CN106676000A - Multi-cavity gene analysis reaction device - Google Patents
Multi-cavity gene analysis reaction device Download PDFInfo
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- CN106676000A CN106676000A CN201611259512.5A CN201611259512A CN106676000A CN 106676000 A CN106676000 A CN 106676000A CN 201611259512 A CN201611259512 A CN 201611259512A CN 106676000 A CN106676000 A CN 106676000A
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Abstract
The invention provides a multi-cavity gene analysis reaction device. The multi-cavity gene analysis reaction device is simple in structure and easy to operate, and is energy-saving and environment-friendly. The multi-cavity gene analysis reaction device is structurally formed by connecting a main pipe a, a branch pipe b, a cavity c and a blockage substance d according to certain order, the cavity c comprises a nucleic acid amplified reaction component, and is used for nucleic acid amplification reaction and gene analysis. The multi-cavity gene analysis reaction device can use one single reaction device to complete high throughput qualitative or quantitative analysis of 10-50 genes at a time, and is suitable for being applied to the fields of fundamental research of molecular biology, clinical molecular diagnosis, food safety gene detection, and bioterrorism attack gene detection.
Description
Technical field
The present invention relates to a kind of multi-chamber gene analysiss reaction unit, belongs to biotechnology and mechano-electronic crossing domain.
Background technology
The multi-fluorescence analysis of gene starts in biology field application from the nineties in last century, it is typical that fluorescence
Polymerase chain reaction（Polymerase Chain Reaction, PCR）Technology, also referred to as real-time fluorescence PCR technology, it is based on
FRET (fluorescence resonance energy transfer)（Fluorescence Resonance Energy Transfer, FRET）Principle, expanded in PCR
Fluorophor or fluorescent probe are added in journey, using the whole PCR processes of fluorescence signal accumulation real-time monitoring, finally to unknown gene
The method that template carries out quantitative or qualitative analyses, carrying out the fluorescent PCR instrument critical piece of fluorescent PCR includes producing thermograde
The analysis of composite nalysis, the excitation source for carrying out fluorescence excitation, the detecting system that launching light is detected and data acquisition
Software workstation.Major fluorescent PCR instrument brand and model on market include the type of Life technologies companies of the U.S. 7500,
7300 types, StepOnePlus, Viia7 type fluorescent PCR instrument, the types of Roche Holding Ag LightCycler 480, LightCycler 96
Type fluorescent PCR instrument, BioRad companies CFX96 type fluorescent PCR instrument, and Agilent Stratagene company Mx3000P types,
Mx3005P type fluorescent PCR instrument.Gene content, single nucleotide polymorphism that these fluorescent PCR instrument are studied in molecular biology mechanism
（Single nucleotide polymorphism, SNP）Or gene mutation determine, in Clinical Laboratory pathogenic microorganism or
Disease-causing gene qualitative and quantitative analysis, the food safety detection in food hygiene quarantine, and some biological species attack of terrorism detections
Field etc., is all widely used.
Above-mentioned market mainstream fluorescent PCR instrument, general reaction tube consumptive material be the single reaction tube of 0.1 or 0.2ml capacity, eight
Pipe or 96 orifice plates, for holding the reactant liquor containing PCR components and gene template and being used for amplification fluorescent signal detection, but
Due to the restriction of instrument fluoroscopic examination wavelength in single reaction tube, generally in 520 ± 15nm（FAM fluorescent reporter groups, SYBR
Green dyestuffs）、558±12nm（VIC, HEX, JOE fluorescent reporter group）、587±10nm（NED, Cy3 fluorescent reporter group）、
623±14nm（ROX, Texas Red fluorescent reporter groups）And 682 ± 14nm（Cy5 fluorescent reporter groups）This 5 wavelength inspections
Survey and detect respectively 1 gene（Under the conditions of amplified production melting curve analysis are not adopted）, therefore at most can most analyze 5 bases
Cause；If the sample there are more genes to need analysis, need additionally to increase reaction tube, increase is manually operated and reduced once
The sample size of detection, this greatly limits application of the fluorescent PCR in more high flux gene analysiss.
The content of the invention
The purpose of the present invention is to overcome above-mentioned prior art not enough, there is provided a kind of simple structure, easily operated, energy-conserving and environment-protective
Multi-chamber gene analysiss reaction unit, it is possible to achieve the high throughput analysis of 10~50 genes, be adapted in molecular biology base
Plinth research, clinical molecular diagnosis, food safety gene test and biological terrorist genoid detection field application.
The above-mentioned purpose of the present invention is achieved through the following technical solutions：
A kind of multi-chamber gene analysiss reaction unit, it is characterised in that the reaction unit includes supervisor a, arm b, chamber c and envelope
Stifled material d structures, 1 supervisor a connects one end of 2~10 arm b, and the other end coupled reaction chamber c of each arm b is propped up
Be equipped between pipe b and chamber c closure material d, each within the chamber component containing nucleic acid amplification reaction, for nucleic acid amplification reaction with
Gene analysiss.
Supervisor a, arm b in the gene analysiss reaction unit, chamber c, are that polypropylene material is prepared or through erosion
The micro-fluidic glass-chip carved, is hollow, can accommodate liquid or solid.
Chamber c in the gene analysiss reaction unit, when nucleic acid amplification reaction and gene analysiss are carried out, different genes
Analyzed using different wavelength of fluorescence, these wavelength are 520 ± 15nm, 558 ± 12nm, 587 ± 10nm, 623 ± 14nm and 682
1~5 in ± 14nm.
Closure material d in the gene analysiss reaction unit, is a kind of measurable micro valve, can make determined volume liquid
Body is by tube chamber, or arm b and chamber c closings are separated.
Nucleic acid amplification reaction component in the gene analysiss reaction unit chamber, containing having primer, fluorescent probe, deoxidation
The metal ion of ribonucleotide triphosphate or ribonucleotide triphosphate, nucleic acid polymerase, nuclease and auxiliary enzyme effect, be
Liquid condition or glassy solid state；Especially, nucleic acid amplification reaction component contains primer, fluorescent probe, deoxyribose core
Guanosine triphosphate（That is dNTPs, including dATP, dGTP, dCTP, dUTP, dTTP）, Taq archaeal dna polymerases, Uracil N Glycosylase
（UNG enzymes）And magnesium ion.
The nucleic acid amplification reaction of the gene analysiss reaction unit, refers to that PCR reacts, or transcript mediated amplification technology
（Transcription-Mediated Amplification, TMA）, chain substitute amplification（Strand Displacement
Amplification, SDA）, ligase chain reaction（Ligase Chain Reaction, LCR）With dependence nucleotide sequence
Amplification（Nucleic-Acid Sequence Based Amplification, NASBA）, ring mediated isothermal amplification (Loop-
Mediated Isothermal Amplification, LAMP), branched nucleic acid signal amplifying system（Branched DNA,
bDNA）, rolling circle amplification（Rolling Circle Amplification, RCA）These nucleic acid amplification methods.
When the gene analysiss reaction unit is used, when gene nucleic acid template under ambient pressure or electric field action from supervisor
Respectively Jing pipe flow mixes in each reaction chamber with nucleic acid amplification reaction component, then blocks material and closes chamber, whole
Individual device is placed on the thermal cycler with fluorescence analysiss function and reacts, the gene nucleic acid template in each chamber in thermal cycle or
Real-time fluorescent analysis are carried out during isothermal duplication, the qualitative or quantitative detection of 5 genes can be realized.Device connects 2 reactions
Chamber can at most analyze 10 genes, and 10 chambers of connection can at most analyze 50 genes.Gene nucleic acid template used is come
Come from animal, plant, microorganism, the DNA that prepared by synthetic or amplification is obtained or RNA.
Beneficial effect
The present invention is according to the technical scheme of above-mentioned design, the multi-chamber gene analysiss reaction dress designed used in gene analysiss
Put, the technological merit having has the beneficial effect that with generation：
（1）High flux gene analysiss：The single reaction tube of conventional fluorescent PCR instrument is limited to wavelength restriction, and one-time detection can only analyze 5
Gene, and reaction unit of the present invention most multipotency analyzes 50 genes, substantially increases the gene analysiss work efficiency of fluorescent PCR.
（2）Energy-conserving and environment-protective：Reaction unit small volume of the present invention, simple structure, in homologous genes analysis quantity, and tradition
Fluorescent PCR compare with it is light the characteristics of, greatly reduce the consumption of reagent and consumptive material, reach the purpose of energy-conserving and environment-protective.
（3）Suitable real-time test（Point-of-care testing, POCT）Detection：Apparatus of the present invention combine sample core
Sour extraction step, can realize all processes from sample to testing result with a step, not only simple to operate, and when reporting result
Between it is short, be adapted to POCT detection.
Description of the drawings
Accompanying drawing 1 is multi-chamber gene analysiss reaction unit schematic diagram, illustrates supervisor a, arm b, chamber c, closing material
The order of connection of d, wherein arm b, chamber c, the number of closing material d at least have respectively 2, be up to 10, i.e., have respectively
One reaction unit has 2~10 reaction chambers.
Specific embodiment
With reference to specific embodiment, the present invention is expanded on further.It should be understood that these embodiments are merely to illustrate the present invention
Rather than restriction the scope of the present invention.In addition, it is to be understood that after the content for having read instruction of the present invention, people in the art
Member can make various technical changes or modification to the present invention by technology general knowledge, and these equivalent form of values equally fall within the application institute
Attached claims limited range.
Embodiment 1：The preparation and application of multi-chamber gene analysiss reaction unit
（1）The preparation of multi-chamber gene analysiss reaction unit
It is responsible for a, arm b in accompanying drawing to specifications, chamber c, the order of connection of closing material d design mould, and using poly- third
Alkene material prepares the gene analysiss reaction unit with 2 chambers.Nucleic acid amplification primers probe is respectively charged in 2 chambers,
DNTPs, reverse transcriptase, nucleic acid polymerase, UNG enzymes and magnesium ion, 2 chamber gene analysiss reaction units of preparation are used for serum
Sample hepatitis B viruss（HBV）, hepatitis C viruss（HCV）Gene type detection.
HBV gene serotype specific primer probe adopts document（J Clin Microbiol. 2010 Apr; 48(4): 1105–
1111）As, Bps, Cps, D primer probe sequence in table 1（4 pairs of HBV gene type primers, 4 HBV gene type probes）, but visit
The end of pin sequence 5 ' is respectively adopted successively FAM, JOE, Cy3, ROX fluorophor labelling, 3 ' ends using BHQ quenching group labellings, point
Not Ji Yu HBV S antigen genes detect common HBV gene type A, B, C, the D of China.HCV genotyping primer probes adopt document
（J Clin Virol. 2005 Oct; 34(2): 108-14）In gene hypotype primed probe（The 4 pairs of HCV genotyping primers,
4 HCV genotype probes）, but the probe Gt1probe~Gt4probe sequences 5 ' end for HCV 1-4 genotype distinguishes successively
BHQ quenching group labellings are adopted using FAM, JOE, Cy3, ROX fluorophor labelling, 3 ' ends, non-volume is held based on HCV genomes 5 '
The code region sequence detection gene hypotypes of HCV 1,2,3,4.Meanwhile, the patent of invention applied using the applicant
（201310742857.6, a kind of highly sensitive Epstein-Barr FLuorescent quantitative PCRs test kit containing internal reference）Middle employing
GAPDH gene primers probe as internal reference primed probe, the end of probe sequence 5 ' Cy5 fluorophor labellings, 3 ' ends adopt BHQ
Quenching group labelling.12 primed probes of the HBV gene typing that above-mentioned design is used, 12 of HCV gene analysiss types draw
Physical prospecting pin and 3 internal reference primed probe commission Shanghai Sangon Biological Engineering Technology And Service Co., Ltd synthesize, and primer is
PAGE purification, probe is HPLC purification.
In the gene analysiss reaction unit of above-mentioned 2 chambers, the 100mM of pH8.3 is separately added in 2 chambers first
Tris-HCl 3ul、500mM KCl 3ul、10mM dNTPs（Containing each 10mM of dATP, dGTP, dCTP, dUTP, dTTP）
0.9ul、100mM MgCl2 1ul, Taq archaeal dna polymerase 3U, UNG enzyme 0.2U；Then in each 10mM of the 1st chamber 8 articles of concentration of addition
The each 10mM HBV genes type of HBV gene type primer 0.9ul, 2 each 0.45ul of 10 mM GAPDH gene primers, 4 concentration is visited
Each 0.6ul, 10mM GAPDH gene probe 0.3ul of pin.2nd chamber addition SuperScript III reverse transcriptase 3U, 8
The each 0.45ul of each 0.9ul of each 10mM HCV genotyping primers of bar concentration, 2 10mM GAPDH gene primers, 4 concentration are each
Each 0.6ul, 10mM GAPDH gene probe 0.3ul of 10mM HCV genotype probes.Said apparatus are put into desk freeze-dryer
（Alpha1-4LSC types, German Christ companies）Lyophilizing, now nucleic acid amplification reaction mixture is dry glass solid, shaped
State, by reaction unit 4 DEG C of preservations are placed in.
（2）Multi-chamber gene analysiss reaction unit is applied in serum HBV/HCV gene types
Collection Jing clinic nucleic acid quantification detections are defined as the patients serum 5 of viral hepatitis, using the applicant's patent of invention
（ZL201010621890.X, a kind of cell cracking agent for biological sample amplifying nucleic acid extraction purification）Middle viral DNA/RNA
Magnetic bead combines extracting method altogether, and every sample extraction obtains nucleic acid 100ul, detected immediately.
The gene analysiss reaction unit of 2 chambers that above-mentioned preparation is preserved is taken, room temperature is equilibrated to, is added in supervisor a above-mentioned
The 100ul of extraction is nucleic acid-templated, corresponding micro- when liquid template separately flows into 2 chambers each 30ul under ambient pressure effect
Type valve is closed, and solid reaction component is dissolved by nucleic acid-templated liquid in chamber, and reaction system is 30ul.Reaction unit is proceeded to into band
The thermal cycler augmentation detection of fluorescence analysiss function, response procedures are after 50 DEG C of 10min, 94 DEG C of 5min, according to 94 DEG C
15sec, 60 DEG C of 1min cyclic amplifications 40 times, and in 60 DEG C of collection five wavelength of FAM, JOE, Cy3, ROX, Cy5 of each circulation
（520 ± 15nm of correspondence, 558 ± 12nm, 587 ± 10nm, 623 ± 14nm and 682 ± 14nm）Fluorescence signal.After reaction terminates
According to software analysis, the positive is judged as with the result for having substantially amplification, presentation S type amplification curves.5 pattern detection result such as tables
Shown in 1, the gene analysiss reaction unit of above-mentioned preparation can intactly in one-time detection to clinical viral hepatitis clinical samples
The measure of HBV, HCV genotypic results is completed, and at present conventional use of fluorescence PCR method compares, with easy to operate, inspection
Survey quick advantage.
The gene analysiss reaction unit of the present invention of table 1 is to serum HBV/HCV gene analysiss results
| As a result | Serum 1 | Serum 2 | Serum 3 | Serum 4 | Serum 5 |
| HBV gene type | C-type | Type B | A types | C-type | D types |
| HCV genotype | 3 types | 1 type | 4 types | 2 types | 1 type |
| Internal reference | It is positive | It is positive | It is positive | It is positive | It is positive |
Embodiment 2：The preparation and application of multi-chamber gene analysiss reaction unit
（1）The preparation of multi-chamber gene analysiss reaction unit
It is responsible for a, arm b, chamber c, the order of connection design mould of closing material d in accompanying drawing to specifications, and adopts glass
Silicon materials and MEMS（MEMS, Micro-Electro-Mechanical System）Technology, the lithography on silicon chip
It is prepared for the micro-fluidic chip gene analysiss reaction unit with 2 chambers.Nucleic acid amplification primers are respectively charged in 2 chambers
Probe, dNTPs, reverse transcriptase, Taq archaeal dna polymerases, UNG enzymes and magnesium ion, the 2 chamber gene analysiss reaction dress of preparation
Put for urogenital tract sample Common STD pathogen（Chlamydia trachomatiss, gonococcuss, Ureaplasma urealyticum, genital branch
Substance, the type of human papillomavirus 6,11,16,18）Detection of nucleic acids detection.
Chlamydia trachomatiss（CT）, gonococcuss（NG）, Ureaplasma urealyticum（UU）, mycoplasma genitalium（Mg）Detection of nucleic acids primer is visited
Pin adopts document（Int J Mol Epidemiol Genet. 2013 Sep 12; 4(3): 167-74）Primer in table 1 is visited
Pin sequence（CT-Plasmid-FP / CT-Plasmid-RP / CT-Plasmid-Probe、NG-FP /NG-RP /NG-
Probe, UP-UU-FP/UP-UU-RP/UU-Probe, MG-FP/MG-RP/MG-Probe, totally 4 pairs of primers, 4 probes）,
But the end of probe sequence 5 ' is respectively adopted successively FAM, JOE, Cy3, ROX fluorophor labelling, 3 ' ends using BHQ quenching group marks
Note, different wave length detects respectively clinical common CT, NG, UU, Mg pathogen.6,/11 two hypotypes of HPV, 16,/18 two hypotypes
Primed probe is respectively adopted the patent of invention of the applicant's application（201511007550.7, a kind of highly sensitive human papillomavirus
6,11 type kit for detecting nucleic acid；201110448224.5, human papillomavirus（HPV）16,18 type multichannel fluorescent PCRs are detected
Method and test kit）Primed probe, wherein 6,/11 two hypotypes share 1 pair of primer, the end of 6 type probe sequence 5 ' flag F AM fluorescence
Group, the end of 11 type probe sequence 5 ' labelling are changed to JOE fluorophors, and 16,/18 two hypotypes respectively have 1 pair of primer and probe, 16 types
The end of probe sequence 5 ' labelling is changed to Cy3 fluorophors, and the end of 18 type probe sequence 5 ' labelling is changed to ROX fluorophors, HPV 6/11/
3 ' the ends of 16/18 4 probes of detection adopt BHQ quenching group labellings.Meanwhile, the patent of invention applied using the applicant
（201310742857.6, a kind of highly sensitive Epstein-Barr FLuorescent quantitative PCRs test kit containing internal reference）Middle employing
GAPDH gene primers probe as internal reference primed probe, the end of probe sequence 5 ' Cy5 fluorophor labellings, 3 ' ends adopt BHQ
Quenching group labelling.12 primed probes of CT, NG, UU, Mg detection of nucleic acids that above-mentioned design is used, HPV 6/11/16/18
Type detects that 10 primed probes and 3 internal reference primed probes entrust Shanghai Sangon Biological Engineering Technology And Service Co., Ltd
Synthesis, primer is PAGE purification, and probe is HPLC purification.
In the micro-fluidic chip gene analysiss reaction unit of above-mentioned 2 chambers, it is separately added in 2 chambers first
The 100mM Tris-HCl 3ul of pH8.3,500mM KCl 3ul, 10mM dNTPs（Containing dATP, dGTP, dCTP, dUTP,
The each 10mM of dTTP）0.9ul、100mM MgCl2 1ul, Taq archaeal dna polymerase 3U, UNG enzyme 0.2U；Then 8 are added in the 1st chamber
The each 0.9ul of each 10mM CT, NG, UU, Mg primer of bar concentration, 2 each 0.45ul of 10 mM GAPDH gene primers, 4 concentration are each
Each 0.6ul, 10mM GAPDH gene probe 0.3ul of 10mM CT, NG, UU, Mg probes, add deionized water to supply volume and arrive
26ul, uses micro valve sealed chamber.2nd chamber adds each 10mM HPV6/11/16/18 genotyping primers of 6 articles of concentration each
The each 10mM HPV6/11/16/18 genotype probes of each 0.45ul of 0.9ul, 2 10mM GAPDH gene primers, 4 concentration are each
0.6ul, 10mM GAPDH gene probe 0.3ul, add deionized water to supply volume to 26ul, use micro valve sealed chamber.
Above-mentioned micro-fluidic chip gene analysiss reaction unit is placed in -20 DEG C of preservations.
（2）Multi-chamber gene analysiss reaction unit is applied in urogenital tract secretion pathogen detection
Clinical 10, clinic attendants urogenital tract secretion rinsing liquid sample is collected, rinsing liquid sample 200ul is taken,
Supernatant is abandoned after 13000rpm centrifugation 10min, adds 50ul to contain 20mM NaOH, the nucleic acid cleavage of 1% polysorbas20 in precipitation
Liquid, 100 DEG C are incubated 10min after whirlpool is mixed, and 13000rpm centrifugation 2min, every sample draws supernatant and obtains nucleic acid extractive about
50ul, is detected immediately.
The gene analysiss reaction unit of 2 chambers that above-mentioned preparation is preserved is taken, room temperature is equilibrated to, is added in supervisor a above-mentioned
The 50ul of extraction is nucleic acid-templated, corresponding miniature when liquid template separately flows into 2 chambers each 4ul under ambient pressure effect
Valve is closed.Reaction unit is proceeded to into the thermal cycler augmentation detection with fluorescence analysiss function, response procedures be 50 DEG C of 2min, 94
After DEG C 5min, according to 94 DEG C of 15sec, 60 DEG C of 1min cyclic amplifications 40 times, and 60 DEG C of collection FAM in each circulation, JOE,
Five wavelength of Cy3, ROX, Cy5（520 ± 15nm of correspondence, 558 ± 12nm, 587 ± 10nm, 623 ± 14nm and 682 ± 14nm）
Fluorescence signal.React after terminating according to software analysis, the positive is judged as with the result for having substantially amplification, presentation S type amplification curves.
10 pattern detection results as shown in table 2, to clinical clinic attendants urinary system give birth to by the gene analysiss reaction unit of above-mentioned preparation
Grow 8 kinds of sexually transmitted disease (STD) pathogen common in sample（Chlamydia trachomatiss, gonococcuss, Ureaplasma urealyticum, mycoplasma genitalium,
The type of human papillomavirus 6,11,16,18）One-time detection obtains information, and at present conventional use of fluorescence PCR method compares, and has
Have that volume is small, the quick, advantage that the report result time is short easy to operate.
The gene analysiss reaction unit of the present invention of table 2 is to urogenital tract secretion pathogen gene analysis result
| Sample | Pathogen | Internal reference | Sample | Pathogen | Internal reference |
| Secretions 1 | CT, UU, HPV6 | It is positive | Secretions 6 | CT, UU, HPV6 | It is positive |
| Secretions 2 | NG, HPV6/11 | It is positive | Secretions 7 | HPV6/11/16/18 | It is positive |
| Secretions 3 | UU, Mg, HPV6/11 | It is positive | Secretions 8 | CT, HPV16 | It is positive |
| Secretions 4 | HPV16/18 | It is positive | Secretions 9 | UU, HPV6/11 | It is positive |
| Secretions 5 | CT, NG, HPV11 | It is positive | Secretions 10 | NG, Mg, HPV18 | It is positive |
Claims (10)
1. a kind of multi-chamber gene analysiss reaction unit, it is characterised in that the reaction unit contain supervisor a, arm b, chamber c and
Closure material d structures, one end of 1 supervisor a 2~10 arm b of connection, the other end coupled reaction chamber c of each arm b,
Closure material d, each within the chamber component containing nucleic acid amplification reaction, for nucleic acid amplification reaction are equipped between arm b and chamber c
And gene analysiss.
2. reaction unit as claimed in claim 1, it is characterised in that supervisor a, arm b in reaction unit, chamber c, is high
Molecularly Imprinted Polymer material is prepared or through the micro-fluidic glass-chip of etching, is hollow.
3. reaction unit as claimed in claim 1, it is characterised in that the chamber c in reaction unit carries out nucleic acid amplification reaction
With during gene analysiss, different genes using different wavelength of fluorescence analyses, these wavelength be 520 ± 15nm, 558 ± 12nm, 587 ±
1~5 in 10nm, 623 ± 14nm and 682 ± 14nm.
4. reaction unit as claimed in claim 1, it is characterised in that the closure material d in reaction unit, is a kind of measurable
Micro valve, can make determined volume liquid by tube chamber, or arm b and chamber c closings are separated.
5. reaction unit as claimed in claim 1, it is characterised in that the nucleic acid amplification reaction component in reaction unit chamber c,
Containing primer, fluorescent probe, dideoxyribonucleotide triphosphate or ribonucleotide triphosphate, nucleic acid polymerase, nuclease and
The metal ion of auxiliary enzyme effect.
6. reaction unit as claimed in claim 1, it is characterised in that the nucleic acid amplification reaction component in reaction unit chamber c,
Containing primer, fluorescent probe, dideoxyribonucleotide triphosphate, Taq archaeal dna polymerases, Uracil N Glycosylase and magnesium ion.
7. reaction unit as claimed in claim 1, it is characterised in that the nucleic acid amplification reaction component in reaction unit chamber c,
For liquid condition or glassy solid state.
8. reaction unit as claimed in claim 1, it is characterised in that the nucleic acid amplification reaction of reaction unit, refers to polymerase
Chain reaction, or the replacement amplification of transcript mediated amplification technology, chain, ligase chain reaction, the amplification for relying on nucleotide sequence, ring Jie
Lead isothermal duplication, branched nucleic acid signal amplifying system, rolling circle amplification these nucleic acid amplification methods.
9. reaction unit as claimed in claim 1, it is characterised in that when reaction unit is used, gene nucleic acid template is in the external world
Mix with nucleic acid amplification reaction component in each reaction chamber c from supervisor's a difference Jing arms b streams under pressure or electric field action,
Then block material d to close chamber, whole device is placed on the thermal cycler with fluorescence analysiss function reacts, each chamber
In gene nucleic acid template real-time fluorescent analysis are carried out during thermal cycle or isothermal duplication, the qualitative of 5 genes can be realized
Or detection by quantitative；When device connects 2~10 reaction chambers, can at most analyze gene has 10~50.
10. reaction unit using method as claimed in claim 9, it is characterised in that gene nucleic acid template is referred to from dynamic
Thing, plant, microorganism, the DNA that prepared by synthetic or amplification is obtained or RNA.
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