • the light emitting portion 60 is configured to emit excitation light to the capillary prefabrication assembly 20
  • the fluorescence detecting member 70 is configured to detect the sample in the capillary prefabrication assembly 20 via the light emitting portion 60. The fluorescence produced by the irradiation.
  • the samples in each layer of the capillary prefabrication assembly 20 can be simultaneously detected, so that the worker can obtain the detection data.
  • the device has a simple structure and is easy to operate. Compared with the conventional thermal cycler shown in FIG. 1, when detecting the same, it is necessary to arrange the light-emitting portion and a part of the fluorescent detecting member in the hollow portion, so that the hollow portion of the thermal cycler must satisfy the light-emitting portion and the fluorescence detection. The size of the components does not allow the thermal cycler to move toward miniaturization.
  • the light-emitting portion 60 and the fluorescence detecting member 70 need only be disposed on one side of the thermal cycle reaction assembly 100, and the size of the thermal cycle reaction assembly 100 may be set according to the PCR reaction. This simplifies the design of the thermal cycle reaction assembly 100.
  • the light emitting portion 60 includes a light source 61 and an excitation band pass filter 62.
  • the excitation light is emitted from the light source 61 to the capillary prefabrication unit 20, the excitation band pass filter 62 is disposed on the optical path of the excitation light, and the wavelength of the excitation light is adjusted by the excitation band pass filter 62.
  • the light source 61 may be a white light source such as a halogen lamp or a xenon lamp, or may be a monochromatic or narrow wavelength light source such as an LED lamp or a laser.
  • the fluorescence detecting section 70 includes a fluorescence condensing lens 71, a detecting sensor 72, and a band pass filter 73.
  • the fluorescent concentrating lens 71 is used to condense the fluorescence generated by the sample in the capillary prefabrication assembly 20, and the detecting sensor 72 is disposed on the optical path of the fluorescence condensed by the condensing condenser lens 71.
  • the band pass filter 73 is disposed on the fluorescent condensing lens 71 and the detecting sensor 72. Between, the band pass filter 73 is used for filtering, and the detecting sensor 72 is for detecting the fluorescence filtered by the band pass filter 73.
  • the fluorescence concentrating lens 71 may be provided as an aspherical lens or an aspherical lens group, and the detecting sensor 72 may be provided as an area CMOS sensor or an area CCD sensor.
  • the band pass filter 73 may be an absorptive filter or a reflective filter.
  • the fourth embodiment provides a real-time detecting device, which differs from the third embodiment only in that the fluorescent detecting component 70 includes a plurality of sets of band pass filters 73 and a plurality of sets of band pass filters.
  • 73 is switchably disposed between the fluorescence condensing lens 71 and the detecting sensor 72, and filters the fluorescence condensed by the condensing lens 71 by any one of the band pass filters 73.
  • the detecting sensor 72 is configured to detect the filtered by the band pass filter 73. Fluorescence.
  • different bandpass filters 73 can be adjusted to obtain fluorescence of different wavelength bands, and fluorescence of different wavelength bands can be detected.
  • the fluorescence detecting component 70 further includes a bracket 74 and a motor 75, wherein the plurality of sets of band pass filters 73 are disposed on the bracket 74, the motor 75 is drivingly coupled to the bracket 74, and the motor 75 drives the bracket 74 to rotate when needed.
  • the plurality of sets of band pass filters 73 are switched.
  • the reaction time of the existing QPCR can be shortened from 1 to 2 hours to 5 to 20 minutes, which can greatly shorten the reaction time and increase the analysis speed.
  • the device has a simple structure and a small overall size, which is convenient for the staff to use.

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Abstract

一种热循环反应组件(100)及具有其的实时检测装置，其中热循环反应组件(100)包括多个柱形加热组件(10)，多个柱形加热组件(10)间隔设置，至少两个柱形加热组件(10)的加热温度不同；毛细管预制组件(20)，毛细管预制组件(20)设置在多个柱形加热组件(10)上，解决了现有技术中不便于对毛细管进行更换的问题。

Description

热循环反应组件及具有其的实时检测装置 技术领域
本发明涉及聚合酶链式反应技术领域，具体而言，涉及一种热循环反应组件及具有其的实时检测装置。
背景技术
聚合酶链式反应(Polymerase Chain Reaction简称为PCR)是一种用于放大扩增特定DNA片段的分子生物学技术。利用该PCR技术，可以在体外由非常少量的DNA通过热循环生成大量的DNA。实时荧光定量核酸扩增检测***(Real-time Quantitative PCR Detecting System简称为QPCR)是在普通PCR体系中加入荧光化学物质对每次热循环后产物的总量进行测定，其结果可以较快取得且数据较为稳定。QPCR装置包括针对PCR反应的热循环器和用于检测反应产物的荧光的荧光计。热循环器由热块、温控电路和散热器构成。其中，热块用于向包含有样品的反应管传导热量。荧光计包括发射激发光的光源、用于选择和传导荧光信号的光学组件以及测量荧光信号强度的光传感器。在QPCR装置中，每一次热循环结束后，在光源的激发下，通过光传感器对样品的荧光值进行测量，从而显示PCR的实时进展。通过实时检测样品的荧光强度，可以精确的反推出待测样品中的特定DNA序列的初始浓度。
如图1所示，现有技术中的热循环器具体包括环状的加热块1和毛细管2，该加热块1在中部具有中空部分，其加热块1的内部被分割成两层，以使加热块1靠外侧的温度与加热块1靠内侧的温度不相同，毛细管2穿过加热块1中空部分并缠绕在该加热块1上，缠绕多圈以对毛细光传导不同的温度，从而能够使其进行热循环加热。
现有技术中利用该热循环器进行PCR反应时，前一个样品在毛细管2内的残留物会对新样品造成污染，导致其反应不准确。因此，在检测新样品前，需要对毛细管进行更换。现有技术中，在对毛细管2进行更换时，需要将每圈毛细管2依次抽出，并重新缠绕新的毛细管2，使得在对毛细管2进行更换时非常不便。
发明内容
本发明提供一种热循环反应组件及具有其的实时检测装置，以解决现有技术中不便于对毛细管进行更换的问题。
根据本发明的一个方面，提供了一种热循环反应组件，热循环反应组件包括多个柱形加热组件，多个柱形加热组件间隔设置，至少两个柱形加热组件的加热温度不同；毛细管预制组件，毛细管预制组件设置在多个柱形加热组件上。
进一步地，毛细管预制组件包括第一段毛细管，第一段毛细管用于缠绕在多个柱形加热组件；第二段毛细管，第二段毛细管用于缠绕在其中一个柱形加热组件上，第一段毛细管与 第二段毛细管相互连通，第一段毛细管和第二段毛细管在多个柱形加热组件上分别缠绕有多层，相邻的两层毛细管之间相互固定在一起。
进一步地，柱形加热组件包括壳体，毛细管预制组件缠绕在壳体的外壁上；加热部，设置在壳体内部；温度控制器，与加热部电连接，温度控制器用于控制柱形加热组件的温度。
进一步地，热循环反应组件还包括推进机构，设置在毛细管预制组件的进口端。
进一步地，热循环反应组件还包括收集装置，设置在毛细管预制组件的出口端。
进一步地，热循环反应组件还包括固定座组件，多个柱形加热组件固定设置在固定座组件上。
进一步地，固定座组件包括固定座，具有多个间隔设置的第一固定孔，多个柱形加热组件的一端一一对应地设置在多个第一固定孔中；限位座，具有多个间隔设置的第二固定孔，第二固定孔的间隔距离大于第一固定孔的间隔距离，多个柱形加热组件的另一端一一对应地设置在多个第二固定孔中。
进一步地，柱形加热组件包括第一柱形加热组件和第二柱形加热组件，第一柱形加热组件和第二柱形加热组件间隔设置，第一柱形加热组件的加热温度与第二柱形加热组件的加热温度不同，毛细管预制组件设置在第一柱形加热组件和第二柱形加热组件上。
根据本发明的另一方面，提供了一种实时检测装置，实时检测装置包括热循环反应组件，热循环反应组件为上述提供的热循环反应组件；发光部，用于向毛细管预制组件发出激发光；荧光检测部件，用于检测毛细管预制组件内样品经发光部照射所产生的荧光。
进一步地，发光部包括光源，用于向毛细管预制组件发出激发光；激发带通滤波器，设置在激发光的光路上，激发带通滤波器用于调节激发光的波长。
进一步地，荧光检测部件包括荧光会聚透镜，用于会聚毛细管预制组件内样品产生的荧光；检测传感器，设置在荧光会聚透镜会聚的荧光的光路上；带通滤波器，设置在荧光会聚透镜与检测传感器之间，带通滤波器用于进行波长选择，检测传感器用于检测经带通滤波器波长选择后的荧光。
进一步地，荧光检测部件包括荧光会聚透镜，用于会聚毛细管预制组件内样品产生的荧光；检测传感器，设置在荧光会聚透镜会聚的荧光的光路上；多组带通滤波器，可切换地设置在荧光会聚透镜和检测传感器之间，通过任意一个带通滤波器对荧光会聚透镜会聚的荧光进行波长选择，检测传感器用于检测经带通滤波器波长选择后的荧光。
进一步地，荧光检测部件还包括支架，多组带通滤波器设置在支架上；电机，电机与支架驱动连接，电机驱动支架转动以对多组带通滤波器进行切换。
应用本发明的技术方案，将毛细管预制组件设置在多个柱形加热组件上，能够在对毛细管预制组件进行替换时，将毛细管预制组件整体从柱形加热组件上取下，再将新的毛细管预 制组件整体对应设置在柱形加热组件上即可完成更换，如此便于工作人员对毛细管预制组件进行更换，提高了检测效率。
附图说明
构成本申请的一部分的说明书附图用来提供对本发明的进一步理解，本发明的示意性实施例及其说明用于解释本发明，并不构成对本发明的不当限定。在附图中：
图1示出了现有技术提供的热循环器的结构示意图；
图2示出了实施例一提供的热循环反应组件的结构示意图；
图3示出了图2中毛细管预制组件的结构示意图；
图4示出了实施例二提供的毛细管预制组件的结构示意图；
图5示出了图2中柱形加热组件以及底座的剖视图；
图6示出了柱形加热组件对毛细管预制组件加热的温度分布示意图；
图7示出了实施例三提供的实时检测装置的结构示意图；
图8示出了实施例四提供的实时检测装置的结构示意图。
其中，上述附图包括以下附图标记：
1、加热块；2、毛细管；10、柱形加热组件；11、壳体；12、加热部；13、温度控制器；20、毛细管预制组件；21、第一段毛细管；22、第二段毛细管；30、推进机构；40、收集装置；51、固定座；52、限位座；100、热循环反应组件；60、发光部；61、光源；62、激发带通滤波器；70、荧光检测部件；71、荧光会聚透镜；72、检测传感器；73、带通滤波器；74、支架；75、电机。
具体实施方式
需要说明的是，在不冲突的情况下，本申请中的实施例及实施例中的特征可以相互组合。下面将参考附图并结合实施例来详细说明本发明。
如图2至图4所示，实施例一是一种热循环反应组件，该热循环反应组件包括：多个柱形加热组件10以及毛细管预制组件20。其中，多个柱形加热组件10间隔设置，并且这些柱形加热组件10的加热温度不相同。毛细管预制组件20设置在多个柱形加热组件10上。柱形加热组件10的数量以及温度可根据试验的不同而具体设置，在PCR反应中，变性阶段的温度在92℃至98℃之间，而在引物结合与DNA延伸阶段，其所需温度在50℃至75℃之间。因此，在本实施例中，设置两个柱形加热组件10，其中一个柱形加热组件10的温度设置在95℃，另一个柱形加热组件10的温度设置在60℃，为便于对方案进行阐述，规定大直径的柱形加热组件10的温度设置在60℃，小直径的柱形加热组件10的温度设置在95℃。通过小直径柱形 加热组件10对样品进行变性，通过大直径柱形加热组件10对样品进行退火和延伸。图6展示出了在实际加热过程中，毛细管预制组件20内样品的温度分布，a处温度为60℃，b处温度为60℃至95℃，c处温度为95℃，d处温度为95℃至60℃。
应用本发明的技术方案，将毛细管预制组件20设置在多个柱形加热组件10上，能够在对毛细管预制组件20进行替换时，将毛细管预制组件20整体从柱形加热组件10上取下，再将新的毛细管预制组件20整体对应设置在柱形加热组件10上即可完成更换，如此便于工作人员对毛细管预制组件20进行更换，提高了检测效率。并且，该缠绕方式便于将毛细管预制组件20制作成一体的环形结构，使得在重新将毛细管预制组件20设置在柱形加热组件10上时，将毛细管预制组件20整体套设在柱形加热组件10上即可。
具体地，毛细管预制组件20缠绕在两个柱形加热组件10之后，可通过粘合剂将毛细管预制组件20粘合成整体结构，也可通过加热的方式使毛细管外壁熔融或软化，以相互粘合。即可使毛细管预制组件20变成整体的环形结构，方便整体取出或安装，从而进一步节省安装时间，提高安装效率。
在本实施例中，将柱形加热组件10设置为圆柱形能够便于毛细管预制组件20的安装，且制作方便。根据需要，也可将该柱形加热组件10设置为棱柱形、截面为椭圆形的柱体等其它柱形结构。
可选地，两个柱形加热组件10的直径分别为10mm和20mm，两者的中心距为40mm，但两个柱形加热组件10的直径以及间隔大小并不限于此，其尺寸以及间隔大小与不同PCR反应所需的时间相关，其柱形加热组件10的直径一般设置在2mm至50mm之间。并且由于各个PCR反应所需的加热循环的次数不同，毛细管预制组件20在柱形加热组件10上缠绕的圈数也不同。因此，针对性地，将柱形加热组件10的长度设置在20mm至200mm之间。
图3和图4展示了两种不同的毛细管预制组件20的结构，其不同之处在于，图3中的毛细管预制组件20包括第一段毛细管21和第二段毛细管22。其中，第一段毛细管21依次绕过多个柱形加热组件10并缠绕在多个柱形加热组件10上，第二段毛细管22缠绕在其中一个柱形加热组件10上。并且，第一段毛细管21和第二段毛细管22在多个柱形加热组件10上分别缠绕有多层，相邻上下两层的毛细管之间通过上述粘和方法相互固定在一起。
在本实施例中，第二段毛细管22缠绕在小直径的柱形加热组件10上。由于在热激活性DNA聚合酶中，通常加入了针对DNA聚合酶反应位点的抗体、抑制剂或其它能够在常温下阻止聚合酶活性的物质。因此，在PCR反应开始前，通常会通过加热的方式来解除对DNA聚合酶的抑制，使PCR反应可以正常进行。其中，为了获得足够的热启动温度，在本实施例中，将第二段毛细管22在小直径柱形加热组件10上缠绕10圈。并且其加热的温度一般在95℃，通过时间在1至10分钟。通过图3中示出的毛细管预制组件20结构，通过将第二段毛细管22缠绕在温度较高的柱形加热组件10上，即可解除对DNA聚合酶的抑制，无需通过另外的加热装置单独对样品进行加热，如此便于PCR反应的进行，且减少了所需的反应装置的数量。
具体地，毛细管预制组件20的材料为聚四氟乙烯，但不限于聚四氟乙烯，可以是聚偏氟乙烯、全氟乙丙烯聚合物、聚丙烯、聚碳酸酯、聚二甲基硅氧烷、熔融石英、硅酸盐玻璃、硼酸盐玻璃或其复合物、共聚物等。亦可以是由以上一种材料制成的毛细管内壁中覆盖有另一种以上材料制成。在本实施例中，该毛细管预制组件20的外径是0.6mm，内径是0.3mm，但不限于此。根据PCR反应的流速、样品量以及导热速率，毛细管预制组件20的外径可以设置在0.2mm至2.0mm之间，毛细管预制组件20的内径可以设置在0.05mm至1.8mm之间。
如图5所示，提供了柱形加热组件10的剖视图，其中，该柱形加热组件10包括：壳体11、加热部12以及温度控制器13。其中，毛细管预制组件20缠绕在壳体11的外壁上，加热部12设置在壳体11内部，温度控制器13与加热部12电连接，通过温度控制器13能够控制柱形加热组件10的加热温度。可选地，该加热部12可以是加热芯，可由电阻丝构成，也可设置电热片、电热管、电热线、电路板、帕尔贴单元等。加热部12可以通过电流产生热辐射来对壳体11进行加热，也可通过热对流的方式，例如加热水或其他液体、气体、或者相变性导热对壳体11进行加热。并且，通过温度控制器13实时检测并调整加热部12产生的热量，能够准确控制对毛细管预制组件20提供的热量，保证PCR反应的正常进行。
其中，该热循环反应组件还包括推进机构30，推进机构30设置在毛细管预制组件20的进口端，通过推进机构30推动样品液体在毛细管预制组件20内移动。在本实施例中，将推进机构30设置在第二段毛细管22的端口，将推进机构30可设置为注射器泵。但不限于此，推进机构30可以是蠕动泵、空气压力泵、或通过压缩气体、电解液体产生气体的方式对液体产生推动，亦或者是利用空气加热体积膨胀等方式实现样品液体在毛细管预制组件20中的单方向或者双方向运动。在本实施例中，推进机构30的推动流速约为0.4uL/s，600s后可以填充至第一段毛细管21的第36圈，即可完成PCR反应。但反应样品的推动速度和推进时间不限于此。根据毛细管预制组件20的内径大小和对PCR反应的热循环数需求，推进机构30的推动流速可以在0.01uL/s到10uL/s之间变化，单个待测样品的推进时间通常在300s至1200s之间。
在本发明实施例一中，该热循环反应组件还包括收集装置40，收集装置40设置在毛细管预制组件20的出口端。在本实施例中，该收集装置40设置在第一段毛细管21的端口处，通过收集装置40收集反应后的反应液。其中，该收集装置40可设置为弹性的气球，也可以设置为其它体积恒定或可变的容器中，例如注射器、塑料瓶等。收集装置40中可填充空气、吸水材料(如海绵)、与水反应的物质(如生石灰、石膏、氯化钴等干燥剂)、DNA吸附材料(如多孔的硅氧化物)等。
在本发明实施例一中，该热循环反应组件还包括固定座组件，其中，多个柱形加热组件10固定设置在固定座组件上。以避免在试验中柱形加热组件10产生晃动，影响试验进行。
可选地，该固定座组件具体包括固定座51和限位座52，其中，固定座51上具有多个间隔设置的第一固定孔，多个柱形加热组件10的一端一一对应地设置在多个第一固定孔中。限位座52上对应也设置多个第二固定孔，多个柱形加热组件10的另一端一一对应地设置在多个第二固定孔中。具体地，第二固定孔的间隔距离大于第一固定孔的间隔距离，以使柱形加 热组件与两个底座之间存在预紧力，进而能够使多个柱形加热组件10与固定座51、限位座52紧固连接。在安装该热循环反应组件时，先将多个柱形加热组件10的一端固定在固定座51上，再将毛细管预制组件20套在多个柱形加热组件10上，然后将限位座52固定设置在多个柱形加热组件10的另一端，由于第二固定孔的间隔距离大于第一固定孔的间隔距离，多个柱形加热组件10会向两侧移动，进而能够撑开毛细管预制组件20，使毛细管预制组件20紧固在多个柱形加热组件10上。
在通过该热循环反应组件进行PCR反应时，如果样品体积较小，会无法充满毛细管预制组件20。此时可选取另一种与样品不互溶的惰性液体作为样品承载的介质，以辅助推动样品在毛细管中前进。在本实施例中可使用硅油作为样品承载的介质，但不限于使用硅油，亦可以是氟代烷烃，氟代醚，液体石蜡等。本实施例中使用了1uL的样品与400uL的硅油混合，样品与承载介质间隔通过，如此可保证PCR反应顺利完成。
如图7所示，本发明实施例三为一种实时检测装置，该实时检测装置包括：热循环反应组件100、发光部60以及荧光检测部件70。其中，热循环反应组件100为上述实施例提供的热循环反应组件100，发光部60用于向毛细管预制组件20发出激发光，荧光检测部件70用于检测毛细管预制组件20内样品经发光部60照射所产生的荧光。
通过该实时检测装置，能够对每层毛细管预制组件20内的样品同时进行检测，便于工作人员获取检测数据。且该装置结构简单、便于操作。相对于传统如图1所示的热循环器，在对其进行检测时，需要将发光部以及荧光检测部件的部分装置设置在中空部分，使得热循环器的中空部分必须满足发光部以及荧光检测部件尺寸的要求，无法使热循环器向小型化方向发展。而本实施例三提供的实时检测装置，只需将发光部60以及荧光检测部件70设置在热循环反应组件100的一侧即可，热循环反应组件100的尺寸根据PCR反应需要设置即可，如此能够简化热循环反应组件100的设计。
具体地，该发光部60包括光源61和激发带通滤波器62。其中，通过光源61向毛细管预制组件20发出激发光，激发带通滤波器62设置在激发光的光路上，通过激发带通滤波器62调节激发光发出的波长。可选地，光源61可以为卤素灯、氙灯等白光光源，也可为LED灯、激光等单色或窄波长光源。
具体地，该荧光检测部件70包括：荧光会聚透镜71、检测传感器72以及带通滤波器73。其中，荧光会聚透镜71用于会聚毛细管预制组件20内样品产生的荧光，检测传感器72设置在荧光会聚透镜71会聚的荧光的光路上，带通滤波器73设置在荧光会聚透镜71与检测传感器72之间，带通滤波器73用于滤波，检测传感器72用于检测经带通滤波器73滤波后的荧光。通过检测传感器72，即可实时获取样品在通过毛细管的荧光强度，通过该荧光强度即可得到特定DNA序列的初始浓度。
可选地，荧光会聚透镜71可以设置为非球面透镜或非球面透镜组，检测传感器72可以设置为面阵CMOS传感器或面阵CCD传感器。带通滤波器73可以是吸收式的滤光片，也可是反射式的滤光片。
如图8所示，实施例四提供了一种实时检测装置，与实施例三的不同之处仅在于，该荧光检测部件70中包括了多组带通滤波器73，多组带通滤波器73可切换地设置在荧光会聚透镜71和检测传感器72之间，通过任意一个带通滤波器73对荧光会聚透镜71会聚的荧光进行滤波，检测传感器72用于检测经带通滤波器73滤波后的荧光。如此可通过调节不同的带通滤波器73以获取不同波段的荧光，并对不同波段的荧光进行检测。
具体地，该荧光检测部件70还包括支架74和电机75，其中，多组带通滤波器73均设置在支架74上，电机75与支架74驱动连接，电机75驱动支架74转动以在需要时，对多组带通滤波器73进行切换。
通过本发明提供的实时检测装置，可将现有QPCR的反应时间由1到2小时缩短至5到20分钟，如此可极大缩短反应时间，提高分析速度。并且该装置结构简单、整体体积小，方便工作人员使用。
以上所述仅为本发明的优选实施例而已，并不用于限制本发明，对于本领域的技术人员来说，本发明可以有各种更改和变化。凡在本发明的精神和原则之内，所作的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。

Claims (13)

  1. 一种热循环反应组件，其特征在于，所述热循环反应组件包括：
    多个柱形加热组件(10)，多个所述柱形加热组件(10)间隔设置，至少两个所述柱形加热组件(10)的加热温度不同；
    毛细管预制组件(20)，所述毛细管预制组件(20)设置在多个所述柱形加热组件(10)上。
  2. 根据权利要求1所述的热循环反应组件，其特征在于，所述毛细管预制组件(20)包括：
    第一段毛细管(21)，所述第一段毛细管(21)用于缠绕在多个所述柱形加热组件(10)；
    第二段毛细管(22)，所述第二段毛细管(22)用于缠绕在其中一个所述柱形加热组件(10)上，所述第一段毛细管(21)与所述第二段毛细管(22)相互连通，所述第一段毛细管(21)和所述第二段毛细管(22)在多个所述柱形加热组件(10)上分别缠绕有多层，相邻的两层毛细管之间相互固定在一起。
  3. 根据权利要求1所述的热循环反应组件，其特征在于，所述柱形加热组件(10)包括：
    壳体(11)，所述毛细管预制组件(20)缠绕在所述壳体(11)的外壁上；
    加热部(12)，设置在所述壳体(11)内部；
    温度控制器(13)，与所述加热部(12)电连接，所述温度控制器(13)用于控制所述柱形加热组件(10)的温度。
  4. 根据权利要求1所述的热循环反应组件，其特征在于，所述热循环反应组件还包括：
    推进机构(30)，设置在所述毛细管预制组件(20)的进口端。
  5. 根据权利要求1所述的热循环反应组件，其特征在于，所述热循环反应组件还包括：
    收集装置(40)，设置在所述毛细管预制组件(20)的出口端。
  6. 根据权利要求1所述的热循环反应组件，其特征在于，所述热循环反应组件还包括：
    固定座组件，多个所述柱形加热组件(10)固定设置在所述固定座组件上。
  7. 根据权利要求6所述的热循环反应组件，其特征在于，所述固定座组件包括：
    固定座(51)，具有多个间隔设置的第一固定孔，多个所述柱形加热组件(10)的一端一一对应地设置在多个所述第一固定孔中；
    限位座(52)，具有多个间隔设置的第二固定孔，所述第二固定孔的间隔距离大于所述第一固定孔的间隔距离，多个所述柱形加热组件(10)的另一端一一对应地设置在多个所述第二固定孔中。
  8. 根据权利要求1所述的热循环反应组件，其特征在于，所述柱形加热组件(10)包括第一柱形加热组件和第二柱形加热组件，所述第一柱形加热组件和所述第二柱形加热组件间隔设置，所述第一柱形加热组件的加热温度与所述第二柱形加热组件的加热温度不同，所述毛细管预制组件(20)设置在所述第一柱形加热组件和所述第二柱形加热组件上。
  9. 一种实时检测装置，其特征在于，所述实时检测装置包括：
    热循环反应组件(100)，所述热循环反应组件(100)为权利要求1至8中任一项所述的热循环反应组件(100)；
    发光部(60)，用于向毛细管预制组件(20)发出激发光；
    荧光检测部件(70)，用于检测所述毛细管预制组件(20)内样品经所述发光部(60)照射所产生的荧光。
  10. 根据权利要求9所述的实时检测装置，其特征在于，所述发光部(60)包括：
    光源(61)，用于向所述毛细管预制组件(20)发出激发光；
    激发带通滤波器(62)，设置在所述激发光的光路上，所述激发带通滤波器(62)用于调节所述激发光的波长。
  11. 根据权利要求9所述的实时检测装置，其特征在于，所述荧光检测部件(70)包括：
    荧光会聚透镜(71)，用于会聚所述毛细管预制组件(20)内样品产生的荧光；
    检测传感器(72)，设置在所述荧光会聚透镜(71)会聚的荧光的光路上；
    带通滤波器(73)，设置在所述荧光会聚透镜(71)与所述检测传感器(72)之间，所述带通滤波器(73)用于进行波长选择，所述检测传感器(72)用于检测经所述带通滤波器(73)波长选择后的荧光。
  12. 根据权利要求9所述的实时检测装置，其特征在于，所述荧光检测部件(70)包括：
    荧光会聚透镜(71)，用于会聚所述毛细管预制组件(20)内样品产生的荧光；
    检测传感器(72)，设置在所述荧光会聚透镜(71)会聚的荧光的光路上；
    多组带通滤波器(73)，可切换地设置在所述荧光会聚透镜(71)和所述检测传感器(72)之间，通过任意一个所述带通滤波器(73)对所述荧光会聚透镜(71)会聚的荧光进行波长选择，所述检测传感器(72)用于检测经所述带通滤波器(73)波长选择后的荧光。
  13. 根据权利要求12所述的实时检测装置，其特征在于，所述荧光检测部件(70)还包括：
    支架(74)，多组所述带通滤波器(73)设置在所述支架(74)上；
    电机(75)，所述电机(75)与所述支架(74)驱动连接，所述电机(75)驱动所述 支架(74)转动以对多组所述带通滤波器(73)进行切换。
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