WO2019052522A1 - 一种核酸测定方法 - Google Patents

一种核酸测定方法 Download PDF

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Publication number
WO2019052522A1
WO2019052522A1 PCT/CN2018/105634 CN2018105634W WO2019052522A1 WO 2019052522 A1 WO2019052522 A1 WO 2019052522A1 CN 2018105634 W CN2018105634 W CN 2018105634W WO 2019052522 A1 WO2019052522 A1 WO 2019052522A1
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Prior art keywords
tubular chamber
tubular
reaction
nucleic acid
nth
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PCT/CN2018/105634
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English (en)
French (fr)
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苏星
吴开原
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星源智（珠海）生物科技有限公司
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Priority to US16/648,317 priority Critical patent/US20200216875A1/en
Publication of WO2019052522A1 publication Critical patent/WO2019052522A1/zh

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    • 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
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/686—Polymerase chain reaction [PCR]
    • 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
    • B01L3/5085—Containers for the purpose of retaining a material to be analysed, e.g. test tubes rigid containers not provided for above for multiple samples, e.g. microtitration plates
    • B01L3/50851—Containers for the purpose of retaining a material to be analysed, e.g. test tubes rigid containers not provided for above for multiple samples, e.g. microtitration plates specially adapted for heating or cooling samples
    • 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
    • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
    • B01L3/502769—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip characterised by multiphase flow arrangements
    • 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
    • B01L3/5085—Containers for the purpose of retaining a material to be analysed, e.g. test tubes rigid containers not provided for above for multiple samples, e.g. microtitration plates
    • B01L3/50853—Containers for the purpose of retaining a material to be analysed, e.g. test tubes rigid containers not provided for above for multiple samples, e.g. microtitration plates with covers or lids
    • 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
    • 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
    • C12M1/24—Apparatus for enzymology or microbiology tube or bottle type
    • 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
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6851—Quantitative amplification
    • 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
    • B01L2300/04—Closures and closing means
    • B01L2300/041—Connecting closures to device or container
    • B01L2300/042—Caps; Plugs
    • 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
    • B01L2300/0861—Configuration of multiple channels and/or chambers in a single devices
    • 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
    • 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
    • C12Q2561/00—Nucleic acid detection characterised by assay method
    • C12Q2561/113—Real time assay
    • 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
    • C12Q2565/00—Nucleic acid analysis characterised by mode or means of detection
    • C12Q2565/10—Detection mode being characterised by the assay principle
    • C12Q2565/101—Interaction between at least two labels

Definitions

  • the invention relates to the field of molecular biology, and in particular to a method for qualitative or quantitative determination of nucleic acids.
  • a typical nucleic acid quantitative amplification reaction is a polymerase chain reaction, ie, PCR.
  • PCR polymerase chain reaction
  • the viral RNA is first purified, and then the RNA is reverse transcribed (RT) into cDNA, and then subjected to cDNA quantitative amplification reaction (RT-PCR).
  • RT-PCR cDNA quantitative amplification reaction
  • Multi-step reactions also include nested PCR.
  • Nested PCR uses a pair of external primers to complete the first step of PCR, and then uses a pair of internal primers to carry out a second step of the product of the first step reaction and quantitatively determine.
  • Multiplex PCR involves the inclusion of multiple pairs of primers in a single reaction system (reaction tube).
  • the multi-step reaction of nested PCR can be combined with multiplex PCR, that is, in the first step of nested PCR, multiplex PCR, and the second step of nested PCR are performed separately for multiple single quantitative PCR.
  • quantitative PCR is to detect the total amount of products after each polymerase chain reaction cycle in a DNA amplification reaction, and the external reference or internal reference is used as a standard, and the PCR starting template is carried out by analyzing the PCR end product or monitoring the PCR process. Quantification of the amount.
  • Nested PCR uses two or more pairs of PCR primers for amplification reactions.
  • the first pair of PCR primers is called the outer primer (pair) and the second pair of primers is called the inner primer (pair). Since the internal primer is incorporated inside the first PCR product, the second PCR amplified fragment is shorter than the first amplification product.
  • the advantage of nested PCR is that if the first amplification produces an erroneous fragment, the probability of primer pairing and amplification on the wrong fragment for the second time is extremely low. Therefore, nested PCR has very high specificity and sensitivity.
  • Multiplex PCR is a PCR reaction in which two or more primers are added to the same PCR reaction system to simultaneously amplify a plurality of nucleic acid fragments. Multiplex PCR can analyze multiple genes in the same PCR reaction system and is highly efficient.
  • RNA single-stranded molecules can be transcribed (RT) by reverse transcriptase into complementary DNA or cDNA molecules.
  • cDNA can be used in PCR reactions as well as ordinary DNA. This process is called RT-PCR.
  • PCR reactions may be carried out alone or in combination with a plurality of PCR reactions.
  • multiple reactors are often used to carry out multi-step reactions separately, which cannot meet the requirements of sealing, especially in the process of product transfer, the product is liable to cause pollution.
  • an object of the present invention is to provide a nucleic acid measuring method which ensures sealing and is easy to operate.
  • nucleic acid qualitative or quantitative assay method that is probe-free, does not rely on polymerase exo-activity and is not limited by the length of the target molecule.
  • the present invention provides a nucleic acid assay method which is carried out by a closable reaction vessel comprising at least two tubular chambers arranged in sequence and at least one passage communicating with at least two tubular chambers
  • the first tubular chamber is provided with a sample and a first reagent
  • the nth tubular chamber is provided with an nth reagent, wherein n is equal to or greater than 2
  • the method comprises: closing the reaction vessel, at first The first set of reactions takes place in a tubular chamber, the product in the first tubular chamber is transported through the passage to the next tubular chamber; the nth tubular chamber receives the product from the previous tubular chamber, at the nth The nth group of reactions are carried out in a tubular chamber; the products in the nth tubular chamber are tested.
  • the present invention employs a closed reaction vessel having at least two tubular chambers and transfers the product of the previous tubular chamber to the latter tubular chamber through a passageway communicating the tubular chamber, the product being in the latter tubular chamber
  • the reaction can be carried out as a substrate, and a multi-step PCR reaction such as nested PCR, RT-PCR, multiplex PCR or a combination of two or more thereof can be realized.
  • the first reaction reagent comprises a first pair of primers of nested PCR or a reverse transcription reaction primer of RT-PCR or a first set of primers of multiplex PCR;
  • the nth reaction reagent comprises nested PCR or RT-PCR or The nth pair of primers of the multiplex PCR or the nth primer of the multiplex PCR and the affinity substance.
  • the product can be qualitatively and quantitatively determined by a detectable signal formed by the combination of an affinity substance and a product.
  • the affinity substance may be any one or more substances which can be directly combined with the product to form a detectable signal.
  • the affinity substance does not depend on the activity of the exonuclease, so that any polymerase suitable for PCR can be used for the quantitative determination of the present invention, which is advantageous for the promotion of quantitative reaction, and is not designed in the reaction design. It is necessary to reserve space for the molecular probe and thus is not affected by the length of the target molecule.
  • the invention does not need to use a fluorescently labeled molecular probe for qualitative or quantitative determination, thereby effectively reducing the cost.
  • the first reaction reagent and the n-th reaction reagent include, in addition to the corresponding primers and an affinity substance which can be added as needed, and other reaction reagents known in the art such as a polymerase, a reverse transcriptase, a nucleotide, a buffer, and the like. Or inorganic substances.
  • the reagents and samples can be added to the corresponding tubular chamber by hand or automated prior to the reaction. It is also possible to add the reaction reagent to the reaction reagent in advance, store it after storage, and transport it, and add the sample to the corresponding tubular chamber during use. After the completion of the addition of the sample and the reaction reagent, the reaction vessel can be closed by physical, mechanical or chemical means to carry out an enzymatic reaction.
  • an external primer or a reverse transcription primer can be placed in the first tubular chamber, and a pair of internal primers can be placed in the second tubular chamber. If necessary, the third tubular chamber can be placed with the corresponding primer.
  • the sample containing the target molecule is only added to the first reaction tube with the external primer.
  • the principle of quantitative measurement using this method is that the time during which the second and subsequent tubular chambers generate signals is in a specific proportional relationship with the number of sample target molecules in the first tubular chamber during the reaction. This is because the product molecules in the former tubular chamber are limitedly transferred to the latter tubular chamber by methods such as molecular diffusion, liquid convection, etc., and become the molecular substrate of the next reaction, and the amount of product molecules transported. The rate is proportional to the amount of product from the previous reaction.
  • the sensitivity of the quantitative detection and the dynamic range of the reaction can be adjusted by adjusting or controlling the probability of transport of molecules between the reaction tubes.
  • a further technical solution is that the method further comprises temperature control of any one of the tubular chambers, the temperature control comprising maintaining a constant temperature gradient within any of the tubular chambers or periodically changing the temperature within any of the tubular chambers.
  • the temperature of the reaction vessel can be controlled by all known methods.
  • the temperature of the reaction unit may be either an overall equilibrium, an overall change, or a temperature differential between the different portions, particularly each reactor tube may maintain a temperature gradient.
  • the temperature control can be by constant heating of a specific part of the tubular chamber, maintaining a constant temperature gradient within the tubular reactor, or using a cyclically varying temperature control to produce a balanced, periodically varying temperature within the tubular reactor. .
  • the effect is to subject the molecules in the tubular chamber to different temperatures, so as to meet the requirements of different enzyme reaction conditions, and achieve the purpose of realizing nucleic acid amplification in the tubular chamber.
  • the temperature control comprises a plurality of tubular chambers for synchronous temperature control.
  • Simultaneously controlling the temperature of the plurality of tubular chambers during the quantitative determination of the nucleic acid for example, synchronously controlling the temperature of the second and subsequent tubular chambers, ensuring that the second and subsequent tubular chambers are receiving the previous tubular lumen
  • the product delivered at the chamber can be reacted at any time to ensure consistent reaction conditions and then quantitatively analyzed by signal detection.
  • a further technical solution is that a medium is arranged in the channel.
  • a further technical solution is that the product obtained by the reaction in any one of the tubular chambers is transferred to the latter tubular chamber by liquid convection or molecular diffusion in the channel.
  • the medium in the channel can be any medium that enables product transfer, such as a liquid.
  • product transfer such as a liquid.
  • the mode of transport can be liquid convection or molecular diffusion, allowing some product molecules to be transported from one tubular chamber to the next, with the amount, speed and time of delivery being determined by the molecular weight of the sample in the previous reaction tube. This can be used as a basis for quantitative measurements.
  • a further technical solution is that the channel is arranged at the upper end of the tubular chamber.
  • the junction of the channel with the tubular chamber can be the upper, middle or lower end of the tubular chamber.
  • the most preferred way of attachment is that the channel communicates with the plurality of tubular chambers at the upper end of the tubular chamber. This communication mode facilitates mixing of substances between different tubular chambers where mixing is undesirable.
  • the tubular chamber is cylindrical or conical, with a straight diameter between 0.1 mm and 10 mm, a wall thickness of 0.05 to 5 mm, and a ratio of the depth of the tubular chamber to the inner diameter. Greater than or equal to 2.
  • the use of a cylindrical or conical tubular chamber facilitates maintaining a uniform temperature or a constant temperature gradient in the tubular chamber.
  • the inner diameter, the wall thickness, and the ratio of the thickness to the inner diameter of the tubular chamber can satisfy the general reaction requirements and maintain safety and stability, and save cost.
  • reaction vessel further comprises an arcuate connection between the adjacent tubular chambers and projecting toward the passage.
  • Adjacent tubular chambers are connected by curved joints to avoid the formation of dead spaces, so that the product molecules between the different tubular chambers are more smoothly transported in the medium of the channels.
  • the affinity substance comprises a dye.
  • the affinity material can be any existing material capable of binding to a nucleic acid product and forming a detectable signal, such as a dye, particularly a fluorescent dye, which, when combined with a nucleic acid product, can form a fluorescent signal.
  • the affinity substance may also be a substance which can be combined with a product such as a surfactant, a metal complex, or a protein.
  • the detectable signal comprises an optical signal or an electrical signal.
  • the optical signal comprises a fluorescent signal, a light absorbing signal, an infrared absorbing signal, a Raman scatter signal or a chemiluminescent signal.
  • Optical signals or electrical signals can be generated by combining different affinity substances with the product.
  • qualitative or quantitative determination by optical signals is advantageous, and real-time quantitative determination can be performed.
  • Optical signals can be detected by existing optical signal detection instruments.
  • the nucleic acid measuring method of the invention does not need a probe, does not depend on the exo-cleavage activity of the polymerase, is not limited by the length of the target molecule, ensures the sealing property, and is convenient and simple to operate.
  • FIG. 1 is a schematic view showing the structure of a reaction vessel used in an embodiment of the measuring method of the present invention.
  • Figure 2 is a schematic view showing the structure of a closed state of the reaction vessel used in the embodiment of the measuring method of the present invention.
  • Figure 3 is a schematic illustration of the reaction process of an embodiment of the assay method of the present invention.
  • Figure 4 is a graph showing the results of an embodiment of the assay method of the present invention.
  • the nucleic acid test method of this example is carried out by a closable reaction vessel.
  • One of the closable reaction vessels is shown in FIG. 1.
  • the reaction vessel 1 includes at least two tubular chambers 11 arranged in order, which may be sequentially named as a first tubular chamber to an nth tubular chamber. Where n is equal to 2 or greater than 2.
  • the different tubular chambers 11 can perform different reactions in a multi-step reaction, and the number of tubular chambers 11 can be determined according to the needs of the actual reaction.
  • the tubular chamber 11 can be communicated by one or more passages 12 at the upper, middle or lower end of the tubular chamber 11, the passage 12 allowing the reaction product to be transported between the tubular chambers 11 under closed conditions to achieve a multi-step enzyme reaction.
  • the plurality of tubular chambers 11 are communicated by a passage 12 at the upper end of the tubular chamber 11, and the manner in which the upper ends communicate is advantageous to prevent unnecessarily mixing the reaction materials between the different tubular chambers 11.
  • the channel 12 can be designed in different shapes as desired, such as tubular, canal, and the like.
  • the inner diameter of the tubular chamber 11 is between 0.1 mm and 10 mm, and the wall thickness is between 0.05 and 5 mm; the ratio of the depth to the inner diameter of the tubular chamber 11 is greater than or equal to two.
  • the size of the tubular chamber 11 is within the above range, which satisfies the needs of the general multi-step biological enzymatic reaction, and is safe and stable, and saves cost.
  • the reaction vessel 1 may also include an opening 13, preferably the opening 13 is located at the upper end of the channel 12.
  • the opening 13 can be closed in any manner known in the art. As shown in FIG. 2, in the present embodiment, the opening 13 can be closedly engaged with the cover body 2, and the cover body 2 is provided with a through hole 21 for loading and sampling.
  • a seal 3 can be inserted into the through hole 21.
  • Each of the seals 3 includes a sealing plug 31 engageable with the through hole 21, and a sealing rod 32 engageable with at least a portion of the tubular chamber 11 may be fixedly or movably coupled to the lower end of the sealing plug 31 as needed.
  • the sealing rod 32 can also be used for sample collection prior to reaction.
  • An arcuate connecting portion 14 projecting into the channel 12 is disposed between adjacent at least two tubular chambers 11.
  • the arcuate connecting portion 14 can avoid dead angles, facilitating the transfer of products between adjacent tubular chambers 11.
  • the storage chamber 15 may be provided with a storage chamber 15.
  • the reaction vessel 1 is made of a transparent material such as plastic, glass, or the like, and thus has a transparent appearance, and the reaction result can be continuously and rapidly detected by an optical method in real time.
  • the reaction vessel 1 can be integrally formed by mechanical molding or injection molding.
  • the first reaction reagent includes, in addition to components known in the art as polymerases, nucleotides, and buffers, a first pair of primers, i.e., an outer primer, of nested PCR.
  • the n-th reaction reagent includes, in addition to components known in the art as a polymerase, a nucleotide, and a buffer, an n-th primer of nested PCR, that is, an internal primer, and an affinity substance.
  • a polymerase a nucleotide
  • a buffer a buffer
  • an n-th primer of nested PCR that is, an internal primer
  • an affinity substance for the quantitative reaction, the sample containing the target molecule is only added to the first reaction tube with the external primer.
  • the reaction reagent can also be pre-stored in the tubular chamber 11, sealed and stored and transported, and only the sample to be tested can be added during use.
  • the tubular chamber 11 can also be filled with reagents and biological samples by manual or automated methods prior to use.
  • the affinity material can be any existing material capable of binding to a nucleic acid product and forming a detectable signal, such as a dye, particularly a fluorescent dye, which, when combined with a nucleic acid product, can form a fluorescent signal.
  • Affinity materials can also be substances such as surfactants, metal complexes, proteins, and the like that bind to the product and produce a detectable signal.
  • Optical signals or electrical signals can be generated by combining different affinity substances with the product.
  • optical signals including fluorescent signals, light absorption signals, infrared absorption signals, Raman scattering signals or chemiluminescent signals, for real-time detection.
  • One or more media are disposed within the passage 12, which may be liquid, and the product in one tubular chamber 11 is conveyed through the media to the latter tubular chamber 11.
  • the transfer mode can be liquid heat convection or molecular diffusion.
  • the product in the nth tubular chamber combines with the affinity material to produce a detectable signal.
  • a detectable signal can be generated stepwise from the second tubular chamber to the fifth tubular chamber, and the detectable signal gradually increases over time.
  • the time in Figure 3 begins with the transfer of product from the first tubular chamber.
  • the biological enzyme reaction requires temperature control.
  • the biological enzyme reaction is generally carried out between 15 ° C and 99 ° C.
  • the currently known methods can be used to control the temperature of the biological enzyme reaction in the tubular reactor, for example, using infrared light, hot/cold air, cold/hot solid or liquid substances, electromagnetic induction, and the like.
  • the closed reaction unit can be inserted into a temperature control instrument for temperature control.
  • any tubular chamber 11 can be subjected to a constant temperature or periodic varying temperature, and there can also be a uniform temperature or gradient temperature within the tubular chamber 11.
  • the temperature of the temperature control instrument is periodically changed under the control of a computer program, such as holding at a certain temperature for several seconds to several minutes. And the tubular chamber 11 is fully inserted into the heating portion of the temperature control instrument during which the temperature of the liquid within the tubular chamber 11 is substantially equal.
  • a constant temperature gradient temperature control method the temperature of the temperature control instrument remains unchanged under the control of a computer program, and the tubular chamber 11 is only partially in contact with the heated portion of the temperature control instrument. When the bottom is heated, the bottom temperature will be higher than the top temperature, at which point the liquid in the tubular chamber 11 will have a temperature gradient.
  • the upper and lower liquids will convect, and the effect is to drive the molecules in the tubular chamber to flow and to withstand different temperatures to meet different enzyme reaction conditions.
  • the purpose of DNA amplification in the tubular chamber 11 is achieved.
  • the tubular structure of the tubular chamber 11 provides more flexibility to the instrument design.
  • the temperature of the tubular chamber should be controlled synchronously to ensure that the second and subsequent tubular chambers can react immediately at any time while receiving the product delivered from the previous tubular chamber, ensuring the reaction conditions. Consistent.
  • the principle of quantitative measurement by this method is that, when the reaction is carried out, the product molecules in the former tubular chamber are transmitted to the latter tubular chamber through the passage, for example, by diffusion, etc., and become the molecular bottom of the latter reaction.
  • the amount and rate of product molecules delivered is directly proportional to the amount of product from the previous reaction.
  • the sensitivity of the quantitative detection and the dynamic range of the reaction can be adjusted by adjusting or controlling the probability of transport of molecules between the reaction tubes.
  • the ratio of signal to time is proportional to the position of the tubular chamber, where the ratio of signal to time is when the signal reaches an unsaturated, ie linear
  • the ratio of the signal to the time used for an ideal value of the growth period, which is expressed in logarithmic form.
  • the method of this example can be applied to qualitative and quantitative testing of a variety of different nucleic acids.
  • the method can be used for human EGFR gene mutation assays. Specifically, the following steps are included:
  • DNA template 200 ⁇ M dNTPs, 20 mM Tris-HCl, 10 mM (NH 4 ) 2 SO 4 , 10 mM KCl, 2 mM MgSO 4 , 0.1% Triton®-X- in the first tubular chamber of the 50 uL reaction volume. 100,1 unit DNA polymerase, and 0.3 ⁇ M of the following external primer pairs:
  • the second tubular chamber was tested for T790 wild type, and the second tubular chamber contained 200 ⁇ M dNTPs, 20 mM Tris-HCl, 10 mM (NH 4 ) 2 SO 4 , 10 mM KCl, 2 mM MgSO 4 , 0.1% Triton ®-X-100, 0.25X Cyber Green dye (AAT Bioquest, USA) and 1 unit DNA polymerase, and nested PCR primer pair 0.3 ⁇ M:
  • EGFR 790R 5'-GTGTT CCCGG ACATA GTCCA -3’.
  • the third tubular chamber was tested for M790 mutation.
  • the third tubular chamber contained 200 ⁇ M dNTPs, 20 mM Tris-HCl, 10 mM (NH 4 ) 2 SO 4 , 10 mM KCl, 2 mM MgSO 4 , 0.1%.
  • EGFR 790R 5'-GTGTT CCCGG ACATA GTCCA -3’.
  • the fourth tubular chamber was tested for L858 wild type, and the fourth tubular chamber contained 200 ⁇ M dNTPs, 20 mM Tris-HCl, 10 mM (NH 4 ) 2 SO 4 , 10 mM KCl, 2 mM MgSO 4 , 0.1% Triton®-X-100, 0.25X Cyber Green dye (AAT Bioquest, USA) and 1 unit DNA polymerase, and nested PCR primer pair 0.3 ⁇ M:
  • EGFR L858F 5'-GTCAA GATCA CAGAT TTTGG GCT-3’
  • EGFR OutR1 5’-GATCG GCCTC TTCAT GCGAA GG -3'.
  • the fifth tubular chamber was tested for R858 mutation.
  • the fifth tubular chamber contained 200 ⁇ M dNTPs, 20 mM Tris-HCl, 10 mM (NH 4 ) 2 SO 4 , 10 mM KCl, 2 mM MgSO 4 , 0.1%.
  • EGFR R858F 5'-GTCAA GATCA CAGAT TTTGG GCG-3’
  • EGFR OutR1 5’-GATCG GCCTC TTCAT GCGAA GG -3'.
  • the second tubular chamber to the fifth tubular chamber were simultaneously subjected to a constant temperature convection reaction for 15 minutes.
  • the nucleic acid determination method combines a closed reaction vessel having a plurality of tubular chambers and channels with a nested PCR reaction for qualitative or quantitative determination, which can prevent product contamination, and the sensitivity and accuracy of the reaction.
  • High in sex no need to use molecular probes, independent of polymerase exo-activity and not limited by target molecule length.
  • the nucleic acid assay method of the present invention can implement a multi-step PCR reaction such as nested PCR, RT-PCR, multiplex PCR or a combination of two or more thereof.

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Abstract

一种核酸测定方法，该方法通过可封闭的反应容器实施，反应容器包括管状腔室以及连通管状腔室的通道；其中，第一个管状腔室内设置有样品和第一反应试剂；第n个管状腔室内设置有第n反应试剂；该方法包括：将反应容器封闭，在第一个管状腔室中进行第一组反应，第一个管状腔室中的产物通过通道传送到后一个管状腔室；前一个管状腔室的产物传送到第n个管状腔室后，在第n个管状腔室中进行第n组反应。

Description

一种核酸测定方法 技术领域
本发明涉及分子生物学领域，具体涉及一种核酸定性或定量测定的方法。
 背景技术
随着生物技术的发展，现代分子生物学技术或基因工程技术正被日益广泛地用到各种生物技术产业中，特别是应用到医疗诊断中。这类技术的应用，往往涉及用分子探针进行定性、定量测定。典型的核酸定量扩增反应是聚合酶链式反应，即PCR。例如，当对RNA病毒进行检测和诊断时，首先需对病毒RNA进行纯化，再将RNA进行逆转录（RT）成cDNA，然后进行cDNA定量扩增反应 （RT-PCR）。可见RT-PCR是两步反应。多步反应还包括巢式PCR (nested PCR) 。巢式PCR是用一对外引物完成第一步PCR后、再用一对内引物对第一步反应的产物进行第二步反应并进行定量测定。多重PCR是在一反应体系(反应管)内含有多对引物。巢式PCR的多步反应可以与多重PCR相结合、即在巢式PCR的第一步进行多重PCR、巢式PCR的第二步分别进行多个单重定量PCR。
其中，定量PCR是在DNA扩增反应中，检测每次聚合酶链反应循环后产物总量，以外参或内参为标准，通过对PCR终产物的分析或PCR过程的监测，进行PCR起始模板量的定量。
巢式PCR使用两对或两对以上的PCR引物分别进行扩增反应。第一对PCR引物称外引物(对)、第二对引物称内引物(对)。由于内引物结合在第一次PCR产物内部，使得第二次PCR扩增片段短于第一次扩增产物。巢式PCR的好处在于，如果第一次扩增产生了错误片断，则第二次能在错误片段上进行引物配对并扩增的概率极低。因此，巢式PCR具有非常有高的特异性和灵敏度。
多重PCR是在同一PCR反应体系中加入两对以上引物，同时扩增出多个核酸片段的PCR反应。多重PCR能够在同一PCR反应体系中对多个基因进行分析，具有高效性。
RNA单链分子可通过逆转录酶转录(RT)成为互补的DNA或称cDNA分子。cDNA可与普通DNA一样可用于PCR反应。这一过程叫做RT-PCR。
上述多种PCR反应可以单独实施，也可以多种PCR反应相互结合。现有技术常常采用多个反应器分别进行多步反应，不能满足密封的要求，特别是在产品转移的过程中，产物容易造成污染。
此外，现有技术中常用实时荧光定量核酸扩增检测对核酸进行定量检测，检测时需要用到荧光标记的分子探针。现有用探针进行定量的方法的缺点是探针需要有二至三个标记物，在设计、制作上成本高。该方法还需要使用具外切酶活性的聚合酶，对试剂的要求高。现有用探针进行定量的方法还要求靶分子有足够的长度，如五十碱基对以上。以上要求在许多应用情况不能被满足。
现有技术中也存在使用非专一性的荧光染料对核酸进行定量测定等方法。这类荧光染料可与双链的DNA结合并产生荧光，产生的信号与DNA量成正比，但与DNA的序列无关，无法测出DNA中的特定序列。
 技术问题
为了解决上述的问题，本发明的目的是提供一种保证封闭性并且操作便捷简单的核酸测定方法。
进一步地，本发明的目的是提供无探针的、不依赖聚合酶外切活性并且不受靶分子长度限制的核酸定性或定量测定方法。
 技术解决方案
为实现上述目的，本发明提供了一种核酸测定方法，该方法通过可封闭的反应容器实施，反应容器包括按序排列的至少两个管状腔室以及连通至少两个管状腔室的至少一个通道；其中，第一个管状腔室内设置有样品和第一反应试剂；第n个管状腔室内设置有第n反应试剂，其中n等于或大于2；该方法包括：将反应容器封闭，在第一个管状腔室中进行第一组反应，第一个管状腔室中的产物通过通道传送到后一个管状腔室；第n个管状腔室接收到前一个管状腔室的产物后，在第n个管状腔室中进行第n组反应；对所述第n个管状腔室内的产物进行检测。
本发明采用封闭式的具有至少两个管状腔室的反应容器，并通过连通管状腔室的通道将前一个管状腔室的产物传送到后一个管状腔室，该产物在后一个管状腔室中作为底物进行反应，可以实现多步PCR反应，例如巢式PCR、RT-PCR、多重PCR或者是它们的两种或以上的组合。
进一步的技术方案是，第一反应试剂包括巢式PCR的第一对引物或RT-PCR的逆转录反应引物或多重PCR的第一组引物；第n反应试剂包括巢式PCR或RT-PCR或多重PCR的第n对引物或多重PCR的第n组引物和亲和性物质。
在每一步扩增反应中，可以通过由亲和性物质与产物结合形成的可检测信号来对产物进行定性、定量测定。其中，亲和性物质可以是现有的任意一种或多种能够直接与产物结合形成可检测信号的物质。与分子探针不同，亲和性物质不依赖于外切酶的活性，使得任何适用于PCR的聚合酶均可以用于本发明的定量测定，有利于定量反应的推广，且在反应设计时不需要给分子探针预留空间，因而不受靶分子长度的影响。本发明无需使用具荧光标记的分子探针进行定性或定量测定，有效降低了成本。
第一反应试剂和第n反应试剂除了包括对应的引物以及可根据需要加入的亲和性物质，还包括本领域公知的其他反应试剂例如聚合酶、 逆转录酶、核苷酸、缓冲液等有机或无机物质。反应试剂和样品可以在反应前通过手工或自动化方法加入到相应的管状腔室中。也可以预先将反应试剂加入反应试剂，封闭后进行保存和运输，使用时在相应的管状腔室中加入样品即可。完成样品和反应试剂的加入后，再通过物理、机械或化学方式可对反应容器进行封闭，进行酶反应。
根据巢式PCR和RT-PCR原理，第一个管状腔室中可以放置有一对外引物或包括逆转录引物，第二个管状腔室中可以放置有一对内引物。如有必要，第三个管状腔室可以放入相应的引物。作定量反应时，含靶分子的样品只加入到有外引物的第一反应管中。
利用该方法进行定量测量的原理是，反应进行时, 第二个及其后的管状腔室产生信号的时间与在第一管状腔室内样品靶分子的数量成特定的比例关系。这是由于前一个管状腔室中的产物分子会有限地通过例如分子扩散、液体对流等方法传送到后一个管状腔室，并成为下一反应的分子底物，所传送的产物分子的量和速度与上一反应的产物量成正比。通过调节或控制反应管之间分子的传输机率，可以调节定量检测的灵敏度和反应的动态范围。
进一步的技术方案是，该方法还包括对任意一个管状腔室进行温度控制，温度控制包括保持任意一个管状腔室内具有恒定的温度梯度或周期性地改变任意一个管状腔室内的温度。
反应容器的温度可通过所有已知方法进行控制。反应装置的温度可以是整体均衡的、整体变化的或是不同部分之间具有温差，特别是每一反应器管可以保持有一温度梯度。温度控制方式可以是对管状腔室的特定部位恒定加热，保持管形反应器内产生恒定的温度梯度，或者是使用周期变化的温度控制，使管形反应器内产生均衡的周期性变化的温度。其效果是使得管状腔室内的分子经受不同的温度，从而满足不同的酶反应条件要求，达到实现管状腔室内核酸扩增的目的。
进一步的技术方案是，温度控制包括多个管状腔室进行同步温度控制。
在进行核酸定量测定时，同步控制多个管状腔室的温度，例如同步控制第二个及其后管状腔室的温度，保证第二个及其后管状腔室在在接收到前一个管状腔室传送的产物的任何时候都能够立即进行反应，从而保证反应条件一致，再通过信号检测进行定量分析。
进一步的技术方案是，通道中设置有介质。进一步的技术方案是，任意一个管状腔室中反应得到的产物在通道中通过液体对流或分子扩散的方式传送至后一个管状腔室。
通道中的介质可以是任何能够实现产物传送的介质，例如可以是液体。当第一管状腔室进行反应后，其产物分子可以通过介质传送到另一管形反应器。传输的方式可以是液体对流或分子扩散的方式，使部分产物分子从一个管状腔室传送到后一个管状腔室，传送的量、速度和时间决定于前一反应管中的样品靶分子量。以此可以作为定量测量的基础。
进一步的技术方案是，通道设置在管状腔室的上端。
通道与管状腔室的连接部位可以是管状腔室的上端、中间或是下端。最理想的连接方式是，通道在管状腔室的上端连通多个管状腔室，这种联通方式有利于抑制不同管状腔室之间的物质在不希望混合的情况下混合。
进一步的技术方案是，管状腔室是柱形或锥形，其内直经在0.1毫米至10毫米之间，管壁厚度在0.05至5毫米之间；管状腔室的深度与内直径的比例大于或等于2。
采用柱形或锥形的管状腔室，有利于保持管状腔室温度均匀或形成恒定的温度梯度。管状腔室的内直径、壁厚以及厚度与内直径的比例在以上范围内即可满足一般的反应需要并保持安全稳定，节省成本。
进一步的技术方案是，反应容器还包括位于相邻的管状腔室之间且向通道凸起的弧形连接部。
相邻的管状腔室之间通过弧形连接部连接，可以避免形成死角，使得不同的管状腔室之间产物分子在通道的介质中传送更加顺畅。
进一步的技术方案是，亲和性物质包括染料。
亲和性物质可以是现有的任何能够与核酸产物结合并形成可检测信号的物质，例如染料，特别是荧光染料，荧光染料与核酸产物结合后可以形成荧光信号。亲和性物质还可以是一些表面活性剂、金属配合物、蛋白质等能与产物结合的物质。
进一步的技术方案是，可检测信号包括光学信号或电学信号。更进一步的技术方案是，光学信号包括荧光信号、光吸收信号、红外吸收信号、拉曼散射信号或化学发光信号。
通过不同的亲和性物质与产物结合，可以产生光学信号或电学信号。对于透明的反应容器，通过光学信号进行定性或定量测定是有利的，可以进行实时定量测定。光学信号可以通过现有的光学信号检测仪器对其进行检测。
 有益效果
本发明的核酸测定方法无需探针，不依赖聚合酶外切活性，不受靶分子长度限制，保证封闭性，操作便捷简单。
 附图说明
图1是本发明测定方法实施例使用的反应容器的结构示意图。
图2是本发明测定方法实施例使用的反应容器的一种封闭方式的结构示意图。
图3是本发明测定方法实施例的反应过程示意图。
图4是本发明测定方法实施例的结果分析图。
 本发明的实施方式
以下结合附图和实施例对本发明的核酸测定方法作进一步说明。
本实施例的核酸测试方法通过可封闭的反应容器实施。其中一种可封闭的反应容器如图1所示，反应容器1包括按序排列的至少两个管状腔室11，可以按次序依次将其命名为第一管状腔室至第n管状腔室，其中n等于2或大于2。
不同的管状腔室11可进行多步反应中不同的反应，管状腔室11的数目可以根据实际反应的需要而定。管状腔室11可以由一个或多个通道12在管状腔室11的上端、中间或下端连通，通道12可使反应产物在封闭条件下在管状腔室11之间传输，以实现多步生物酶反应。具体在本实施例中，多个管状腔室11在管状腔室11的上端由一个通道12连通，这种上端连通的方式有利于防止不同管状腔室11之间的反应物料不必要地混合。通道12可以根据需要设计成不同的形状，例如管状、渠状等。
在本实施例中，管状腔室11的内直径在0.1毫米至10毫米之间，管壁厚度在0.05至5毫米之间；管状腔室11的深度与内直径的比例大于或等于2。管状腔室11的尺寸在上述范围内，即可满足一般多步生物酶反应的需要，且安全稳定，节省成本。
反应容器1还可以包括开口13，优选地开口13位于通道12的上端。开口13可以采用现有的任何方式进行封闭。如图2所示，在本实施例中，开口13可以与盖体2封闭配合，盖体2上设有可用于加样和取样的通孔21。通孔21中可插入密封件3。每一个密封件3都包括可与通孔21配合的密封塞31，并且可以根据需要在密封塞31下端固定地或可活动地连接有可与管状腔室11的至少一部分配合的密封杆32，密封杆32还可以在反应前用于样品采集。
相邻的至少两个管状腔室11之间设置有向通道12内凸起的弧形连接部14，弧形连接部14可以避免死角，有利于相邻的管状腔室11之间产物的传送。此外，为了便于放置反应试剂或样品，反应容器1中还可以设置存储腔室15。
优选地，反应容器1采用透明材料例如塑料、玻璃等制成，因此具有透明的外观，反应结果可以连续实时迅速地通过光学方法进行检测。反应容器1可以采用机械加工成型或注塑一体成型。
将上述的反应容器1应用于核酸检测时，需要在第一个管状腔室内放置样品和第一反应试剂，在第n个管状腔室内放置第n反应试剂，其中n是由2开始的一个或多个正整数。根据巢式PCR的原理，第一反应试剂除了包括本领域公知的聚合酶、核苷酸以及缓冲液等成分，还包括巢式PCR的第一对引物，即外引物。第n反应试剂除了包括本领域公知的聚合酶、核苷酸以及缓冲液等成分，还包括巢式PCR的第n对引物，即内引物，以及亲和性物质。作定量反应时，含靶分子的样品只加入到有外引物的第一反应管中。
反应试剂也可以预先存于管状腔室11内，封闭后进行保存和运输，使用时只须加入待测样品即可。也可以在使用前，再通过手工或自动化方法对管状腔室11填充反应试剂及生物样品。
亲和性物质可以是现有的任何能够与核酸产物结合并形成可检测信号的物质，例如染料，特别是荧光染料，荧光染料与核酸产物结合后可以形成荧光信号。亲和性物质还可以是一些表面活性剂、金属配合物、蛋白质等能与产物结合并产生可检测信号的物质。
通过不同的亲和性物质与产物结合，可以产生光学信号或电学信号。对于透明的反应容器，通过光学信号进行定性或定量测定是有利的，光学信号包括荧光信号、光吸收信号、红外吸收信号、拉曼散射信号或化学发光信号等，可以进行实时检测。
通道12内设置有一种或多种介质，所述介质可以是液体，一个管状腔室11中的产物通过介质传送到后一个管状腔室11。传送方式可以是液体热对流或分子扩散。
在进行核酸测定时，首先将反应容器1封闭，避免产物造成污染。然后控制各个管状腔室的温度进行巢式PCR反应。具体地，在第一个管状腔室中进行巢式PCR的第一扩增反应，第一扩增反应可以独立地进行，再将所得到的产物传送至第二个管状腔室（n=2）。如图3所示，第二个管状腔室（n=2）中进行巢式PCR的第二扩增反应，随着反应的进行，产物的量逐渐增加，产物还可以逐渐地自动地通过通道传送到第三个管状腔室（n=3）中，继续进行下一步扩增反应，以此类推，直至完成所需的多步反应。第n个管状腔室中的产物与亲和性物质结合，产生可检测信号。从第二个管状腔室至第五个管状腔室可以依次逐步地产生可检测信号，且可检测信号随时间逐渐增强。图3中时间是从第一个管状腔室中产物的传送开始算起。
生物酶反应需要进行温度控制。生物酶反应一般在15 ℃至99 ℃之间进行。可使用目前已知的方法来对管形反应器内的生物酶反应进行温度控制，例如利用红外光、热/冷风、冷/热固体或液体物质、电磁感应等。可以把封闭后的反应装置插入温度控制仪器中进行温度控制。根据反应的要求，任一管状腔室11可经受恒定温度或周期变化温度，管型腔室11内也可有均衡的温度或梯度温度。例如，与传统PCR温度控制相似地，采用周期性均温的温控方法时，温度控制仪器的温度在电脑程序的控制下进行周期性变化，如在某一温度下保持数秒钟至数分钟，且管状腔室11完全插入温度控制仪器的加热部分中，在这变温过程中管状腔室11内的液体温度基本是均衡的。又例如，在温度恒定的梯度温度控温方法中，温度控制仪器的温度在电脑程序的控制下保持不变，且管状腔室11仅有部分与温度控制仪器的加热部分接触。当底部被加热时，底部温度会高于顶部温度，此时管状腔室11内的液体会有一温度梯度。由于上部温度低的液体有相对高的密度或比重，上部与下部的液体会产生对流，其效果是带动管状腔室内的分子流动，且经受不同的温度，从而满足不同的酶反应条件要求，达到实现管状腔室11内DNA扩增的目的。管状腔室11的管状结构给仪器设计带来更多的灵活性。
在进行核酸定量测定时，应当同步控制管状腔室的温度，保证第二个及其后管状腔室在任何时候在接收到前一个管状腔室传送的产物时都能够立即进行反应，保证反应条件一致。
利用该方法进行定量测量的原理是，反应进行时，由于前一个管状腔室中的产物分子会有限地通过通道例如通过扩散等方式传送到后一个管状腔室，并成为后一反应的分子底物，所传送的产物分子的量和速度与前一反应的产物量成正比。通过调节或控制反应管之间分子的传输机率，可以调节定量检测的灵敏度和反应的动态范围。
如图4所示，在连续反应的多个管状腔室中，信号与时间的比值和管状腔室的位置之间呈正比，其中信号与时间的比值是指当信号达到在非饱和、即线性增长期的一理想值时信号与所用的时间的比例，该比值采用对数方式表示。当一号样品斜率大于二号样品斜率时，一号样品的靶分子数大于二号样品靶分子数。
本实施例的方法可以应用于各种不同的核酸的定性和定量测试。
例如，可以将该方法用于人类EGFR基因突变测定。具体地，包括以下步骤：
（1）在第一管状腔室中进行巢式PCR外围初步反应，利用DNA聚合酶及一对外引物扩增EGFR酪氨酸激酶域 （TKI domain）突变片段，具体地，使用外围引物对扩增覆盖多类突变序列的exon18-22序列。
在50uL反应体积的第一个管状腔室内加入DNA template，200 µM dNTPs，20 mM Tris-HCl，10 mM (NH 4) 2SO 4，10 mM KCl, 2 mM MgSO4，0.1% Triton®-X-100，1 unit DNA聚合酶，及0.3 µM 下列外引物对：
EGFR OutF1:  5’-CAATG CCATC CACTT GATAG G-3’
EGFR OutR1:  5’-GATCG GCCTC TTCAT GCGAA GG-3’
然后，在第一个管状腔室内先进行同管恒温热对流DNA扩增。同管恒温对流反应15分钟。
（2）通过通道传送第一轮扩增反应产物到第二个管状腔室，并且依次将前一个管状腔室中的产物传送到后一个管状腔室，各个相继的管状腔室内含有特定突变基因测试内引物对 （以T790R 和L858R突变为例）及检测亲合物：
第二个管状腔室进行T790野生型检测，第二管状腔室内含有200 µM dNTPs, 20 mM Tris-HCl, 10 mM (NH 4) 2SO 4, 10 mM KCl, 2 mM MgSO 4, 0.1% Triton®-X-100, 0.25X Cyber Green dye (AAT Bioquest， USA) 及1 unit DNA聚合酶，以及巢式PCR内引物对0.3 µM：
EGFR T790F：5’-ACCTC CACCG TGCAG CTCAT CAC-3’
EGFR 790R： 5’-GTGTT CCCGG ACATA GTCCA -3’。
第三个管状腔室进行M790突变型检测，第三个管状腔室内含有200 µM dNTPs, 20 mM Tris-HCl, 10 mM (NH 4) 2SO 4, 10 mM KCl, 2 mM MgSO 4, 0.1% Triton®-X-100, 0.25X Cyber Green dye (AAT Bioquest，USA) 及1 unit DNA聚合酶，以及巢式PCR内引物对0.3 µM：
EGFR M790F：5’-ACCTC CACCG TGCAG CTCAT CAT-3’
EGFR 790R：5’-GTGTT CCCGG ACATA GTCCA -3’。
第四个管状腔室进行L858野生型检测，第四个管状腔室内含有200 µM dNTPs, 20 mM Tris-HCl, 10 mM (NH 4) 2SO 4, 10 mM KCl, 2 mM MgSO 4, 0.1% Triton®-X-100, 0.25X Cyber Green dye (AAT Bioquest， USA) 及1 unit DNA聚合酶，以及巢式PCR内引物对0.3 µM：
EGFR L858F：5’-GTCAA GATCA CAGAT TTTGG GCT-3’
EGFR OutR1：5’-GATCG GCCTC TTCAT GCGAA GG -3’。
第五个管状腔室进行R858 突变型检测，第五个管状腔室内含有200 µM dNTPs, 20 mM Tris-HCl, 10 mM (NH 4) 2SO 4, 10 mM KCl, 2 mM MgSO 4, 0.1% Triton®-X-100, 0.25X Cyber Green dye (AAT Bioquest， USA) 及1 unit DNA聚合酶，巢式PCR内引物对0.3 µM：
EGFR R858F：5’-GTCAA GATCA CAGAT TTTGG GCG-3’
EGFR OutR1：5’-GATCG GCCTC TTCAT GCGAA GG -3’。
以上第二个管状腔室至第五个管状腔室同步进行恒温对流反应15分钟。
（3）对第二个管状腔室至第五个管状腔室信号进行连续检测，可以实现对样品的靶分子数进行定量测定。并以野生型和突变型比较来判断样品的基因型。
最后需要强调的是，以上仅为本发明的优选实施例，并不用于限制本发明，对于本领域的技术人员来说，本发明可以有各种变化和更改，凡在本发明的精神和原则之内，所做的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。
 工业实用性
由上可见，本发明提供的核酸测定方法将具有多个管状腔室和通道的封闭式反应容器与巢式PCR反应相结合进行定性或定量测定，能够防止产物造成污染，且反应的灵敏度和准确性高，无需使用分子探针，不依赖聚合酶外切活性并且不受靶分子长度限制。本发明的核酸测定方法可以实现多步PCR反应，例如巢式PCR、RT-PCR、多重PCR或者是它们的两种或以上的组合。

Claims (10)

  1. 一种核酸测定方法，其特征在于：
    所述方法通过可封闭的反应容器实施，所述反应容器包括按序排列的至少两个管状腔室以及连通所述管状腔室的至少一个通道；
    其中，第一个管状腔室内设置有样品和第一反应试剂，第n个管状腔室内设置有第n反应试剂，其中n等于或大于2；
    所述方法包括：将所述反应容器封闭，在所述第一个管状腔室中进行第一组反应，所述第一个管状腔室中的产物通过所述通道传送到后一个管状腔室；所述第n个管状腔室接收到前一个管状腔室的产物后，在所述第n个管状腔室中进行第n组反应；对所述第n个管状腔室内的产物进行检测。
  2. 根据权利要求1所述的一种核酸测定方法，所述第一反应试剂包含巢式PCR的第一对引物或RT-PCR的逆转录反应引物或多重PCR的第一组引物；所述第n反应试剂包含巢式PCR或RT-PCR或多重PCR的第n对引物和亲和性物质。
  3. 根据权利要求1或2所述的一种核酸测定方法，其特征在于：
    所述方法还包括对所述管状腔室进行温度控制，所述温度控制包括保持所述管状腔室内恒定的温度梯度或周期性地改变所述管状腔室的整体温度。
  4. 根据权利要求3所述的一种核酸测定方法，其特征在于：
    所述温度控制包括对多个所述管状腔室进行同步温度控制。
  5. 根据权利要求1至4任一项所述的一种核酸测定方法，其特征在于：
    所述通道内设置有一种或多种介质。
  6. 根据权利要求5所述的一种核酸测定方法，其特征在于：
    任意一个所述管状腔室中反应得到的产物在所述通道中通过液体对流或分子扩散的方式传送至后一个管状腔室。
  7. 根据权利要求1至6任一项所述的一种核酸测定方法，其特征在于：
    所述管状腔室是柱形或锥形，其内直经在0.1毫米至10毫米之间，管壁厚度在0.05至5毫米之间；所述管状腔室的深度与内直径的比例大于或等于2。
  8. 根据权利要求1至7任一项所述的一种核酸测定方法，其特征在于：
    所述反应容器还包括位于相邻的所述管状腔室之间且向所述通道内凸起的弧形连接部。
  9. 根据权利要求2所述的一种核酸测定方法，其特征在于：
    所述亲和性物质包括染料。
  10. 根据权利要求1至9任一项所述的一种核酸测定方法，其特征在于：
    可检测信号包括光学信号或电学信号；所述光学信号包括荧光信号、光吸收信号、红外光吸收信号、拉曼散射信号或化学发光信号。
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