CN107190002B - Portable pathogenic bacteria nucleic acid-extracting apparatus and detection method - Google Patents
Portable pathogenic bacteria nucleic acid-extracting apparatus and detection method Download PDFInfo
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- CN107190002B CN107190002B CN201710375817.0A CN201710375817A CN107190002B CN 107190002 B CN107190002 B CN 107190002B CN 201710375817 A CN201710375817 A CN 201710375817A CN 107190002 B CN107190002 B CN 107190002B
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N15/1013—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers by using magnetic beads
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- H02J7/0029—Circuit arrangements for charging or depolarising batteries or for supplying loads from batteries with safety or protection devices or circuits
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Abstract
The invention discloses a kind of portable pathogenic bacteria nucleic acid-extracting apparatus and detection methods, according to the working principle of nucleic acid-extracting apparatus, the device is mainly the extraction for carrying out nucleic acid, it altogether include three functional units: constant temperature unit, provide a system to suitable isothermal reaction environment, pathogenic bacteria are enabled quickly to hydrolyze, elute, the constant temperature unit of design can provide a system to settable, high-precision temperature environment；Man-machine interaction unit provides the user with operating function, sets and displays including temperature value, reaction time, reaction channel；Power supply unit, meets the needs of outdoor detection, and paper design power supply adaptor power supply and battery are powered two kinds of power supply modes.
Description
Technical field
The present invention relates to automation control area more particularly to a kind of portable pathogenic bacteria nucleic acid-extracting apparatus and detection sides
Method.
Background technique
In recent years, food safety affair takes place frequently, the listeria spp disease event, sramana's germ contamination thing in the U.S. of such as France
Part, the bird flu event in Hong Kong and H1N1 event popular recently etc., food-safety problem has become the very important world
Property problem.Production is flowed into check the primary food of disease carrying germ from source, and is checked on the food that will be come into the market from terminal, this
Just there is an urgent need to Fast Detection Technique and rapid detection systems, in particular for the portable quick detection system of base's detection
System.In the development of portable rapid detection system, a more fatal problem is then that germ nucleic acid-extracting apparatus is difficult to transport
It uses in on-site test.More and more quick detection devices now, such as the laboratory LT- of Japan's Rong Yan chemistry development
16alpha constant-temperature amplification gene detection system, the Genie II isothermal duplication fluorescence detection system that Opitigene company, Britain releases
System, this kind of detection system are free from germs pretreatment function, can be only done the work of nucleic acid reproduction and detection of nucleic acids.Do not have core
The detection system of sour abstraction function can only carry out nucleic acid extraction in laboratory, then do field experiment again.This just hinders scene
The development of rapid detection system.
Desk-top nucleic acid-extracting apparatus used in laboratory is large-scale automatic extracting device, and the plant automation degree is high,
Extraction efficiency is high, but quality is big, and volume is big, does not have battery for electric energy, is not suitable for the germ nucleic acid extraction at scene.For
The development for pushing domestic portable quick detection device, helps domestic equipment to tie up the market share, it is necessary to develop a kind of operation
Simply, quality is small, small in size, can battery powered nucleic acid-extracting apparatus.
Summary of the invention
The present invention is directed at least solve the technical problems existing in the prior art, especially innovatively have developed a kind of portable
Pathogenic bacteria extraction element.
Present invention design realizes a kind of portable pathogenic bacteria nucleic acid-extracting apparatus, the principle which is adsorbed based on magnetic bead
To extract nucleic acid, the constant high temperature hydrolysis environment and elution environment for being suitable for are provided to pathogenic bacteria, cracking, washing, elution can be passed through
Nucleic acid is extracted in equal operations, and whole process does not need instrument centrifugally operated, suitable for outdoor nucleic acid-extracting apparatus.Furthermore this is mentioned
Take device quality small, it is small in size, can battery power supply, and designed, designed extraction magnetic frame, the nucleic acid extraction at the scene of can carrying out.
In order to realize above-mentioned purpose of the invention, the invention discloses the function lists of portable pathogenic bacteria nucleic acid-extracting apparatus
Member, it is critical that including: power supply unit, microcontroller chip, temperature control unit, human-computer interaction module.
The system power supply structure is as shown in Figure 1, power supply unit includes three main modules, and battery charging module is to electricity
Pond charging, electric power detection module real-time detection battery capacity, switching module select to supply by having detected whether adapter access
Electric mode；The power input of the output end connection constant temperature unit of power supply unit, power supply unit output end are connected by voltage stabilizing chip
Connect the power input of main control unit and man-machine interaction unit.The control signal input of the constant temperature unit is connected to control core
The control signal output of piece, the heating sheet of constant temperature unit are connected to drain electrode and the source electrode of MOSFET, control the heating of heating sheet
Whether.The man-machine interaction unit includes digital tube display module and key display module, and control is signally attached to control core
The port I/O of piece.
Having the beneficial effect that for above scheme can be realized germ nucleic acid extraction by above-mentioned control circuit, can be by man-machine
Interactive unit realizes interactive function.
Invention additionally discloses the designs of a kind of structure of portable pathogenic bacteria nucleic acid-extracting apparatus and magnetic frame, crucial
It is, comprising: thermal insulation board 1, aluminum heating domain 2, control circuit board 4,96 orifice plate fixinig plates 6,96 orifice plates 7, handle 8, elastic force are solid
Clamp 9, heat-resistance polypropylene pipe sleeve 10, magnetic bar 11.
Heating sheet is fixed in heating domain 2 and thermal insulation board 1 groove, and temperature detector is fixed on the aperture in heating tank
Interior 3,96 orifice plates 7 are fixed on heating domain by fixinig plate 6.It is added in 96 orifice plates 7 and extracts reagent extraction germ nucleic acid.Handle
8 lower end mounting plates, the installation of fixed plate lower end are used for the elastic force fixed frame 9 of fixing heat-resistant polypropylene pipe sleeve 10, magnetic bar 11
It is threadedly secured on mounting plate.The nucleic acid extraction magnetic frame of appeal probes into 96 orifice plates, utilizes magnetic-adsorption little magnetic bead
Method extracts nucleic acid.
Having the beneficial effect that for above-mentioned technical proposal is effectively added by the structure of portable pathogenic bacteria nucleic acid-extracting apparatus
Heat, setting, nucleic acid extraction；It is reasonable in design, it is easy to carry.
The portable pathogenic bacteria nucleic acid-extracting apparatus, it is preferred that further include: the design of thermal insulation board 1
There is sulculus in the thermal insulation board 1, heating sheet, mountable heating domain 2 on heating sheet can be fixed.One side thermal insulation board 1
Scald when can prevent user from installing 96 orifice plate 7 keeps constant the temperature for heating domain.On the other hand, thermal insulation board 1 will control electricity
Road plate 4 is isolated with heating domain 2, reduces the interference of control circuit board 4.
Above-mentioned technical proposal has the beneficial effect that so that the more stable safety of constant temperature unit and control circuit board.
The portable pathogenic bacteria nucleic acid-extracting apparatus, it is preferred that further include: the design of magnetic frame
The magnetic frame that the present invention designs is handheld magnetic frame, the elastic force including handle 8 and fixed polypropylene pipe sleeve 10
Fixing clamp 9 enables a user to extract nucleic acid extraction using magnetic frame, does not need instrument centrifugally operated, suitable for open air
Nucleic acid-extracting apparatus.
Above-mentioned technical proposal has the beneficial effect that so that nucleic acid extraction is simple to operation.
Invention additionally discloses a kind of detection methods for portable pathogenic bacteria nucleic acid-extracting apparatus, it is critical that packet
It includes:
Step 1, germ and little magnetic bead are put into the 1st column and the 7th column of 96 orifice plates, and a certain amount of lysate is added and splits
Solve enzyme.And two column are heated to 90 DEG C, it is stirred up and down with magnetic frame, cracks pathogenic bacteria.Time about 15 minutes.
Step 2, cleaning solution is added in 2-5 column and 8-11 column, and the little magnetic bead of cracking nucleic acid will be had with magnetic frame
Washing column are successively transferred to, up and down concussion washing.Each column wash time 2 minutes.
Step 3, elution solution is added in the 6th column and the 12nd column, and the temperature of two column is heated to 80 DEG C.Magnetic frame is used again
The little magnetic bead with cracking nucleic acid of washes clean is put into elution column and carries out high-temperature elution, the germ core of purification can be obtained
Acid.
In conclusion by adopting the above-described technical solution, the beneficial effects of the present invention are:
A kind of portable pathogenic bacteria nucleic acid-extracting apparatus of the present invention, by the combination of biotechnology and automatic technology, its benefit
The principle being easily adsorbed on magnetic bead with nucleic acid extracts germ nucleic acid by the operation of germ cracking, washing, elution.It
Hardware configuration is relatively easy, and software is also to be write using portable higher C language, which can extract two kinds simultaneously every time
A kind of germ nucleic acid, extractable 8 samples of germ nucleic acid, improves extraction efficiency.The nucleic acid-extracting apparatus meets outdoor inspection
The demand of survey, by comparing with similar product currently on the market, the extraction element invented herein has following characteristics:
Portability
The size of entire extraction element is 230mm*200mm*60mm, compared to desk-top nucleic acid-extracting apparatus, its body
Product is smaller, and quality is small, easy to operate, easy to carry.Desk-top pathogenic bacteria nucleic acid-extracting apparatus must provide the alternating current of 220V
Source, and excessively than cumbersome, which has limited it to use in laboratory, is unable to complete outdoor nucleic acid extraction work.This
The DC power supply that the power supply adaptor used is 9V is invented, two pieces of concatenated lithium batteries can meet its work requirements, and again
For amount in 4kg or so, this allows it smoothly to complete outdoor extraction work.
Economy
From the device of economic the angle changing rate present apparatus and desk-top same type, its superiority can be clearly obtained: same
150,000 yuan of price, the full-automatic core of GenePurePlus needed for Auto-Pure series Full automatic instrument for extracting nucleic acid in the product of type
Sour 200,000 yuan of extraction purification instrument price, 180,000 yuan of Full automatic instrument for extracting nucleic acid price of the release of Hui Yan company；Of the invention is portable
The cost of formula pathogenic bacteria nucleic acid-extracting apparatus is insufficient its 1/10th.And desk-top nucleic acid extraction instrument is difficult to utilization on site in fact
It tests, is difficult to match in portable rapid detection system, so that the applicability of device is wider.
Additional aspect and advantage of the invention will be set forth in part in the description, and will partially become from the following description
Obviously, or practice through the invention is recognized.
Detailed description of the invention
Above-mentioned and/or additional aspect of the invention and advantage will become from the description of the embodiment in conjunction with the following figures
Obviously and it is readily appreciated that, in which:
Fig. 1 is system power supply schematic diagram；
Fig. 2 is the structure chart of portable pathogenic bacteria nucleic acid-extracting apparatus；
Fig. 3 is the design diagram of magnetic frame；
Fig. 4 is the working principle diagram of portable pathogenic bacteria nucleic acid-extracting apparatus；
Fig. 5 is the circuit design drawing of the digital tube display module in man-machine interaction unit；
Fig. 6 A-6B is the circuit design drawing of the key setup module of man-machine interaction unit；
Fig. 7 is the charging circuit design drawing of power supply unit；
Fig. 8 is the battery charger design drawing of power supply unit；
Fig. 9 is the linear fit curve graph for heating domain temperature and environment temperature；
Figure 10 is the main circuit design figure of power supply unit；
Figure 11 is the result analogous diagram of the Sliding mode variable structure control algorithm of constant temperature unit；
Figure 12 is the main flow chart of portable pathogenic bacteria nucleic acid-extracting apparatus.
Specific embodiment
The embodiment of the present invention is described below in detail, examples of the embodiments are shown in the accompanying drawings, wherein from beginning to end
Same or similar label indicates same or similar element or element with the same or similar functions.Below with reference to attached
The embodiment of figure description is exemplary, and for explaining only the invention, and is not considered as limiting the invention.
In the description of the present invention, it is to be understood that, term " longitudinal direction ", " transverse direction ", "upper", "lower", "front", "rear",
The orientation or positional relationship of the instructions such as "left", "right", "vertical", "horizontal", "top", "bottom" "inner", "outside" is based on attached drawing institute
The orientation or positional relationship shown, is merely for convenience of description of the present invention and simplification of the description, rather than the dress of indication or suggestion meaning
It sets or element must have a particular orientation, be constructed and operated in a specific orientation, therefore should not be understood as to limit of the invention
System.
In the description of the present invention, unless otherwise specified and limited, it should be noted that term " installation ", " connected ",
" connection " shall be understood in a broad sense, for example, it may be mechanical connection or electrical connection, the connection being also possible to inside two elements can
, can also indirectly connected through an intermediary, for the ordinary skill in the art to be to be connected directly, it can basis
Concrete condition understands the concrete meaning of above-mentioned term.
Portable pathogenic bacteria nucleic acid-extracting apparatus is to extract object with pathogenic bacteria, by high temperature by germ nucleic acid cleavage, and
Nucleic acid washing, elution will be cracked using magnetic bead absorption principle, the working principle of system is as shown in Figure 4.
Cracking in figure washs, is eluted in 96 orifice plates and completes, wherein pathogenic bacteria cracking and Nucleic Acid Elution need to exist
It is carried out under hot environment.It in cracking link, is moved up and down with magnetic frame, destroys the biological structure of cell, discharge nucleic acid, magnetic bead meeting
In conjunction with nucleic acid, under the influence of a magnetic field, the magnetic bar on nucleic acid-extracting apparatus siphons away magnetic bead, thus reaches extraction core
The purpose of acid.Hot environment in figure is settable, high-precision constant temperature unit.Entire nucleic acid extraction does not need centrifugation behaviour
Make, can manually complete, greatly facilitate the extraction of outdoor nucleic acid.
As shown in Figures 2 and 3, comprising: thermal insulation board 1, aluminum heat domain 2, control circuit board 4,96 orifice plate fixinig plates 6,96 holes
Plate 7, handle 8, elastic force fixing clamp 9, heat-resistance polypropylene pipe sleeve 10, magnetic bar 11.
Heating sheet is fixed in heating domain 2 and thermal insulation board 1 groove, and temperature detector is fixed on the aperture in heating tank
Interior 3,96 orifice plates 7 are fixed on heating domain by fixinig plate 6.It is added in 96 orifice plates 7 and extracts reagent extraction germ nucleic acid.Handle
8 lower end mounting plates, the installation of fixed plate lower end are used for the elastic force fixed frame 9 of fixing heat-resistant polypropylene pipe sleeve 10, magnetic bar 11
It is threadedly secured on mounting plate.The nucleic acid extraction magnetic frame of appeal probes into 96 orifice plates, utilizes magnetic-adsorption little magnetic bead
Method extracts nucleic acid.
Having the beneficial effect that for above-mentioned technical proposal is effectively added by the structure of portable pathogenic bacteria nucleic acid-extracting apparatus
Heat, setting, nucleic acid extraction；It is reasonable in design, it is easy to carry.
The portable pathogenic bacteria nucleic acid-extracting apparatus, it is preferred that further include: the design of thermal insulation board 1
There is sulculus in the thermal insulation board 1, heating sheet, mountable heating domain 2 on heating sheet can be fixed.One side thermal insulation board 1
Scald when can prevent user from installing 96 orifice plate 7 keeps constant the temperature for heating domain.On the other hand, thermal insulation board 1 will control electricity
Road plate 4 is isolated with heating domain 2, reduces the interference of control circuit board 4.
Above-mentioned technical proposal has the beneficial effect that so that the more stable safety of constant temperature unit and control circuit board.
The portable pathogenic bacteria nucleic acid-extracting apparatus, it is preferred that further include: the design of magnetic frame
The magnetic frame that the present invention designs is handheld magnetic frame, the elastic force including handle 8 and fixed polypropylene pipe sleeve 10
Fixing clamp 9 enables a user to extract nucleic acid extraction using magnetic frame, does not need instrument centrifugally operated, suitable for open air
Nucleic acid-extracting apparatus.
The man-machine interaction unit includes:
1. key module
The operation input of key module realization nucleic acid-extracting apparatus.It is there are two types of circuit connecting mode: non-matrix keyboard and
Matrix keyboard.Wherein what the I/O interface of occupancy system was few is the connection type of matrix keyboard；Rather than the I/O that matrix keyboard occupies
Interface resource is more；But matrix keyboard programming is complicated, suitable for more, the I/O interface by bond number and system more in short supply.Nucleic acid extraction
Key 4 needed for device need 2 I/O excuses using matrix press-key mode, rather than matrix keyboard mode needs 4 I/O interfaces, connects
Mouth number is not much different, therefore selects to program relatively simple non-matrix keyboard mode.Its circuit design drawing, as shown in figures 6 a-6b.
2. digital tube display module
The present invention uses common cathode charactron, due to the common end grounding of common cathode charactron, so selection three pole of NPN type
Pipe amplifies electric current.Charactron with control chip pin connect there are two types of connection type: static connection type and Dynamic link library side
Formula.Need charactron that there are 8 I/O interfaces in the case where static state connection, system needs 4 charactrons to show time and temperature
Value, therefore 32 I/O interfaces are needed, I/O interface overhead is larger.Same 4 charactrons, the mode of Dynamic link library lower needs 12
A I/O interface significantly reduces the expense of I/O interface, therefore the design of this charactron connection is by the way of Dynamic link library.Its
Circuit design drawing is as shown in Figure 5.
By the way of Dynamic link library, then its software is required to control number in such a way that circulation is lighted by sunset glow effect
The display of code pipe.
Said supply unit includes:
Power supply unit includes three charging module, battery capacity detection module, switching circuit, main circuit parts.
1. charging module
The charging module of battery is a critically important module in device.Consider what is selected first in actual design
Charging management chip it is safer reliable: the charging of battery is not simple constant-voltage charge, according to the characteristic of battery, charging
Preferably it is divided into three modes, the trickle charge under deep discharge conditions, the charging current under the mode is generally constant-current charge mould
15% under formula, it prevents from impacting battery excessive；It then is constant-current charge, the charging current under the mode generally can artificially be set
It sets；Final stage is constant-voltage charge, and charging current at this time is also smaller, reduces the impact to battery.Ferric phosphate lithium cell is in work
Temperature may increase during work, and temperature it is excessively high battery potential danger it is larger, it is therefore desirable to a temperature threshold is set
Value stops charging after battery temperature is more than this threshold value.Paper selects the thermistor of an abutting battery, when temperature is more than
Threshold value, charging pause, when battery temperature is restored in operating temperature, charging continues.Its circuit design drawing is as shown in Figure 7.
2. the design of electric power detection module
The size of cell voltage is not only related to battery state in which and related with the remaining electricity of battery.Battery is in and puts
When electricity condition, with the consumption of battery capacity, cell voltage reduces therewith.Pertinent literature shows cell voltage and battery capacity
In certain proportional relation.Remaining capacity can be predicted with cell voltage, discharge, recorded in discharge process after battery is full of
The relationship of remaining capacity and voltage, data and curves are put into program, then characterize residue by measuring the variation of cell voltage
The variation of electricity.Hardware can design a battery voltage detection circuit, detect the variation of cell voltage, and paper uses differential amplification
The method of circuit detects the real-time voltage of battery, by reading the detection voltage for amplifying circuit of checking the mark, to calculate the use of battery
Electric situation.The voltage measured and electricity relation table are then put into the ROM of controller by software, and electricity is obtained by way of tabling look-up
Situation.
3. switching module
Extraction element is divided into two parts, high-current module and low current module by size of current is flowed through.High current mould
Block is constant temperature unit, and low current module is man-machine interaction unit and main control chip and adaptor detection circuit.Battery directly to
Low current module for power supply has detected whether adapter access by the adaptor detection circuit in low current module, if there is adapter
Access is then directly powered by adapter to high-current module；If detecting no adapter access, main control chip controls MOSFET
It opens, is powered by MOSFET to high-current module by battery.Wherein, high-current module is thermostat module, remaining is low current
Module.Adapter can charge the battery in the case where accessing automatically by charging circuit.Electrical schematic is as shown in Figure 8.
4. main circuit
Low current module described in switching module is directly powered by battery.Wherein, the work of main control chip, charactron, key
Making voltage is 3.3V, and the operating voltage of isolated drive circuit is 12V.So needing cell voltage switching to 3.3V and 12V.Its electricity
Road principle is as shown in Figure 10.
DC-DC1 is WRB0505YD-6W isolated power supply, and 4.5-9V voltage can be converted to 5V, and electric current is full up to 1.2A
The current requirements of sufficient main control chip and charactron.And isolated power supply is isolated by low current module with high-current module, is reduced big
Interference of the current module to low current module.The 5V voltage of input is switched to by 3.3V by AMS1117-3.3 again, gives main control chip
And charactron and key are powered.5V voltage is switched to 12V and is powered to isolation drive chip by D1209NXS chip.
The constant temperature unit includes:
The essence of constant temperature unit is that a degeneration factor using temperature difference e as the input quantity of system passes through certain control
Algorithm processed calculates control amount u.The working principle of constant temperature unit is as shown in Figure 8.
Temperature sensor detects real-time temperature data T, is transmitted to microcontroller, and microcontroller is by collected temperature number
It is compared according to preset temperature value T0, obtains temperature error e, by certain control algolithm, controller provides control signal
U, the driving heater heating of control signal or stopping heating.By constantly detecting real time temperature, continuous adjustment control letter
Number, so that temperature difference going to zero as far as possible, and then reach the control higher constant temperature effect of precision.
Thermostatic control algorithm
Constant temperature unit is a unit being even more important in the present invention, and the germ of nucleic acid-extracting apparatus hydrolyzes, needed for elution
Constant high temperature environment be closely related with the unit.Therefore the selection of temperature control algorithm is particularly significant.
The model foundation of 1 constant temperature system
Heater of the paper using ceramic heating flake as constant temperature system, by heating sheet in METAL HEATING PROCESS domain,
Real time temperature is detected by the temperature sensor in heating domain, then temperature is controlled by the control signal u of controller.
According to the principle of thermodynamics and thermal conduction study, the mathematical model in heating domain is established.
Wherein, c is the specific heat capacity for heating domain, m is the quality for heating domain, the electricity that I is heated current, R is ceramic heating flake
Resistance, a are heat transfer coefficient, T0For environment temperature locating for heating domain.For the increased heat in heating domain, I2R is heating
The heat flow of piece generation, α A (T-T0) it is that the heat in environment is passed in heating domain.
Since nucleic acid-extracting apparatus is closed heating space, environment temperature cannot be considered as to ideal not alternating temperature
Degree.In airtight heating space, environment temperature can rise with heating domain temperature and be increased, and by largely testing, obtain ring
The relationship of border temperature and heating domain temperature is as shown in the table.
1 environment temperature of table and heating domain temperature
Environment temperature T in order to obtain0With heating domain temperature T mathematical relationship, we can the data according to table 3.1,
Data are fitted under MATLAB environment.
Since the temperature range of detection system constant temperature unit concentrates between 45 DEG C Dao -100 DEG C, in this section, data
Linear relationship is more obvious, and straight line fitting can greatly facilitate the design of controller, therefore paper selects the side of straight line fitting
Formula is fitted T0-T.Its matched curve is as shown in Figure 9.
Shown in Fig. 9, between 45 DEG C to 100 DEG C, temperature more rule is distributed in matched curve two sides.The fitting obtained
Equation are as follows:
T0=kT ± b
Wherein k=0.5885；B=-1.1346.
Therefore the mathematical model in domain is heated are as follows:
3.3 derivation of formula is obtained:
The mathematical model of constant temperature unit is a relatively simple second-order linear system.
2 Sliding mode variable structure control algorithms
Relatively common algorithm has expert, fuzzy, neural network, PID etc. in temperature control system, and this kind of algorithm does not need
The mathematical model of controlled device and be nonlinear control algolithm, however but need a large amount of engineering experience that can just design control
Device processed.Wherein algorithm the simplest and common is PID and fuzzy self-turning PID control algorithm, which realizes simply, but temperature
Degree overshoot is difficult to avoid that, in the wider situation of the temperature-control range of especially demand, then more seldom realizes the control of overshoot.Portable
In pathogenic bacteria nucleic acid-extracting apparatus, since the activity of enzyme is easy to inactivate at high temperature, excessive overshoot may or lead to experiment
Failure, being difficult to control based on overshoot and the deficiency of experience, select overshoot small, the Sliding mode variable structure control based on modeling
Algorithm.
The algorithm is substantially a kind of control algolithm that control structure is variable, and control structure is changeable, and works as system
It is highly stable when being moved on sliding mode.Secondly, this algorithm overshoot is small, system response is fast, regulating time is short, robust
Property it is good, similar even if the respective performances of different work dot systems, and to plant model error and external disturbance
Have the advantages that fabulous insensitive, is well suited for the temperature requirements of food-borne pathogens filed detection system constant temperature unit.Therefore originally
Control algolithm of the Sliding mode variable structure control algorithm as constant temperature unit is selected in invention.
The basic principle of Sliding mode variable structure control.In the control process of system, the changed system of control structure
Variable structure system is done, for example, expert system, Fuzzy PID Control System etc..Sliding mode variable structure control algorithm is also that a kind of structure can
The control algolithm of change according to the current state of controlled system, purposefully converts control structure, so that being in control process
System is moved according to preset " sliding mode " track.Due to sliding formwork dynamic can designed, designed and with object parameters and outer
Boundary's disturbance is unrelated, therefore control algolithm has many advantages, such as that response quickly, insensitive to Parameters variation and disturbance, robustness is good.?
In the design of algorithm, need to find a stable diverter surface first, allow on system motion to the diverter surface, then make system along
Sliding surface tends to origin.Since the diverter surface of design is inherently stable, do not need to analyze its stability.
From the above mentioned, the design of Sliding mode variable structure control algorithm.It mainly include the selection of switching function and setting for controller
Count two aspects.
1. the selection of switching function,
In single input system, switching function only one.By switching control in s=c1x1+x2On function, sliding die
State equation is
In multiple entry system, there are multiple switching functions, and since constant temperature unit is single-input single-output system, then this is small
Section only discusses the case where single-input single-output.
2. controller is sought
On the basis of determining switching function, its control can be acquired
Wherein ui -(x)≠ui +(x), so that design object below is achieved:
There are Fault slip rates for 1 diverter surface；
2 all phase paths will reach diverter surface in finite time；
3 sliding formworks move Asymptotic Stability and have good dynamic quality.
Sliding formwork movement includes two motion processes: approach movement and sliding formwork movement.When system gradually tends to from original state
Diverter surface, the as process of s → 0, this is approach procedure.In controller design, the method that Reaching Law can be used becomes to improve
The quality closely moved.For Reaching Law, acceleration type Reaching Law, constant value type Reaching Law and proportional-type Reaching Law can use.
The advantage and disadvantage of Sliding mode variable structure control algorithm.A advantage: when controlled system reach diverter surface after, will automatically along
Diverter surface movement, moves unrelated with parameter of system itself；The input signal of consecutive variations can preferably be tracked；Robustness
By force.B disadvantage: realize control algolithm, need to obtain whole state variables of system, this be it is relatively difficult, when control object is
When high order system, difficulty especially, therefore algorithm is difficult to apply to high order system, can only simplify systematic education；The process of switching
Middle switching switch is undesirable, easily generates shake, removes difficulties.
The design of 3 Sliding Mode Controllers
The system known to the modeling analysis of 3.5.1 is the second-order linear system of a single-input single-output, therefore it is slided
The switching function of mould variable structure control algorithm are as follows:
Wherein s is sliding formwork amount, T0For the preset value of temperature, e is temperature error,For the derivative of temperature error.
Then system once enters sliding formwork dynamic and should meet:
With guarantee system origin can be run to along sliding-mode surface.The solution of the differential equation are as follows:
Obviously, only work as c1For timing, variable structure system is stablized.
In order to improve the dynamic quality of system approach movement, paper selects exponentially approaching rule to design controller:
ζ > 0, k1> 0
Wherein ζ and k1All be can any value positive number, eliminate and buffet while in order to guarantee to fast approach, emulating
When, it should increasing k1When, reduce ζ.
Controller function has just been determined in this way:
Wherein c is the specific heat capacity for heating domain, value 880J/Kg；M is the quality for heating domain, value 0.055Kg；I is
Heated current, value 25A；R is the resistance of ceramic heating flake, and value is 1 Ω；A is the heat transfer coefficient for heating domain, and value is
237w/(m2.k)；A is the heat transfer area for heating domain, and being measured its value is 0.004408m2；c1, k1, ζ is can voluntarily value be just
Number.Bring parameter into controller, obtained constant temperature controller are as follows:
4 algorithm simulatings
It has obtained the mathematical model and controller model of constant temperature unit, then can carry out MATLAB/Simulink emulation.Each ginseng
Number is k1=30, c1=5；ζ=30.It is emulated with the s_function module in Simulink, result figure is amplified such as
Shown in Figure 11.
It was found from figure, on the one hand, Sliding mode variable structure control algorithm can control heating domain temperature well, and can be by temperature control essence
Degree control is at ± 0.2 DEG C；On the other hand, control system does not have temperature overshoot, this is even more important to detection system, excessive overshoot
It may result in reaction enzymes inactivation.From analogous diagram this it appears that the response of constant temperature unit is fast, error is small, enzyme overshoot excellent
Point, but as it can be seen that temperature has high-frequency slight jitter from the figure of amplification, this is one of Sliding mode variable structure control algorithm solid
There is feature.
The software configuration of portable nucleic acid-extracting apparatus
The main flow chart of portable pathogenic bacteria nucleic acid-extracting apparatus is as shown in figure 12, after nucleic acid-extracting apparatus booting, into
Enter system initialization link, to register, the initialization such as parameters amount and counter in the link；Initialization is completed
Afterwards, into parameter setting link, heating temperature, heating time and heating channel are set；Click and start again, into heating link,
And real-time display heating temperature and heating time；Heating can ends with system after terminating.
In the description of this specification, reference term " one embodiment ", " example ", " is specifically shown " some embodiments "
The description of example " or " some examples " etc. means specific features, structure, material or spy described in conjunction with this embodiment or example
Point is included at least one embodiment or example of the invention.In the present specification, schematic expression of the above terms are not
Centainly refer to identical embodiment or example.Moreover, particular features, structures, materials, or characteristics described can be any
One or more embodiment or examples in can be combined in any suitable manner.
Although an embodiment of the present invention has been shown and described, it will be understood by those skilled in the art that: not
A variety of change, modification, replacement and modification can be carried out to these embodiments in the case where being detached from the principle of the present invention and objective, this
The range of invention is defined by the claims and their equivalents.
Claims (4)
1. a kind of portable pathogenic bacteria nucleic acid-extracting apparatus characterized by comprising aluminum heats domain (2), thermal insulation board (1), control
Circuit board (4) processed, 96 orifice plates (7), 96 orifice plate fixinig plates (6)；Power supply unit, temperature control unit, man-machine interaction unit；
The power supply unit includes power supply adaptor and battery two kinds of power supply modes of power supply；The input of power supply adaptor offer 9V
Voltage, battery use the concatenated ferric phosphate lithium cell of two sections, provide the input voltage of 5V-7V；The output end of power supply unit connects
The power input of constant temperature unit, the power supply unit power output end switch to 3.3V by isolated from power chip and voltage stabilizing chip
The power input of connection control chip, while being also connected to the power input of man-machine interaction unit；
Heating sheet is fixed on the groove of (1) in heating domain (2) and thermal insulation board, and temperature detector is fixed on the aperture in heating tank
Interior (3), 96 orifice plates (7) are fixed on heating domain by fixinig plate (6)；It is added in 96 orifice plates (7) and extracts reagent extraction germ
Nucleic acid；
The nucleic acid extraction magnetic frame includes: handle (8), elastic force fixing clamp (9), heat-resistance polypropylene pipe sleeve (10), magnetic bar
(11)；
Handle (8) lower end mounting plate, elastic force of the fixed plate lower end installation for fixing heat-resistant polypropylene pipe sleeve (10) are fixed
Frame (9), magnetic bar (11) are threadedly secured on mounting plate；The nucleic acid extraction magnetic frame of appeal probes into 96 orifice plates, utilizes
The method of magnetic-adsorption little magnetic bead extracts nucleic acid；
Further include: heat domain (2) in four tunnels；
The heating domain (2) shares four tunnels, and 96 orifice plates (7) have 12 rows, and extraction operation includes cracking, washing 1, washing 2, washing
3, washing 4, elution, wherein cracking and elution need to be heated at high temperature, and wash room temperature, and then, one time extraction operation needs two
Domain is heated on road, and nucleic acid-extracting apparatus can once carry out the extraction of two kinds of germs；
The man-machine interaction unit further include: digital tube display module and key setup module, the display module and setting mould
Block is installed on shell, is connected on circuit board, for data and display to be arranged.
2. portable pathogenic bacteria nucleic acid-extracting apparatus according to claim 1, which is characterized in that the temperature control unit
It include: temperature sensor, ceramic heating flake and metal-oxide-semiconductor,
One end connection metal-oxide-semiconductor drain electrode of the heating sheet, the other end connect positive pole, and the power cathode connects metal-oxide-semiconductor source
Pole, the control signal that the metal-oxide-semiconductor grid connection control chip PWM is issued, the temperature sensor temperature signal input inspection
Heating domain temperature is surveyed, the temperature sensor temperature signal output end connection controller AD detects port, input temp signal.
3. portable pathogenic bacteria nucleic acid-extracting apparatus according to claim 1, which is characterized in that said supply unit packet
Include: charging module completes the charging to ferric phosphate lithium cell；Electric power detection module detects the electricity of battery；Switching module,
Free switching between battery power supply and adapter power supply.
4. a kind of extracting method for the described in any item portable pathogenic bacteria nucleic acid-extracting apparatus of claim 1-3, special
Sign is, comprising:
Step 1, corresponding liquid is added in 96 orifice plates:
1 to 6 column are separately added into lysate, cleaning solution, cleaning solution, cleaning solution, cleaning solution, eluent in 96 orifice plates, and the
Germ bacterium solution is added in one column；
Step 2, germ is heated by heater, completes the operation of germ cracking and elution, method for heating and controlling are as follows:
Temperature sensor detects real-time temperature data T, is transmitted to microcontroller, microcontroller by collected temperature data with
Preset temperature value T0 is compared, and obtains temperature error e, calculates control amount, controller by Sliding mode variable structure control algorithm
Provide control signal u, the driving heater heating of control signal or stopping heating；By constantly detecting real time temperature, constantly
Adjustment control signal so that temperature difference going to zero as far as possible, and then reach the control higher constant temperature effect of precision；
Step 3, the extraction to nucleic acid is completed using magnetic bead absorption principle,
The 1st column and the 6th column of 96 orifice plates are heated, and little magnetic bead is added in first row, using the magnetic frame in the 1st column
It moves up and down, destroys the biological structure of cell, released dna, magnetic bead can be in conjunction with DNA, under the influence of a magnetic field, nucleic acid extraction dress
The magnetic bar set siphons away magnetic bead, thus reaches the purpose for extracting nucleic acid；After extraction, small magnetic is washed in 2-5 column
Pearl, and in the 6th column elution, the germ nucleic acid that can must finally purify.
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|---|---|---|---|---|
| CN1431290A (en) * | 2003-01-22 | 2003-07-23 | 广州市光机电工程研究开发中心 | Device for automatic extracting nucleic acid |
| CN203174009U (en) * | 2013-01-15 | 2013-09-04 | 北京金麦格生物技术有限公司 | Working platform system for bioactive substance extraction instrument |
| CN103897987A (en) * | 2014-02-18 | 2014-07-02 | 中国农业大学 | Automatic extraction device and method of nucleic acid based on nano magnetic beads |
| CN104914045A (en) * | 2015-05-29 | 2015-09-16 | 重庆大学 | Portable type pathogenic bacterium rapid detection system and device |
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Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1431290A (en) * | 2003-01-22 | 2003-07-23 | 广州市光机电工程研究开发中心 | Device for automatic extracting nucleic acid |
| CN203174009U (en) * | 2013-01-15 | 2013-09-04 | 北京金麦格生物技术有限公司 | Working platform system for bioactive substance extraction instrument |
| CN103897987A (en) * | 2014-02-18 | 2014-07-02 | 中国农业大学 | Automatic extraction device and method of nucleic acid based on nano magnetic beads |
| CN104914045A (en) * | 2015-05-29 | 2015-09-16 | 重庆大学 | Portable type pathogenic bacterium rapid detection system and device |
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