The method and its equipment of a kind of very fast nucleic acid amplification and application
Technical field
The present invention relates to for fields such as biology, medicine gene tester and its equipment and application, and in particular to
The method and its equipment of a kind of very fast nucleic acid amplification and application.
Background technique
Polymerase chain reaction (Polymerase Chain Reaction) abbreviation PCR, has become the normal of detection of nucleic acids
Rule technology.One typical PCR program is usually to prolong response sample by high-temperature denaturation-low temperature renaturation-thermophilic repeatedly
Three fundamental reaction steps are stretched to constitute, i.e., 90-95 DEG C high-temperature process 10-30 seconds or so, be then cooled to 50- in different ways
72 DEG C or so maintenances 30-60 seconds are warming up to after 65 DEG C, maintenance about 10-30 seconds, and so on 30-40 period.According to " molecule gram
Grand experiment guide " the tri- step cyclic amplification process procedures of typical PCR recorded in a book are usual are as follows: 94 DEG C 30 seconds, 55 DEG C 30 seconds,
72 DEG C 1 minute, recycle 30 times.The amplification method of two step circulations can also be carried out for short-movie section, such as 94 DEG C 30 seconds, 60 DEG C 30
Second.In entire reaction process, the actual experiment time further includes the change of time needed for reaction block heating and cooling process, current amplification instrument
Warm rate is mostly within 3 DEG C/sec, and a heating and cooling circulation is taken time altogether altogether on about 30 seconds, usual one complete PCR process
It is more than hour to generally require one.And in fact, wherein to reacting liquid temperature in PCR reaction itself most importantly PCR pipe, only
Normal reaction can be carried out by reaching temperature in the PCR pipe of requirement.
There are many present heating and cooling modes, such as the heating of gas heating, water and electric heating block, semiconductor piece heating and cooling etc., and
And by semiconductor, automatic amplification and detection are realized in heating and cooling to mainstream PCR instrument repeatedly, but most fast temperature rate does not also surpass
3 DEG C/sec are crossed, and the rate temperature change of inner reaction tube liquid is then lower, therefore be restricted on PCR all times.
It is widely applied as round pcr is continuously available, different types of pattern detection and sample is examined using round pcr
Measurement increases, so that more stringent requirements are proposed to analysis efficiency and cost.Therefore, further shorten the PCR amplification time, hair
Fast PCR technology is opened up, the timeliness of quick detection and particular sample such as clinical sample for large sample size has particularly significant
Meaning.
In hospital or laboratory, PCR detection method is the very effective means for being frequently necessary to use.Under special circumstances,
It is higher and higher due to requiring the timeliness of diagnosis especially in hospital, more require PCR it is efficient in time.But due to mesh
Preceding PCR process is longer there are still the reaction time, and needs batch reaction i.e. period longer problem, to wait a batch anti-
The reaction that should complete could to start next batch, causes PCR instrument service efficiency not high in this way, and influences very much work effect
Rate.It therefore, is the realization of the method for adaptation rapid amplifying, it is also desirable to be equipped with suitable equipment.
Summary of the invention
To overcome above-mentioned technological deficiency, the object of the present invention is to provide a kind of method of very fast nucleic acid amplification and its equipment and
Using.By reacting the larger temperature difference of goal response temperature in deblocking temperature and PCR pipe outside setting PCR pipe, and directly pass through machinery
PCR pipe is transferred on respective reaction block by movement, in this way, promoting temperature conduction in reaction tube to accelerate by the big temperature difference, is contracted significantly
Gradient of temperature temperature rate in short PCR pipe, time needed for making PCR process greatly shorten, and improve the utilization rate of PCR instrument;And
The chain type arrangement and the design of progressive mode that pore is reacted by thermal module avoid that a collection of PCR can only be carried out, thus real
Sample and reaction now can be directly added at any time.
To achieve the above object, it adopts the following technical scheme that:
The method of very fast nucleic acid amplification of the invention is that the reaction tube equipped with reaction mixture is placed in superhigh temperature reaction zone
With ultralow temperature reaction zone, 30-45 circulation, the temperature of the superhigh temperature reaction zone are carried out are as follows: one in 100 DEG C -150 DEG C is solid
Determine temperature or temperature range, temperature spot is selected according to the difference of tubing, to ensure inner reaction tube liquid in pipe actual temperature 88
DEG C -95 DEG C of sections can maintain that DNA fragmentation double-strand is made within 1-3 seconds to complete denaturation;The temperature of the ultralow temperature reaction zone is 10 DEG C -40
A fixed temperature or temperature range in DEG C, temperature spot select the difference according to tubing, to ensure to react liquid in pipe practical
Temperature can maintain the 2-4 seconds renaturation for completing template and primer in 55 DEG C of -65 DEG C of sections, while complete during rapid temperature rise and drop
DNA chain extends and fluorescence detection;The program of the circulation are as follows: reacted 4-10 seconds in the superelevation warm area, then described
It reacts 4-10 seconds ultra-low temperature region;Carry out 30-45 circulation, completion nucleic acid amplification detected in ultra-short Time in 8-15 minutes
Journey.
Preferably, the reaction tube is polymerase chain reaction (PCR) pipe of polypropylene PP material or the PCR of glass material
Reaction tube.
Preferably, the reaction mixture includes polymerase, amplimer, dNTP, PCR buffer and sterilizing distilled water.
Second object of the present invention is to disclose a kind of reactor for very fast nucleic acid amplification method, the reactor
Including superhigh temperature reaction zone and ultralow temperature reaction zone;The temperature regulating range of the superhigh temperature reaction zone is in 100 DEG C -150 DEG C
A fixed temperature or temperature range;The temperature regulating range of the ultralow temperature reaction zone is that one in 10 DEG C -40 DEG C is solid
Determine temperature or temperature range.
Preferably, the ultralow temperature reaction zone and superhigh temperature reaction zone are by two deblocking reaction conduit saddle bath temperature control metal module structures
At the assembly model of the two temperature-controlling metals module is that concentric loop, parallel stick or gear sample intersect mutually embedding mode, described anti-
It answers and is provided with reaction pore on conduit saddle bath temperature control metal module, the quantity of the reaction pore is 1-45, and reaction tube is in difference
It moves in temperature control module and is automatically performed with manipulator, fluorescence detection program is completed in low temperature.Reactor include manually, half from
It is dynamic, full-automatic.
Preferably, the reactor includes low-temp reaction ring, pyroreaction disk, heat insulating belt, cover board, rotary shaft and stepping electricity
Machine;The low-temp reaction ring and the same center of circle of pyroreaction disk;It is anti-that the diameter of the low-temp reaction ring is greater than the high temperature
The diameter that should be coiled;The outer edge of the pyroreaction disk is gear sample;The inside edge of the low-temp reaction ring is gear sample;It is described
It is mutually separated with the heat insulating belt and be mutually twisted to form complete circular discs between low-temp reaction ring and the pyroreaction disk,
The low-temp reaction ring forms gear sample lines of occlusion with the pyroreaction disk place of occlusion;Along disk circumference in the lines of occlusion
Same circumference on distribution samples pore;Sample pore sum can be 60-90.The low-temp reaction ring and the high temperature
The position of reaction tray is fixed;The cover board is connected with the rotary shaft, the axle center of the rotary shaft and the low-temp reaction ring or
The center of circle concentric of pyroreaction disk;The cover board is connect by the rotary shaft with the stepper motor;The low-temp reaction
Ring and pyroreaction disk are located at the lower section of the cover board;One in 10 DEG C -40 DEG C of the temperature setting of the low-temp reaction ring
Fixed temperature or temperature range;A fixed temperature or temperature of the temperature setting of the pyroreaction disk in 100 DEG C -150 DEG C
Spend section.
Preferably, the side perimeters of the cover board are provided with T-slot open card, the T-slot open card and the sample
Pore is corresponding.
Preferably, the bottom of the sample pore is provided with the hole location for installing laser diode;The low-temp reaction
Sample pore on ring is provided with the hole location for installing optical fiber.
Third object of the present invention is to disclose a kind of detection of nucleic acids equipment, and the equipment includes above-mentioned reactor.
Fourth object of the present invention is to disclose the nucleic acid amplification being used in above-mentioned nucleic acid amplification method based on PCR
In analysis method.Preferably, above-mentioned application includes real-time fluorescence quantitative PCR, multiplex PCR, RT-PCR, nest-type PRC and sequencing
Deng.
5th purpose of the invention be disclose a kind of operating system for above-mentioned very fast nucleic acid amplification method, including
High-temperature control component, low temperature control unit and fluorescence reading control unit and computerized control system, the computerized control system point
It is not connect with the high-temperature control component, low temperature control unit, fluorescence reading control unit.
Compared with prior art, nucleic acid amplification method of the invention has carried out innovative improvement to traditional nucleic acid amplification,
Change the triphasic reaction pattern of normal PCR, be utilized in advance fix required temperature and can batch reaction mode, proposition
The PCR operating mode of pipeline system improves the utilization rate of PCR equipment, saves the time of user, realizes with to make with survey
The system of obtaining has greater flexibility and applicability.It uses extreme temperature difference rank to improve rate temperature change, is considerably reduced
Inner reaction tube liquid reaches the time required for optimum temperature, the purpose of Rapid nucleic acid detection is realized, so that nucleic acid detection technique
Wider application can be obtained.
Detailed description of the invention
Fig. 1 show length be respectively the amplified fragments Standard PCR of 70bp, 100bp, 150bp, 180bp and at 100 DEG C and
The pcr amplification product electrophoresis result figure that 10 DEG C of extreme ambient temperatures mediate, wherein M swimming lane is molecular weight marker, swimming lane
1 is Standard PCR product electrophoresis result, and swimming lane 2 is the PCR product electrophoresis result that extreme ambient temperature mediates;
Fig. 2 show length be respectively the amplified fragments Standard PCR of 70bp, 100bp, 150bp, 180bp and at 130 DEG C and
The pcr amplification product electrophoresis result figure that 20 DEG C of extreme ambient temperatures mediate, wherein M swimming lane is molecular weight marker, swimming lane
1 is Standard PCR product electrophoresis result, and swimming lane 2 is the PCR product electrophoresis result that extreme ambient temperature mediates;
Fig. 3 shows that length is respectively the Standard PCR of the amplified fragments of 70bp, 100bp, 150bp, 180bp and at 150 DEG C
The pcr amplification product electrophoresis result figure mediated with 40 DEG C of extreme ambient temperatures, wherein M swimming lane is molecular weight marker, swimming
Road 1 is Standard PCR product electrophoresis result, and swimming lane 2 is the PCR product electrophoresis result that extreme ambient temperature mediates;
Fig. 4 is the schematic top plan view of a preferred embodiment of very fast nucleic acid amplification reactor of the invention;
Fig. 5 is the schematic side view of a preferred embodiment of very fast nucleic acid amplification reactor of the invention.
Specific embodiment
To keep the present invention easier to understand with reference to specific embodiments the present invention is further explained.It should be understood that this
A little examples are only for illustrating the present invention and not for limiting the scope of the present invention, unmentioned specific experiment in the following example
Method is usually carried out according to routine experiment method.
The preliminary test of influence of 1 high ambient temperatures of embodiment to the warm variable Rate of heating
It carries out environment temperature and the preliminary test that efficiency influences is become on temperature, concrete operations are as follows:
The temperature that PCR instrument reaction hole slot is arranged is 100 DEG C, with the initial temperature of point thermometer test PCR reaction liquid in pipe
It is 60 DEG C, this reaction tube is placed in PCR instrument reaction hole slot, it is time-consuming when the temperature of PCR reaction liquid in pipe reaches 92 DEG C
14 seconds.
The temperature that PCR instrument reaction hole slot is arranged is 95 DEG C, and the initial temperature with point thermometer test PCR reaction liquid in pipe is
60 DEG C, this reaction tube is placed in PCR instrument reaction hole slot, when the temperature of PCR reaction liquid in pipe reaches 92 DEG C, time-consuming 22
Second.
The temperature of special heating instrument module reaction hole slot is set as 130 DEG C, with point thermometer test PCR reaction liquid in pipe
Initial temperature is 60 DEG C, this reaction tube is placed in PCR instrument reaction hole slot, when the temperature of PCR reaction liquid in pipe reaches 92
DEG C when, time-consuming 6 seconds.
The temperature of special heating instrument module reaction hole slot is set as 150 DEG C, with point thermometer test PCR reaction liquid in pipe
Initial temperature is 60 DEG C, this reaction tube is placed in PCR instrument reaction hole slot, when the temperature of PCR reaction liquid in pipe reaches 92
DEG C when, time-consuming 5 seconds.
By above-mentioned test, it is concluded that in the case where 150 DEG C of environment temperatures, when being risen to by 60 DEG C to 92 DEG C,
Warm variable Rate is 6.4 DEG C/sec;In the case where 130 DEG C of environment temperatures, when being risen to by 60 DEG C to 92 DEG C, warm variable Rate is 5.3
DEG C/sec;In the case where 100 DEG C of environment temperatures, when being risen to by 60 DEG C to 92 DEG C, warm variable Rate is 2.13 DEG C/sec;In 95 DEG C of rings
In the case where the temperature of border, when being risen to by 60 DEG C to 92 DEG C, warm variable Rate is 1.45 DEG C/sec.
Conclusions illustrate when the reaction solution to identical initial temperature carries out the heating of same gradient, environment temperature and anti-
Answer liquid initial temperature difference bigger, then the warm variable Rate actually to heat up in reaction solution is faster.
The preliminary test of influence of the 2 low temperature environment temperature of embodiment to the warm variable Rate of cooling
It carries out environment temperature and the preliminary test that efficiency influences is become on temperature, concrete operations are as follows:
The temperature that PCR instrument reaction hole slot is arranged is 10 DEG C, and the initial temperature with point thermometer test PCR reaction liquid in pipe is
92 DEG C, this reaction tube is placed in PCR instrument reaction hole slot, when the temperature of PCR reaction liquid in pipe drops to 60 DEG C, time-consuming 4
Second.
The temperature that PCR instrument reaction hole slot is arranged is 20 DEG C, and the initial temperature with point thermometer test PCR reaction liquid in pipe is
92 DEG C, this reaction tube is placed in PCR instrument reaction hole slot, when the temperature of PCR reaction liquid in pipe drops to 60 DEG C, time-consuming 5
Second.
The temperature that PCR instrument reaction hole slot is arranged is 40 DEG C, and the initial temperature with point thermometer test PCR reaction liquid in pipe is
92 DEG C, this reaction tube is placed in PCR instrument reaction hole slot, when the temperature of PCR reaction liquid in pipe drops to 60 DEG C, time-consuming 8
Second.
The temperature that PCR instrument reaction hole slot is arranged is 45 DEG C, and the initial temperature with point thermometer test PCR reaction liquid in pipe is
92 DEG C, this reaction tube is placed in PCR instrument reaction hole slot, when the temperature of PCR reaction liquid in pipe drops to 60 DEG C, time-consuming 11
Second.
The temperature that PCR instrument reaction hole slot is arranged is 60 DEG C, and the initial temperature with point thermometer test PCR reaction liquid in pipe is
92 DEG C, this reaction tube is placed in PCR instrument reaction hole slot, when the temperature of PCR reaction liquid in pipe drops to 60 DEG C, time-consuming 45
Second.
By above-mentioned test, it is concluded that in the case where 10 DEG C of environment temperatures, when being down to by 92 DEG C to 60 DEG C,
Practical temperature variable Rate is 8 DEG C/sec;In the case where 20 DEG C of environment temperatures, when being down to by 92 DEG C to 60 DEG C, practical temperature variable Rate is
6.4 DEG C/sec;In the case where 40 DEG C of environment temperatures, when being down to by 92 DEG C to 60 DEG C, practical temperature variable Rate is 4 DEG C/sec;45
In the case where DEG C environment temperature, when being down to by 92 DEG C to 60 DEG C, practical temperature variable Rate is 2.9 DEG C/sec;In 60 DEG C of environment temperatures
In the case of, when being down to by 92 DEG C to 60 DEG C, practical temperature variable Rate is 0.7 DEG C/sec.
Conclusions illustrate when the reaction solution to identical initial temperature carries out the cooling of same gradient, when environment temperature with
Reaction solution initial temperature difference is bigger, then the warm variable Rate to cool down is faster.
One preferred embodiment of the comparative experiments of the very fast PCR method of embodiment 3 and conventional PCR method
It is expanded using four groups of difference short-movie sections of hepatitis B (HBV) gene as target fragment, using the very fast side PCR
Method is compareed with conventional PCR method.
1, four groups of target amplification sequences and corresponding forward primer and reverse primer sequences are as follows:
First group of target amplification sequence SEQ ID NO:1:
5’-CCCCAACCTCCAATCACTCACCAACCTCTTGTCCTCCAATTTGT
CCTGGTTATCGCTGGATGTGTCTGCG-3’
First group of forward primer SEQ ID NO:2:5 '-CCCCAACCTCCAATCACTCA-3 '
First group of reverse primer SEQ ID NO:3:5 '-CGCAGACACATCCAGCGATAAC-3 '
Second group of target amplification sequence SEQ ID NO:4:
5’-CCCCAACCTCCAATCACTCACCAACCTCTTGTCCTCCAATTTGT CCTGGTTATCGCTGGATGTG
TCTGCGGCGTTTTATCATCTTCCTCTTCAT CCTGCT-3’
Second group of forward primer SEQ ID NO:5:5 '-CCCCAACCTCCAATCACTCA-3 '
Second group of reverse primer SEQ ID NO:6:
5’-AGCAGGATGAAGAGGAAGATGATAAA-3’
Third group target amplification sequence SEQ ID NO:7:
5’-CCCCAACCTCCAATCACTCACCAACCTCTTGTCCTCCAATTTGT CCTGGTTATCGCTGGATGTG
TCTGCGGCGTTTTATCATCTTCCTCTTCAT CCTGCTGCTATGCCTCATCTTCTTGTTGGTTCTTCTGGACTACCA
AGGT ATGTTGC-3’
Third group forward primer SEQ ID NO:8:5 '-CCCCAACCTCCAATCACTCA-3 '
Third group reverse primer SEQ ID NO:9:
5’-GCAACATACCTTGGTAGTCCAGAAG-3’
4th group of target amplification sequence SEQ ID NO:10:
5’-CCCCAACCTCCAATCACTCACCAACCTCTTGTCCTCCAATTTGT CCTGGTTATCGCTGGATGTG
TCTGCGGCGTTTTATCATCTTCCTCTTCAT CCTGCTGCTATGCCTCATCTTCTTGTTGGTTCTTCTGGACTACCA
AGGT ATGTTGCCCGTTTGTCCTCTACTTCCAGGAACATCAA-3’
4th group of forward primer SEQ ID NO:11:5 '-CCCCAACCTCCAATCACTCA-3 '
4th group of reverse primer SEQ ID NO:12:
5’-TTGATGTTCCTGGAAGTAGAGGACA-3’
2, PCR reaction system reaction solution is formulated as follows, wherein PCR reaction buffer is selected from the product of TAKARA company:
3, routine PCR reaction program is as follows:
4, the PCR experiment operation that extreme ambient temperature mediates is as follows:
The reaction environment temperature bracket for using independent high temperature module, refrigerated module to react as PCR, wherein high temperature mould
The temperature setting of block is 100 DEG C, and the temperature setting of refrigerated module is 10 DEG C.
The temperature that reaction solution in PCR pipe is measured in PCR reaction tube is placed in point thermometer.
PCR pipe is placed in independent 100 DEG C of high temperature slots by the experimental result that reference implementation example 1 and 2 obtains, and measures PCR
Inner reaction tube liquid temperature rises to 92 DEG C (± 3 DEG C) from 60 DEG C (± 3 DEG C), needs time-consuming 14s;PCR pipe is placed in independent 10
In DEG C cryostat, reacting liquid temperature is measured in PCR pipe from 92 DEG C (± 3 DEG C) and is down to 60 DEG C (± 3 DEG C), needs time-consuming 4s.Therefore,
The response procedures for the PCR experiment that the extreme ambient temperature that we design mediates are as follows:
The temperature of reaction solution in PCR pipe during PCR pipe is placed in 100 DEG C of independent high temperature slots, the highest temperature be 92 DEG C (±
3 DEG C), during being placed in 10 DEG C of independent high temperature slots, lowest temperature is 60 DEG C (± 3 DEG C).
Move PCR reaction tube between high temperature slot and cryostat, by manually mode, so that anti-in PCR reaction tube
Answer liquid actual temperature iterative cycles, the amplification to realize target gene segment in 60-92 DEG C (± 3 DEG C).
Experimental result shows, with conventional PCR method, the used time 25 minutes about 1 hour, and the PCR mediated with extreme ambient temperature
Reaction, the used time about 10 minutes.The expanding effect of two methods is detected with 2% agarose gel electrophoresis, and electrophoresis result is shown in figure
1, Fig. 1 shows that length is respectively the amplified fragments Standard PCR of 70bp, 100bp, 150bp, 180bp and in 100 DEG C and 10 DEG C of poles
The pcr amplification product electrophoresis result figure for holding environment temperature to mediate, wherein M swimming lane is molecular weight marker, and swimming lane 1 is normal
PCR product electrophoresis result is advised, swimming lane 2 is the PCR product electrophoresis result that extreme ambient temperature mediates.
By Fig. 1 result as it can be seen that the PCR amplification detection process that extreme ambient temperature mediates can be i.e. complete at 11 minutes or so
At, and its expanding effect can achieve standard PCR amplification and detect similar effect.
Second preferred embodiment of the comparative experiments of the very fast PCR method of embodiment 4 and conventional PCR method
Reagent system, template, primer, Standard PCR control experiment are the same as embodiment 3.
The response procedures slight changes of experiment are as follows:
The temperature of reaction solution in PCR pipe during PCR pipe is placed in 130 DEG C of independent high temperature slots, the highest temperature be 92 DEG C (±
3 DEG C), when being placed in during 20 DEG C of independent cryostat,s, lowest temperature is 60 DEG C (± 3 DEG C).
Move PCR reaction tube between high temperature slot and cryostat, by manually mode, so that anti-in PCR reaction tube
Answer liquid actual temperature iterative cycles, the amplification to realize target gene segment in 60-92 DEG C (± 3 DEG C).
Experimental result is shown, with conventional PCR method, the used time 25 minutes about 1 hour, is mediated with this condition extreme ambient temperature
PCR reaction, the used time about 8 minutes.The expanding effect of two methods is detected with 2% agarose gel electrophoresis, electrophoresis result
See that Fig. 2, Fig. 2 show that length is respectively the amplified fragments Standard PCR of 70bp, 100bp, 150bp, 180bp and at 120 DEG C and 25
The pcr amplification product electrophoresis result figure that DEG C extreme ambient temperature mediates, wherein M swimming lane is molecular weight marker, swimming lane 1
For Standard PCR product electrophoresis result, swimming lane 2 is the PCR product electrophoresis result that extreme ambient temperature mediates.
By Fig. 2 result as it can be seen that the PCR amplification detection process that extreme ambient temperature mediates can be completion in 8 minutes, and
And its expanding effect can achieve standard PCR amplification and detect approximate effect.
The third preferred embodiment of the comparative experiments of the very fast PCR method of embodiment 5 and conventional PCR method
Reagent system, template, primer, Standard PCR control experiment are the same as embodiment 3.
The response procedures slight changes of experiment are as follows:
The temperature of reaction solution in PCR pipe during PCR pipe is placed in 150 DEG C of independent high temperature slots, the highest temperature be 92 DEG C (±
3 DEG C), when being placed in during 20 DEG C of independent cryostat,s, lowest temperature is 60 DEG C (± 3 DEG C).
Move PCR reaction tube between high temperature slot and cryostat, by manually mode, so that anti-in PCR reaction tube
Answer liquid actual temperature iterative cycles, the amplification to realize target gene segment in 60-92 DEG C (± 3 DEG C).
Experimental result shows, with conventional PCR method, the used time 25 minutes about 1 hour, and the PCR mediated with extreme ambient temperature
Reaction, the used time about 11.5 minutes.The expanding effect of two methods is detected with 2% agarose gel electrophoresis, and electrophoresis result is shown in
Fig. 3, Fig. 3 show that length is respectively the Standard PCR of the amplified fragments of 70bp, 100bp, 150bp, 180bp and at 100 DEG C and 30
The pcr amplification product electrophoresis result figure that DEG C extreme ambient temperature mediates, wherein M swimming lane is molecular weight marker, swimming lane 1
For Standard PCR product electrophoresis result, swimming lane 2 is the PCR product electrophoresis result that extreme ambient temperature mediates.
By Fig. 3 result as it can be seen that the PCR amplification detection process that extreme ambient temperature mediates can be completed at 11.5 minutes,
And its expanding effect can achieve standard PCR amplification and detect approximate effect.
By the result of embodiment 3-5 as it can be seen that the PCR method that mediates of conventional PCR method and extreme ambient temperature is to Bu Tong big
Small short genetic fragment can effectively realize PCR amplification;Extreme ambient temperature mediates PCR method that can realize height in 8-12min
Effect, supper-fast amplification, used time can shorten within about 1/10th of Standard PCR.It can invent accordingly, in specific experiment
Selection temperature and time is combined with placement duration according to high and low warm area temperature spot to combine, and reaches control reaction inner reaction tube
Liquid temperature 60-92 DEG C (± 3 DEG C) and realize target fragment amplification purpose.
One preferred embodiment of the very fast nucleic acid amplification reactor of the invention of embodiment 6
The present embodiment is a preferred embodiment of very fast nucleic acid amplification reactor of the invention, structure such as Fig. 4 and Fig. 5
It is shown.Fig. 4 is the schematic top plan view of a preferred embodiment of very fast nucleic acid amplification reactor of the invention;Fig. 5 is the present invention
Very fast nucleic acid amplification reactor a preferred embodiment schematic side view.From fig. 4, it can be seen that nucleic acid amplification of the invention
Reactor includes low-temp reaction ring 1, pyroreaction disk 2, heat insulating belt 3, cover board 4, rotary shaft 5 and stepper motor 6;The low temperature is anti-
Answer ring 1 and the same center of circle of the pyroreaction disk 2;The diameter of the low-temp reaction ring 1 is greater than the straight of the pyroreaction disk 2
Diameter;The outer edge of the pyroreaction disk 2 is gear sample;The inside edge of the low-temp reaction ring 1 is gear sample;The low temperature is anti-
It answers mutually isolated with the heat insulating belt 3 between ring 1 and the pyroreaction disk 2 and is mutually twisted to form complete circular discs, institute
It states low-temp reaction ring 1 and forms gear sample lines of occlusion with 2 place of occlusion of pyroreaction disk;Along disk circumference in the lines of occlusion
Same circumference on distribution samples pore 7;The position of the low-temp reaction ring 1 and pyroreaction disk 2 is fixed;The cover board 4
It is connected with the rotary shaft 5, the axle center of the rotary shaft 5 is located at the center of circle of the low-temp reaction ring 1 or pyroreaction disk 2;Institute
It states cover board 4 and is connect by the rotary shaft 5 with the stepper motor 6;The low-temp reaction ring 1 and pyroreaction disk 2 are located at institute
State the lower section of cover board 4;A fixed temperature or temperature range of the temperature setting of the low-temp reaction ring 1 in 10 DEG C -40 DEG C;
A fixed temperature or temperature range of the temperature setting of the pyroreaction disk 2 in 100 DEG C -150 DEG C.The cover board 4
Side perimeters are provided with T-slot open card 41, and the T-slot open card 41 is corresponding with the sample pore 7.The sample cell
The bottom in hole 7 is provided with the hole location 8 for installing laser diode;The sample pore 7 is provided with for installing optical fiber
Hole location 9.
Sample pore 7 can be 60-90, in this way, adjacent sample cup aperture is physically located on different humidity provinces.Such as
As soon as one sample of fruit is moved into another adjacent hole from hole, from a humidity province to another humidity province.If
This sample circumferentially moves 60 hole locations, then this sample just experienced the period of 30 high and low temperatures variation, also
It is to say the overall process for completing a PCR reaction environment.
Sample cup for liquid circumferentially successively from a hole to adjacent hole movement be by lid rotatable and moving up and down
Plate is realized.Fig. 5 is the schematic side view of a preferred embodiment of very fast nucleic acid amplification reactor of the invention, in conjunction with figure
5, which further includes cover board 4 and rotary shaft 5 in addition to low-temp reaction ring 1, pyroreaction disk 2 and heat insulating belt 3, cover board 4 and rotation
Shaft 5 is connected, and rotary shaft 5 is located at the center of circle of low-temp reaction ring 1 or pyroreaction disk 2;Low-temp reaction ring 1 and pyroreaction disk 2
Positioned at the lower section of cover board 4;The side perimeters of cover board 4 are provided with 40 T-slot open cards 41, and sample cell 71 is open by T-slot
Card 41 is stuck on cover board 4.T-slot open card 41 is corresponding with sample pore 7;Sample cell 71 is mounted on the T of 4 side perimeters of cover board
In type groove open card 41, cover board 4 is followed to do circumference stepwise operation and up and down action, after 1-6 seconds, cover board 4 is moved up, will
All samples pipe 71 extracts sample pore 7.Cover board 4 rotates a hole location, moves still further below.In this way, all samples pipe 71 is with regard to one
It rises and has replaced temperature environment.Moving up and down for cover board 4 can be driven by stepper motor 6 by preset program with stepping moving in rotation.
Cover board 4 carries sample cell 71 and makees to act once with circular motion, every five seconds up and down, and it is the equal of 20 that 40 movements, which rotate a circle,
Temperature cycles.After this sample cell 71 has been completed 30 temperature cycles, it will be removed from cover board.
7 bottom of sample pore is provided with 30 hole locations 8 for installing laser diode;Sample on low-temp reaction ring 1
The side in quality control hole 7 is provided with 30 hole locations 9 for installing optical fiber, for reading signal.It is stopped in sample cell 71
When in low-temperature sample hole, laser tube issues exciting light, and the sample in stimulated samples pipe 71 issues possible fluorescence, possible glimmering
Light is captured by optical fiber and is transmitted to photoelectric detection part.
Finally, it should be noted that the above embodiments are merely illustrative of the technical solutions of the present invention rather than protects to the present invention
The limitation of range is protected, although the invention is described in detail with reference to the preferred embodiments, those skilled in the art should
Understand, it can be with modification or equivalent replacement of the technical solution of the present invention are made, without departing from the essence of technical solution of the present invention
And range.
Sequence table
<120>a kind of method and its equipment and application of very fast nucleic acid amplification
<160> 12
<210> 1
<211> 70
<212> DNA
<213> Artificial Sequence
<400> 1
ccccaacctc caatcactca ccaacctctt gtcctccaat ttgtcctggt tatcgctgga 60
tgtgtctgcg 70
<210> 1
<211> 20
<212> DNA
<213> Artificial Sequence
<400> 2
ccccaacctc caatcactca 20
<210> 1
<211> 22
<212> DNA
<213> Artificial Sequence
<400> 3
cgcagacaca tccagcgata ac 22
<210> 1
<211> 100
<212> DNA
<213> Artificial Sequence
<400> 4
ccccaacctc caatcactca ccaacctctt gtcctccaat ttgtcctggt tatcgctgga 60
tgtgtctgcg gcgttttatc atcttcctct tcatcctgct 100
<210> 1
<211> 20
<212> DNA
<213> Artificial Sequence
<400> 5
ccccaacctc caatcactca 20
<210> 1
<211> 26
<212> DNA
<213> Artificial Sequence
<400> 6
agcaggatga agaggaagat gataaa 26
<210> 1
<211> 150
<212> DNA
<213> Artificial Sequence
<400> 7
ccccaacctc caatcactca ccaacctctt gtcctccaat ttgtcctggt tatcgctgga 60
tgtgtctgcg gcgttttatc atcttcctct tcatcctgct gctatgcctc atcttcttgt 120
tggttcttct ggactaccaa ggtatgttgc 150
<210> 1
<211> 20
<212> DNA
<213> Artificial Sequence
<400> 8
ccccaacctc caatcactca 20
<210> 1
<211> 25
<212> DNA
<213> Artificial Sequence
<400> 9
gcaacatacc ttggtagtcc agaag 25
<210> 1
<211> 180
<212> DNA
<213> Artificial Sequence
<400> 10
ccccaacctc caatcactca ccaacctctt gtcctccaat ttgtcctggt tatcgctgga 60
tgtgtctgcg gcgttttatc atcttcctct tcatcctgct gctatgcctc atcttcttgt 120
tggttcttct ggactaccaa ggtatgttgc ccgtttgtcc tctacttcca ggaacatcaa 180
<210> 1
<211> 20
<212> DNA
<213> Artificial Sequence
<400> 11
ccccaacctc caatcactca 20
<210> 1
<211> 25
<212> DNA
<213> Artificial Sequence
<400> 12
ttgatgttcc tggaagtaga ggaca 25