CN1325028A - Real-time quantitative analysis method and instrument for piezoelectric gene diagnosis - Google Patents
Real-time quantitative analysis method and instrument for piezoelectric gene diagnosis Download PDFInfo
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- CN1325028A CN1325028A CN 01108612 CN01108612A CN1325028A CN 1325028 A CN1325028 A CN 1325028A CN 01108612 CN01108612 CN 01108612 CN 01108612 A CN01108612 A CN 01108612A CN 1325028 A CN1325028 A CN 1325028A
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Abstract
The present invention adopts piezoelectric gene diagnosis chip and frequency detection instrument and utilizes multiple PCR technique to make temp. circulation of hydridization-extension-denaturation to amplify, target gene, monitor and obtain the linear relationship of frequency change and target gene concentration, then uses external standard curve to make quantitative analysis. Said apparatus comprises shell body, power supply and its connected circuit, and main body module, analysis, software, sample preparation module, positioning liquid flow module and piezoelectric gene diagnosis chip detection module. Said invention is suitable for clinical diagnosis and molecular mechanism research, monitoring epidemic disease and infections disease spreading and harmful microbial genesis and diffusion.
Description
Technical field
The invention belongs to the instrument of outer-gene detection method and this method of enforcement.
Background technology
The generation development of most diseases is all relevant with patient's genetic background or its change, and gene diagnosis is meant the method for using genetic analysis disease to be made diagnosis.The object of gene diagnosis at first is the intrusion of causal organism, directly the inhereditary material that detects causal organism can improve the susceptibility and the specificity of diagnosis greatly, and in the time can't obtaining commercialization antibody, gene diagnosis just becomes the detection cause pathogeny imcrobe infection, especially the only resource of virus infections; Next is the congenital heredity illness, the disease that the heredity illness that some pathogenic factors are determined and other cause of disease it be unclear that all may with the holding or change relevant of certain or some genes; Moreover be the disease that gene mutation will cause the day after tomorrow, for example the cell infinite multiplication of undergoing mutation and causing owing to individual cells gene such as tumor suppressor gene or oncogene can be tentatively thought in the generation of tumour; Other is as dna fingerprint, individual identification, parent identification, forensic etc.Gene diagnosis is not only to clinical diagnosis, and to the cause of disease of disease and pathogenetic research, for patient detects curative effect, analyze more after, the objective health status of correctly estimating human body all has great importance.The gene diagnosis The Application of Technology makes the diagnosis of many difficult disease become easy, accurate, avoided some illness clinically to instruct the blindness of treatment by experience, reached the earliest, the most effective, the most most economical therapeutic purposes.
But the shortage of gene samples is a big obstacle that carries out gene diagnosis.The appearance of PCR (PCR) technology provides effective means for this reason.Round pcr is to utilize heat-staple DNA synzyme-Taq polymerase in external synthetic DNA fragment, by round-robin DNA sex change, annealing and extension repeatedly, target DNA is increased in a large number.The pcr amplified dna fragment is an important means, the detection and the analysis of amplified fragments are only purpose, therefore, mainly adopt following two kinds of analytical approachs according to the different of research object and purpose behind the pcr amplification: gel electrophoresis analysis method: by Ago-Gel or polyacrylamide gel electrophoresis, behind the electrophoresis with the dyeing of forming sediment of bromination second, observations and taking pictures under the UV lamp.Gel electrophoresis can be identified the size of product, detects the situation of amplification.Spot hybridization: at first the DNA with amplification is fixed on nylon membrane or the nitrocellulose filter, again with the probe hybridization of radioactivity or non-radioactive marker's mark.Spot hybridization helps to detect the mutation type of mutant DNA, is used for the somatotype of the sick diagnosis of human inheritance and some gene.
Round pcr is used for the quantitate gene diagnosis also need adopt specific method." Chinese laboratory medicine magazine " 2000,23 (2): " progress of quantitative polyase chain reaction and clinical practice " article that 120-121 delivers, several traditional quantifying PCR methods have been summarized, mainly comprise: internal standard method: in different PCR reaction tubes, add in the quantitative synthetic mark and-to primer (one is a fluorescence labeling), in template amplification, interior mark also is amplified.In the PCR product, because interior mark is different with the length of template, the amplified production of the two can come with electrophoretic separation, measures its fluorescence intensity respectively, with the interior contrast detection by quantitative template that is designated as.The PCR-ELISA method: utilize labeled primers such as digoxin or biotin, amplified production is by probe institute combination special on the solid phase plate, marks anti-digoxin with the enzyme that adds again or antibiotin combines, and final enzyme makes the substrate colour developing.Conventional PCR-ELISA method is a qualitative experiment, if mark in adding is made typical curve, also can realize the detection by quantitative purpose.But above-mentioned traditional quantivative approach all is an end point determination, and promptly PCR detects after arriving plateau, and PCR during plateau, detects the reappearance extreme difference through logarithmic phase amplification arrival.Behind the mark, can partly eliminate the quantitative inaccuracy that causes of end-product in adding.But even so, owing in testing sample, add the interior mark of known initial copy number, interference and competition in existing in the PCR reaction between mark and the template, especially when both initial copy numbers differed bigger, this competition can show more significantly.In addition because the initial copy number of testing sample is unknown, so the known template that can't add suitable quantity is as interior mark.Therefore, though traditional quantivative approach is marked in adding, in fact still be a kind of semiquantitative method.
For this reason, the real-time fluorescence quantitative PCR technology proposed in 1996, because this technology has not only realized the leap of PCR from qualitative to quantitative, and compared with conventional PCR, it have specificity stronger, effectively solve characteristics such as PCR pollution problem, automaticity height, be used widely at present.Disclosed " the DNA/RNA Real-Time Quantitative PCR-Rev.A﹠amp in 1999 of U.S. Applied Biosystems company; B " report, introduced the principle and the characteristics of real-time fluorescence quantitative PCR technology.So-called real-time fluorescence quantitative PCR technology is meant in the PCR reaction system to add fluorescence probe, utilizes the fluorescence signal accumulation whole PCR process of monitoring in real time, the method for by typical curve unknown template being carried out quantitative test at last.A key of quantitative fluorescent PCR success is to adopt special fluorescence probe to realize that the formation of the accumulation of fluorescence signal and PCR product is synchronous fully.The real-time fluorescence quantitative PCR technology has solved traditional limitation that quantitatively can only end point determination effectively, has realized that each takes turns the intensity that circulation all detects the first order fluorescence signal.In this technology, with preceding 15 round-robin fluorescence signals of PCR reaction be the fluorescence background signal as fluorescence thresholding, the period (C that is experienced when the fluorescence signal of (being each template) arrives thresholding in each reaction tube
t) there is linear relationship with the logarithm of the initial copy number of this template, initial copy number is many more, C
tBe worth more little.Utilize the standard items of known initial copy number can make typical curve, therefore, as long as obtain the C of unknown sample
tBe worth, can calculate the initial copy number of this sample from typical curve.Compare with the interior scalar quantity method of conventional P CR, real-time fluorescence quantitative PCR is the outer typical curve quantitative methods of a kind of employing.Because circulating in, PCR arrives C
tWhen being worth the period at place, just entered the real index amplification phase (logarithmic phase), this moment, slight error was amplified as yet, so C
tThe reappearance of value is better, has improved quantitative accuracy and reappearance greatly.
In addition, the biochip technology that occurs in recent years also is expected to be applied to gene diagnosis.Biochip technology is hybridized with the sample molecule of mark after being meant and being fixed in a large amount of gene probe molecules (gene probe array) on the holder, by hybridization signal intensity that detects each probe molecule and then quantity and the sequence information that obtains sample molecule, realize to gene accurately, fast, the detection of large information capacity.Deficiencies such as biochip technology has solved traditional nucleic acid blot hybridization technique trivial operations, automaticity is low, sequence of operation quantity is few, detection efficiency is low.And, by designing different probe arrays, using specific analytical approach can make this technology have multiple different gene diagnosises application." bioengineering progress " 1999, " biochip technology and applied research progress " article that Vol.19 (No.4): 45-51 delivers has been summarized both at home and abroad biochip technology in the main achievement in research aspect processing and preparing, the function and application.The existing biochip technology preparation of genetic chip on the one hand mainly is to adopt solid phase original position synthetic technology synthetic, stationary probe molecular array on carriers such as glass sheet in conjunction with photolithography; The detection of genetic chip mainly is to utilize labels targets gene such as fluorescein on the other hand, detects and analyzes by laser co-focusing microscan technology or the Charge Coupled Device (CCD) camera technique fluorescence signal to hybridization target gene on the genetic chip.
Above-mentioned real-time fluorescence quantitative PCR and biochip technology, on the one hand, all be to adopt fluoroscopic examination, need large complicated, accurate expensive detection equipment, need the long period simultaneously (3-4 hour), promptly need after (more than 15 times) amplification cycles repeatedly, or detect after finishing hybridization reaction, and impact analysis the factor of accuracy is more as a result, being difficult to control holds, easily produce false positive results, and need target gene or probe are carried out fluorescence labeling, increased the consumption reagent cost; On the other hand, analytic process comprises the multistep operation, comprises that especially sample pretreatment, target gene obtain with mark etc., complex operation, and that whole analytic system is difficult to is integrated, robotization and miniaturization.These defectives are the obstacles that are difficult to go beyond that popularize the gene diagnosis technology at present.
Summary of the invention
Given this, the objective of the invention is the problems referred to above of existing at existing gene diagnosis technology, provide a kind of accurate sensitivity, rapidly and efficiently, piezoelectric gene diagnosis real-time quantitative analysis method and instrument easy, with low cost automatically.
For reaching above-mentioned purpose, the present invention utilizes the multiple PCR technique multiple target gene that increases simultaneously, the piezoelectric gene diagnosis chip that adopts the gene probe diagnostic techniques to combine with the piezoelectric sensor chip technology, the multiple target gene of identification hybridization simultaneously, and obtain the reaction information of each probe one target gene hybridization in real time, with accurate sensitivity, rapidly and efficiently, the easy analysis of carrying out gene diagnosis inexpensively; On the other hand, the present invention is integrated in a system with each module of gene diagnosis analytic processes such as sample preparation, sample detection, interpretation of result, make up piezoelectric gene diagnosis real-time quantitative analysis instrument, adopt microcomputer and specific program, carry out each link of gene diagnosis quantitative test with automatic control, and dynamically show each link information, implement gene diagnosis analytical approach of the present invention.
Piezoelectric gene diagnosis real-time quantitative analysis method of the present invention, gene diagnosis quantitative test to certain class disease, employing has the piezoelectric gene diagnosis chip of the probe of at least a disease gene, to there be the sample of at least a target gene, corresponding primer, Taq-DNA polymerase, deoxy-ribonucleoside triphosphate to contact with piezoelectric gene diagnosis chip, hybridize-extend-temperature cycles of sex change, hybridization temperature is 30-70 ℃, and elongating temperature is 65-80 ℃, and denaturation temperature is 85-99 ℃; The temperature cycles starting point is denaturation temperature 10-60 second, and then hybridization temperature 10-60 second, terminal point is elongating temperature 10-60 second; At crossing phase, the probe of each target gene in the sample and corresponding disease gene and primer based on sequence complementation hybridize combination; In the extension stage, the probe of hybridization and primer are that template is carried out polymerase chain reaction,PCR with the target gene under the effect of Taq-DNA polymerase, make probe and primer obtain extending, and be identical with target gene until both chain lengths; In the sex change stage, probe-target gene, primer-target gene heterocomplex dissociate, and be the thresholding that background noise is provided with frequency change according to the frequency change of first crossing phase, when frequency change surpasses thresholding, stop circulation, reach the hit logarithm of mrna concentration of the required cycle index of thresholding and sample according to frequency change again and have linear relationship, carry out the quantitative test of target gene with outer typical curve.
Above-mentioned probe, primer are 20-30 base, sequence and target gene complementation and the unduplicated oligonucleotides of both sequences; Background noise (G) is the standard deviation of frequency change in the preset time, or be the extreme difference (maximum frequency and minimum frequency poor) of frequency change in the preset time, frequency variable domain value (f
R) be made as background noise (G) 2-10 doubly; Frequency change reaches the required cycle index (N of thresholding
t) with the sample mrna concentration (C that hits
T) the pass be N
t=AlogC
T+ B, A and B to a certain sensor and target gene under same temperature cycles are and C in the formula
TIrrelevant constant; Outer typical curve is by measuring 2-10 standard solution that contains the concentration known target gene, measuring each corresponding N
tTo logC
TThe typical curve that mapping draws; The concentration of target gene is to reach the required cycle index of thresholding according to typical curve and sample to record in the sample.
The piezoelectric gene diagnosis real-time quantitative analysis instrument of implementing gene diagnosis analytical approach of the present invention is (referring to accompanying drawing 1,2), comprise housing (10), control panel on the housing, display, power supply in the housing (20) and joining circuit thereof, the mainboard module of microcomputer (60), analysis software is characterized in that having in the housing and circuit and the joining specimen preparation module (30) that can once extract at least one clinical DNA sample of mainboard module, select and carry the location flow module of sample, the piezoelectric gene diagnosis chip detection module (50) of amplification and detection target gene.
It is 0.2-2cm that above-mentioned specimen preparation module (30) has by 2-100, volume
3The microwell plate (31) that constitutes of the micropore of placement test tube, the temperature controller (32) that is made of heating and refrigeration device and joining temperature sensor thereof is arranged.
Above-mentioned location flow module, the mechanical arm (41) that is driven by driving machine and the suction spindle (42) of linking to each other thereof are arranged, suction spindle, peristaltic pump (43), piezoelectric gene diagnosis chip detection module (50), waste liquid storage bottle (47) are communicated with successively, and the import of piezoelectric gene diagnosis chip detection module and outlet have liquid level sensor (44,45) respectively.
Above-mentioned piezoelectric gene diagnosis chip detection module (50) has the long-pending circulation type detection cell (52) of microbody and is encapsulated in wherein the piezoelectric gene diagnosis chip (51) that is made of piezoelectric gene sensor array, links to each other with circuit and the middle piezoelectric gene diagnosis chip testing circuit of mainboard module (60).The long-pending circulation type detection cell (52) of microbody has semiconductor heating and refrigeration device and the joining temperature controller (53) thereof on detection cell.
Use piezoelectric gene diagnosis real-time quantitative analysis instrument of the present invention (referring to accompanying drawing), at first, according to the clinical diagnosis object, select corresponding piezoelectric gene diagnosis detection module and gene diagnosis reagent test tube, the piezoelectric gene diagnosis chip detection module is inserted in the piezoelectric gene diagnosis real-time quantitative analysis instrument, add gene diagnosis reagent test tube and insert the specimen preparation module being equipped with good sample solution and standard solution.Then, by the set up standard parameter of solution concentration, sample sequence and target gene group to be checked thereof and specimen preparation, location liquid stream and piezoelectric gene diagnosis chip detection module of analysis software.At last, automatically implement the state of also dynamically show sample preparation, location liquid stream and piezoelectric gene diagnosis chip detection module by programmed control, and the detection signal of piezoelectric gene diagnosis chip (frequency change), when frequency change reaches the frequency variable domain value of setting, stop circulation, the piezoelectric gene diagnosis chip detection module remains on the sex change stage, begin to draw cleaning solution simultaneously, after the every dot frequency of piezoelectric gene diagnosis chip indicates each site to recover to detect preceding frequency, emptying detection cell solution is measured next sample.After finishing all samples and measuring, enter the data analysis interface, provide the typical curve and the linear dimensions (slope, intercept and related coefficient) thereof of each target gene, and the concentration of each target gene to be measured in each sample.
The present invention and existing gene diagnosis technology relatively have following advantage and effect.
At first, piezoelectric gene diagnosis real-time quantitative analysis method of the present invention, the piezoelectric gene diagnosis chip that adopts the piezoelectric sensor chip to combine with the gene diagnosis technology, has the unique advantage of probe-target gene hybridization reaction on highly sensitive, the real-time chip monitoring of Piezoelectric sensing technique on the one hand, realized on piezoelectric gene diagnosis chip, carrying out simultaneously hybridization reaction and two steps of input, made analytical approach of the present invention simple and efficient; On the other hand, have gene diagnosis chip large information capacity, high efficiency advantage again, analytical approach of the present invention can once be detected a plurality of target genes simultaneously.
Secondly, piezoelectric gene diagnosis real-time quantitative analysis method of the present invention on the one hand, is based upon on the basis of dynamic tracking analysis probe-target gene hybridization reaction, eliminated in the end-point detection method impact analysis factor of accuracy as a result, made analytical approach of the present invention more accurately and reliably; On the other hand, determine target gene,,, also guaranteed the reliability of analysis result of the present invention so avoided false positive because of the sequence of probe has specificity by probe hybridization.
Once more, piezoelectric gene diagnosis real-time quantitative analysis method of the present invention, on the one hand, used probe and target gene do not need mark, and the thing of also not labelling had in addition both reduced the reagent expense, again environmentally safe; On the other hand, need be expanded to the 50-100ng order of magnitude with common PCR with the fluoroscopic examination gene and compare, only need be expanded to the pg order of magnitude, significantly reduced cycle index, shorten detection time, also significantly reduced the consumption of reagent such as Taq enzyme simultaneously, saved the consumption costs of diagnostic check.
At last, the piezoelectric gene diagnosis real-time quantitative analysis instrument that the present invention makes up, on the one hand, the detection of piezoelectric gene diagnosis chip is a frequency measurement accuracy height and interlock circuit is simple, and is inexpensive and be easy to miniaturization; On the other hand, the The whole analytical process that will comprise specimen preparation, gene magnification and detection, data analysis is integrated, easy and simple to handle, automatic high speed, can in a system, short time, finish from primary sample and operate, and the integrated analysis system that makes simultaneously of total analysis process is tightly isolated with extraneous to a complete set of obtaining required analysis result, effectively avoided pollution, environment need not strict clean requirement to external world.
The present invention is suitable for research, monitoring epidemic disease and infectious disease diffusion, the generation of monitoring harmful microbe and the diffusion etc. of clinical disease diagnosis and molecule mechanism, is with a wide range of applications.
Below, with embodiment and accompanying drawing thereof the present invention is further described again.
Description of drawings
Brief description of drawings.
Fig. 1 is the structural representation of a kind of piezoelectric gene diagnosis real-time quantitative analysis instrument of the present invention.
Fig. 2 is the structure principle chart of Fig. 1.
The temperature cycles figure of piezoelectric gene diagnosis detection module when Fig. 3 is to use Fig. 1 to carry out the gene diagnosis analysis.
Site frequency change-time chart corresponding of piezoelectric gene diagnosis chip when Fig. 4 is to use Fig. 1 to carry out the gene diagnosis analysis with Fig. 3.
Frequency change-cycle index graph of a relation of when Fig. 5 is to use Fig. 1 to carry out the gene diagnosis analysis same target gene concentration repeatedly being measured.
Fig. 6 is the canonical plotting of a kind of target gene of piezoelectric gene diagnosis real-time quantitative analysis method of the present invention.
Embodiment
Embodiment 1
A kind of piezoelectric gene diagnosis real-time quantitative analysis instrument of the present invention is shown in accompanying drawing 1,2.Constitute by housing 10, power supply 20, specimen preparation module 30, location flow module, piezoelectric gene diagnosis chip detection module 50, circuit and mainboard module 60 and piezoelectric gene diagnosis real-time quantitative analysis instrument software etc.
Above-mentioned specimen preparation module 30, the DNA extraction of a plurality of clinical samples is promptly once finished in preparation when realizing a plurality of sample, is made of microwell plate 31 and temperature controller 32.Above-mentioned microwell plate 31 usefulness heat conductance good metal materials as gold, silver, copper, aluminium etc., are made rectangle or circular slab, open on the plate and are shaped on a plurality of micropores, and micro pore volume is 0.2-2cm
3, number cells is 2-100, the quantity of micropore and volume, and the size of fixed therefrom plate are decided on the needs of practical application.Above-mentioned temperature controller 32 is made of the heating made from semiconductor and refrigeration device and temperature sensor, link to each other with related circuit in mainboard and the circuit module 60, controlled by subprograms corresponding, the precision of said temperature control is below ± 0.1 ℃, the heating and refrigerating speed 1.5 ℃/more than the sec, the fluctuation of per minute is less than ± 0.1 ℃ during constant temperature.
Above-mentioned location flow module realizes selecting automatically sample introduction, selectedly draws solution to be measured and also sends into detection cell, by mechanical arm 41, suction spindle 42, peristaltic pump 43, the liquid level sensor 44,45 of common structure, connect sebific duct 46 and constitute.Above-mentioned mechanical arm 41 comprises X, Y two coordinates are numerical control linked and Z coordinate driving device structure, link to each other with related circuit in mainboard and the circuit module 60, controlled by subprograms corresponding.Above-mentioned suction spindle 42 with material heat-resisting, resistance to chemical attack, is made as stainless steel, teflon etc., is fixed on the mechanical arm 41.Above-mentioned peristaltic pump 43 adopts the housing and the structure of common peristaltic pump, links to each other with related circuit in mainboard and the circuit module 60, controlled by subprograms corresponding.Above-mentioned liquid level sensor 44,45 adopts photo-electric liquid position sensor, is placed in the front and back of piezoelectric gene diagnosis chip detection cell 52 respectively, links to each other with related circuit in circuit and the mainboard module 60.Above-mentioned connection sebific duct 46 adopts resilient material heat-resisting, heat-resisting, resistance to chemical attack, makes as teflon etc., links to each other with detection cell 52 with suction spindle 42, leads to waste liquid storage bottle 47.
Above-mentioned piezoelectric gene diagnosis chip detection module 50 is realized the amplification and the detection of target gene, is made of piezoelectric gene diagnosis chip 51, the long-pending circulation type detection cell 52 of microbody, temperature controller.Above-mentioned piezoelectric gene diagnosis chip 51 is made of piezoelectric gene sensor array, and above-mentioned piezoelectric gene diagnosis chip is packaged in the long-pending circulation type detection cell 52 of microbody, links to each other with piezoelectric gene diagnosis chip testing circuit in circuit and the mainboard module 60.Above-mentioned microbody amasss circulation type detection cell 52, by detection cell 52 be installed in semiconductor heating on the detection cell and refrigeration device and temperature controller 53 thereof constitute, links to each other with control circuit with temperature detection in circuit and the mainboard module 60.The precision of above-mentioned piezoelectric gene diagnosis chip testing circuit frequency measurement is that 1Hz is following, the fluctuation of per minute is less than ± 1Hz, the precision that said temperature is measured is below ± 0.1 ℃, the heating and refrigerating speed 1.5 ℃/more than the sec, the fluctuation of per minute is less than ± 0.1 ℃ during constant temperature.
Foregoing circuit and mainboard module 60, for each module provides operating circuit and interface circuit, and micro-computer function, circuit and computer motherboard by above-mentioned each module constitute, computer motherboard adopts more than the Pentium II, good stable and extensibility is arranged, as the standard motherboard P3V133 in 2000 of Asus.Each modular circuit links to each other with computer motherboard by interface.
Above-mentioned piezoelectric gene diagnosis real-time quantitative analysis software is realized system's master control and data analysis.Analysis software is made of master control interface and data analysis interface.Above-mentioned master control interface by with above-mentioned each module subprogram communication, carry out system's master control, system initialization and detecting pattern setting, each functions of modules shows, detects subroutines such as data call and performance graph demonstration and constitutes.Above-mentioned data analysis interface provides typical curve and linear dimensions (slope, intercept and related coefficient) and target gene concentration.
The course of work of using above-mentioned piezoelectric gene diagnosis real-time quantitative analysis instrument of the present invention to implement piezoelectric gene diagnosis real-time quantitative analysis method of the present invention is as follows:
At first, in cleaning, common test tube such as Ependoff pipe, add 10-100 ready standard solution series of μ l and sample solution.Standard solution series comprises the solution of blank (target gene concentration is 0) and concentration known target gene group to be checked for two or more, is 0,0.1 * 10 as target gene concentration
-5Two standard solution of mol/L, or 0,0.1 * 10
-5, 1 * 10
-5Three standard solution of mol/L, or 0,0.1 * 10
-5, 0.5 * 10
-5, 1 * 10
-5Four standard solution of mol/L etc.In above-mentioned each solution test tube, add the gene diagnosis reagent that contains primer, Taq-DNA polymerase, deoxy-ribonucleoside triphosphate, target gene to be checked is contacted with piezoelectric gene diagnosis chip with the sample of corresponding primer, Taq-DNA polymerase, deoxy-ribonucleoside triphosphate, subsequently, put into each micropore of the microwell plate of specimen preparation module respectively.Then, by the input of master control interface and control panel set up standard solution concentration, sample sequence and target gene group to be checked thereof, the temperature and time of specimen preparation and the parameters such as temperature and time of temperature cycles.
Then, implement down and the dynamic the following step that shows by the programmed control of computer motherboard:
1. by temperature controller control, heated sample prepares module and prefabricated sample, and promptly heating-up temperature 85-99 ℃, and retention time 10-100 second.
2. mechanical arm moves to suction spindle in the sample tube in the predetermined microwell plate, opens peristaltic pump and draws sample and also inject detection cell, after liquid level sensor indication detection cell is full of sample, stops to draw, stops to inject.
3. by hybridizing-extend-temperature cycles of sex change, the temperature and time of temperature cycles is: 85-99 ℃ of sex change stage, 10-60 second, crossing phase 30-70 ℃, 10-60 second, extend stage 65-80 ℃, 10-60 second, as shown in Figure 3.In temperature cycles, on display, show in real time piezoelectric gene diagnosis chip each with the predetermined corresponding site frequency change-time curve of target gene group to be checked, as shown in Figure 4, and determine the frequency variable domain value in this site in the frequency change of first crossing phase according to each site, as 10-100Hz, when every dot frequency variation meets or exceeds the frequency variable domain value of setting, stop temperature cycles, and the cycle index when determining that each site reaches variable domain value frequently.
4. with detection cell heating and remain on denaturation temperature, begin to draw cleaning solution by suction spindle simultaneously and clean detection cell, treat the every dot frequency of piezoelectric gene diagnosis chip indicate each site to recover to detect before after the frequency, emptying detection cell solution is also delivered to the waste liquid storage bottle.
Repeat 2.～4. step operation, measure all the other samples.
5. after finishing the detection of all solution, by the data analysis interface, program processing data, result by standard solution series draws the typical curve that each target gene detects cycle index-target gene concentration logarithm, as shown in Figure 6, more per sample the cycle index that detects of target gene draw the concentration of sample target gene.
As previously mentioned, the present invention only needs less cycle index, usually be less than 10 times, just can reach target gene and detect required frequency change, as shown in Figure 5, solution to same concentration target gene, repeatedly measure the relation of frequency change and temperature cycles number of times, the result shows that reaching frequently, the cycle index of variable domain value has fabulous reappearance, but after this with the increase of temperature cycles number of times, the frequency change under different the mensuration differs big more.Therefore, comparing more than 15 times usually with real-time fluorescence quantitative PCR technology cycle index, gene quantification analysis of the present invention has higher accuracy and reappearance, and shorter advantage of time.
Claims (6)
1, the piezoelectric gene diagnosis real-time quantitative analysis method, it is characterized in that adopting piezoelectric gene diagnosis chip and frequency tester, on the electrod-array of piezoelectric gene diagnosis chip, be fixed with the probe of at least a disease gene, the primer that contains at least a target gene will be added, the Taq-DNA polymerase, the sample of the gene diagnosis reagent of deoxy-ribonucleoside triphosphate contacts with piezoelectric gene diagnosis chip, hybridize-extend-temperature cycles of sex change, its hybridization temperature is 30-70 ℃, elongating temperature is 65-80 ℃, denaturation temperature is 85-99 ℃, the starting point of temperature cycles is denaturation temperature 10-60 second, then hybridization temperature 10-60 second, terminal point is elongating temperature 10-60 second; At crossing phase, target gene and probe, primer based on sequence complementation hybridize combination, in the extension stage, the probe and the primer of hybridization are that template is carried out polymerase chain reaction,PCR with the target gene under the catalytic action of Taq-DNA polymerase, make probe and primer obtain extending, in the sex change stage, probe-target gene, primer-target gene heterocomplex dissociates, increase with the temperature cycles number of times, the sample mrna concentration that hits increases, the target gene amount amplification that combines with probe, electrode surface quality is increased and the frequency of each detection site changes, monitoring frequency changes in the temperature cycles overall process, the frequency change of first crossing phase is the thresholding that background noise is set to frequency change, when frequency change surpasses thresholding, stops temperature cycles, reach the required cycle index of thresholding and the sample linear relationship that the logarithm of mrna concentration exists that hits according to frequency change, target gene is carried out quantitative test with outer typical curve.
2, piezoelectric gene diagnosis real-time quantitative analysis method according to claim 1 is characterized in that said probe and primer are 20-30 base, sequence and target gene complementation and the unduplicated oligonucleotides of both sequences; Background noise is that G is the standard deviation of frequency change in the preset time, or is the maximum frequency of frequency change in the preset time and the extreme difference between the minimum frequency; The thresholding of frequency change is f
RFor the 2-10 of background noise doubly; It is N that frequency change reaches the required cycle index of thresholding
tWith the sample mrna concentration that hits be C
TThe pass be N
t=AlogC
T+ B, A and B are constant in the formula; Outer typical curve is that 2-10 contains each N of the standard solution gained correspondence of concentration known target gene after measured
tTo logC
TThe curve that mapping draws.
3, implement the piezoelectric gene diagnosis real-time quantitative analysis instrument of claim 1 or 2 described methods, comprise housing (10), control panel on the housing, display, the mainboard module (60) of power supply in the housing (20) and joining circuit thereof, microcomputer, analysis software is characterized in that having in the housing and circuit and the joining piezoelectric gene diagnosis chip detection module (50) that can once extract the specimen preparation module (30) of at least one clinical DNA sample, the location flow module (40) of selecting and carry sample, amplification and detect target gene of mainboard module.
4, piezoelectric gene diagnosis real-time quantitative analysis instrument according to claim 3 is characterized in that it is 0.2-2cm that said specimen preparation module (30) has by 2-100, volume
3The microwell plate (31) that constitutes of the micropore of placement test tube, the temperature controller (32) that is made of heating and refrigeration device and joining temperature sensor thereof is arranged.
5, piezoelectric gene diagnosis real-time quantitative analysis instrument according to claim 3, it is characterized in that said location flow module has the mechanical arm (41) that is driven by driving machine and the suction spindle (42) of linking to each other thereof, suction spindle, peristaltic pump (43), piezoelectric gene diagnosis chip detection module (50), waste liquid storage bottle (47) are communicated with successively, and the import of piezoelectric gene diagnosis chip detection module and outlet have liquid level sensor (44,45) respectively.
6, piezoelectric gene diagnosis real-time quantitative analysis instrument according to claim 3, it is characterized in that said piezoelectric gene diagnosis chip detection module (50) has the long-pending circulation type detection cell (52) of microbody and is encapsulated in wherein the piezoelectric gene diagnosis chip (51) that is made of piezoelectric gene sensor array, has semiconductor heating and refrigeration device and joining temperature controller (53) thereof on detection cell.
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| CN101403724B (en) * | 2008-10-08 | 2011-12-21 | 湖南大学 | Instrument and reagent for fast detection of microbe in blood specimen, and preparation method thereof |
| CN102604826A (en) * | 2012-03-16 | 2012-07-25 | 盛司潼 | Gene sequencing equipment |
| CN106681224A (en) * | 2017-01-24 | 2017-05-17 | 中南大学 | Method and system for quantitatively extracting solution |
| WO2022068937A1 (en) * | 2020-09-30 | 2022-04-07 | 富佳生技股份有限公司 | Nucleic acid testing device |
| CN114317221A (en) * | 2020-09-30 | 2022-04-12 | 富佳生技股份有限公司 | Nucleic acid detection host and nucleic acid detection equipment |
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2001
- 2001-07-10 CN CN 01108612 patent/CN1325028A/en active Pending
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| CN101403724B (en) * | 2008-10-08 | 2011-12-21 | 湖南大学 | Instrument and reagent for fast detection of microbe in blood specimen, and preparation method thereof |
| CN102604826A (en) * | 2012-03-16 | 2012-07-25 | 盛司潼 | Gene sequencing equipment |
| CN102604826B (en) * | 2012-03-16 | 2015-12-16 | 盛司潼 | A kind of gene sequencing equipment |
| CN106681224A (en) * | 2017-01-24 | 2017-05-17 | 中南大学 | Method and system for quantitatively extracting solution |
| CN106681224B (en) * | 2017-01-24 | 2019-04-19 | 中南大学 | A kind of method and system quantitatively extracting solution |
| WO2022068937A1 (en) * | 2020-09-30 | 2022-04-07 | 富佳生技股份有限公司 | Nucleic acid testing device |
| CN114317221A (en) * | 2020-09-30 | 2022-04-12 | 富佳生技股份有限公司 | Nucleic acid detection host and nucleic acid detection equipment |
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