CN109797097A - A kind of top illuminated nucleic acid isothermal amplification detection portable instrument - Google Patents

Abstract

The invention discloses a kind of top illuminated nucleic acid isothermal amplifications to detect portable instrument.Including fluorescence detector, reagent memory block, liquid-transfering gun the storage box, consumptive material the storage box, battery case, above-mentioned five part is placed in suitcase；Reagent memory block is connected with reagent storage box cover by reagent the storage box and is formed, and multiple centrifugation pores is provided in the reagent the storage box, reagent the storage box top is provided with reagent the storage box water filling port, and side wall is provided with reagent the storage box water level limit hole, using water as refrigerant；Fluorescence detector includes tool housing and sample frame module, control circuit module, fluorescence detection module, heating module and upper computer module.The present invention is directed to the fluorescence analysis method quantitative detection biological sample based on nucleic acid isothermal amplification technology, realize amplification and detection integration and high throughput, required sample to be tested is few, whole system is small in size easy to carry, measurement accuracy is high, detect the field quick detection use that speed is fast, suitable for food safety.

Description

A kind of top illuminated nucleic acid isothermal amplification detection portable instrument

Technical field

The present invention relates to a kind of isothermal duplication detectors in food safety mesophytization sample testing instrument field, especially relate to And a kind of top illuminated nucleic acid isothermal amplification detects portable instrument, combines mechanical electronic control to be formed nucleic acid isothermal amplification technology A set of automatic detection device system.

Background technique

Polymerase chain reaction (Polymerase chain reaction, PCR) is used as most common molecular biology skill One of art can have many advantages, such as high specificity, high sensitivity, simplicity, therefore wide for micro up to a million times of target DNA amplification It is general to be applied to field of detection of food safety.But Standard PCR technology has the disadvantage that expensive equipment, volume are very much not portable, It is not suitable for on-site test, Yi Yinqi non-specific amplification etc..

In recent years the nucleic acid isothermal amplification technology that new development is got up, either in terms of practical operation or instrument requirements, all More more simple and convenient than round pcr, it gets rid of the dependence to superior equipment, shows in food safety field quick detection Its good application prospect.Common isothermal amplification technique has: ring mediated isothermal amplification (Loop-mediated isothermal Amplification, LAMP), rely on amplification of nucleic acid sequences (Nucleic acid sequence-based Amplification, NASBA), rolling circle amplification (Rolling circle amplification, RCA), rely on untwist Enzymatic amplification technology (Helicase-dependent isothermal DNA amplification, HDA), recombinase polymerase Expand (Recombinase Polymerase Amplification, RPA) etc..Its reaction process of isothermal amplification technique is tieed up always It holds in stationary temperature, compared with round pcr, instrument requirements is greatly simplified, the time greatly shortens, and is more suitable for research and development just Formula instrument is taken for field quick detection.

After the completion of nucleic acid amplification, needs just to be capable of determining whether really to obtain by strict inspection expected specific amplification and produce Object, common method are gel electrophoresis (including agarose gel electrophoresis method and polyacrylamide gel electrophoresis), but should The method detection that needs to uncap easily causes Aerosol Pollution, has certain harm to human body.Numerous scholars explore new method such as: micropore Plate sandwich hybridization, fluorescence analysis, branched DNA etc., wherein fluorescence analysis has high sensitivity, spy compared to other methods The opposite sex is good and detects the advantages that quick and favors by researcher.

The phenomenon that fluorescence is a kind of luminescence generated by light.When light source irradiates Cucumber, that is, fluorescein, fluorescein can be issued not The light of co-wavelength；After removing light source, the light that fluorescein issues can also disappear therewith.In fluorescence analysis based on nucleic acid amplification Fluorescein mainly have fluorescent dye, fluorescence probe: (1) fluorescent dye such as SYBR series dyes, DAPI fluorescent dye, SYTOX Series dyes, EvaGreen, FAM etc.；(2) fluorescence probe such as Taqman technology, molecular beacons technology, complex probe etc..

Existing fluorescence detector often use xenon lamp, deuterium lamp, mercury lamp, light emitting diode (LED), laser diode etc. as swash Light emitting source, wherein xenon lamp, deuterium lamp, mercury lamp spectral bandwidth, energy are high, but bulky, expensive, and LED, laser diode Although small in size easy of integration, luminescence band is single to be difficult to meet multiple fluorescent substance excitation requirement, and currently based on the glimmering of LED The fixation of optical detector luminous power can not be adjusted, it is difficult to meet the excitation of various concentration determinand.The receiver of fluorescence detector Have many advantages, such as that detection is sensitive, response is fast frequently with photomultiplier tube (Photomultiplier, PMT), conventional PMT, but its body Product is larger, is not suitable for multichannel, portable inspectiont, expensive although having miniaturization PMT at present, substantially increases Instrument cost.In addition, most multichannel fluorescence detector eliminates the noise in different channels using same benchmark at present, but It is different channels since differences, the noises such as detecting instrument sensitivity, detection position, signal processing circuit have little bit different, Same benchmark is unfavorable for the consistency of multi-channel detection.

Summary of the invention

The purpose of the present invention is in view of the deficiencies of the prior art, to provide a kind of based on nucleic acid isothermal amplification technology, combination The fluorescence analysis method of fluorescent dye or fluorescence probe as marker, in conjunction with mechano-electronic and computer software control technology, It realizes the apparatus system being used for quickly detecting to one or more samples, has the characteristics that portable, inexpensive, easy to operate.

To achieve the above object, the technical solution adopted by the present invention mainly includes:

The present invention includes fluorescence detector, reagent memory block, liquid-transfering gun the storage box, consumptive material the storage box and battery case, above-mentioned Five parts are placed in suitcase；Wherein the reagent memory block is connected with reagent storage box cover by reagent the storage box and is formed, and is tried It is adsorbed between agent the storage box and reagent storage box cover by the flat thin magnet that two sides are installed；Be provided in the reagent the storage box it is multiple from Heart pore, for placing the centrifuge tube for holding low temperature storing reagent, pore size can carry out centrifugation pore according to the specification of centrifuge tube Adjustment；Reagent the storage box top is provided with reagent the storage box water filling port, and side wall is provided with reagent the storage box water level limit hole, with water As refrigerant.

The fluorescence detector includes tool housing and the sample frame module, the control circuit mould that are installed in tool housing Block, fluorescence detection module, heating module and upper computer module, tool housing top surface are provided with gap slot, install sample at gap slot Frame module, sample frame module upper end are in gap slot, and setting shell shading cover covers on gap slot, and sample frame module connects fluorescence Detection module, fluorescence detection module, heating module are electrically connected with control circuit module.

The sample frame module includes aluminum sample pond and sample container；Lower section at gap slot is fixed in aluminum sample pond, Aluminum sample pond top surface is equipped with multiple sample ports along linear interval arrangement, and each sample port is for placing sample container, sample Container upper end is open, and fills sample to be tested in sample container；Each sample port is the bellmouth that diameter reduces from top to bottom, aluminium Sample preparation product bottom of pond portion side be provided with it is multiple along linear interval arrangement light-conductive optic fibre holes, each light-conductive optic fibre hole respectively with each sample Product mouth, which is correspondingly connected with, to be communicated, and light-conductive optic fibre hole horizontal is deeply conducting in itself corresponding sample port bottom.

The heating module includes heating element fixing seat, heating element and temperature sensor, heating element fixing seat Built-in heating element, heating element geometric center are overlapped with heating element fixing seat geometric center, heating element fixing seat and aluminium The connection of sample preparation product pond underrun fastener；Temperature sensor location hole, temperature sensor positioning are additionally provided on aluminum sample pond Mounting temperature sensor at hole, temperature sensor, which detects the temperature in aluminum sample pond and then controls heating element heats, adjusts sample The sample container temperature of mouth.

The fluorescence detection module includes fluorescence excitation light source, fluorescent emission optical filter, light-conductive optic fibre and fluorescence detection Device；Fluorescence excitation light source fixed disk is fixed on shell shading cover bottom surface, fluorescence excitation light source fixed disk bottom surface peace by fastener Equipped with multiple fluorescence excitation light sources along linear interval arrangement, each fluorescence excitation light source is corresponding with each sample port respectively, glimmering Phot-luminescence source is located at right above itself corresponding sample port；Pass through the first fiber laser arrays head mounting base at each light-conductive optic fibre hole One end of a light-conductive optic fibre is connected, the first fiber laser arrays head mounting base at each light-conductive optic fibre hole is by the first light-conductive optic fibre Fixed disk is fixed on aluminum sample pond side；Fluorescent emission optical filter is installed in light-conductive optic fibre hole, emits filter center wavelength Value is selected according to fluorescence analysis method；In tool housing bottom be fixedly mounted control circuit board, each light-conductive optic fibre other end and The second light-conductive optic fibre fixed disk in control circuit board connects, and installs in the control circuit board in the second light-conductive optic fibre fixed disk glimmering Photodetector, light-conductive optic fibre end are fixed simultaneously by the second fiber laser arrays head mounting base for being embedded in the second light-conductive optic fibre fixed disk It is directed at fluorescence detector photosurface.

Existing nucleic acid isothermal amplification detection generallys use photomultiplier tube, and the present invention is glimmering using photodiode excitation Light emitting fluorescent light source is detected, and fluorescence detector is located in control circuit board 1.

Two threaded holes are additionally provided on the aluminum sample pond, fastener passes through two threaded holes for the aluminum sample Gap slot is fixed in pond；Fluorescence excitation light source fixed disk positioning spiro pit is additionally provided in the fluorescence excitation light source fixed disk, Fluorescence excitation light source fixed disk positioning spiro pit is for the fixed use of fluorescence excitation light source fixed disk and the aluminum sample pond.

The aluminum sample bottom of pond portion side opens up the first light-conductive optic fibre fixed disk positioning groove, and the first light-conductive optic fibre is solid Price fixing is in the first light-conductive optic fibre fixed disk positioning groove.

The fluorescence excitation light source fixed disk is equipped with multiple fluorescence excitation light source setting circles along linear interval arrangement Hole, installs fluorescence excitation light source at each fluorescence excitation light source positioning round orifice, the fluorescence excitation light source peak wavelength according to Fluorescence analysis method selection, it is appropriate that watt level can be carried out according to measured object concentration by selection driving current by operator It adjusts.

The tool housing includes upper shell and lower casing, is connected between upper shell and lower casing by shell fixation fastener It connects fixed in one.

The fluorescence detector uses photodiode, and each fluorescence detector detects the corresponding aluminum sample pond respectively In each sample port channel, different sample port channels according to detection wavelength of fluorescence select different fluorescence detectors, Huo Zhexuan Select the fluorescence detector of wide detection wave band.

The fluorescent emission optical filter is selected with fluorescence excitation light source fluorescence analysis according to used by reality Select replacement.

The excitation light source uses direct insertion LED, also can be adopting surface mounted LED.

The temperature sensor can be thermistor, platinum resistance, thermocouple etc.；The heating element can be ceramic heat Piece, Electric radiant Heating Film, heating wire, power resistor, nano indium tin metal oxide (Indium Tin Oxides, ITO) etc..

The control circuit module includes power module, main control module, fluorescence excitation light source drive module, fluorescence intensity letter Number conditioning module, temperature control modules, overheating protection module and communication module；Power module and main control module, fluorescent exciting Source drive module, fluorescence intensity signals conditioning module, temperature control modules, overheating protection module and communication module be electrically connected into Row power supply, main control module respectively with fluorescence excitation light source drive module, fluorescence intensity signals conditioning module, temperature control modules, Overheating protection module, communication module electrical connection, fluorescence excitation light source drive module are electrically connected fluorescence excitation light source, fluorescence intensity letter Number conditioning module is electrically connected fluorescence detector, and temperature control modules are electrically connected temperature sensor and heating element, overheating protection mould Block is installed on heating element, and communication module is connect with upper computer module.

The main control module further includes instrument running LED, and instrument running LED is installed on tool housing Outer surface is used for display working condition.

The invention has the advantages that:

1, the present invention realizes the one of amplification and testing process in the fluorescence analysis based on nucleic acid isothermal amplification technology Change, fluorescence detector and required auxiliary tool are integrated in a suitcase, real-time live detects, is simple and convenient.

2, the fluorescence analysis that the present invention uses is relatively simple, and detects time-consuming shorter；It can be to expansion after the completion of detection Increase production object and carry out solubility curve analysis, avoids Aerosol Pollution caused by carrying out product verifying of uncapping in biochemistry detection, and institute It is disposable to state eight connecting leg of PCR, improves the accuracy of detection.

3, the present invention is using LED as excitation light source, small in size, low in energy consumption, long working life, while excitation light source power Adjustable, suitable for various concentration gradient determinand；It selects photodiode to replace PMT as fluorescence detection device, drops significantly Low cost reduces equipment instrument, is suitable for field quick detection；The optical path system that excitation module and receiving module group are orthogonal simultaneously System improves instrument signal to noise ratio.

4, optical filter and excitation light source can carry out selection change according to fluorescence analysis in fluorescence detection module of the present invention, can To realize various samples while detect, detection efficiency is improved.

5, upper computer module of the present invention is not limited solely to PC machine, it is possible to use mobile phone, single-chip microcontroller with touch screen etc. are set It is standby, widen use scope of the invention.

For synthesis, the present invention is directed to the fluorescence analysis method quantitative detection biochemistry sample based on nucleic acid isothermal amplification technology Product realize amplification and detection integration and high throughput, and required sample to be tested is few, whole system is small in size easy to carry, measurement Precision is high, and detection speed is fast, and the field quick detection suitable for food safety uses.

Detailed description of the invention

Fig. 1 is the schematic diagram of the mechanical structure module of fluorescence detector of the present invention

Fig. 2 is the structural front view in the aluminum sample pond of fluorescence detector of the present invention.

Fig. 3 is the structure top view in the aluminum sample pond of fluorescence detector of the present invention.

Fig. 4 is the fluorescence excitation light source fixed plate top view of fluorescence detector of the present invention.

Fig. 5 is the main view of fluorescence detector of the present invention.

Fig. 6 is the frame construction drawing of fluorescence detector of the present invention.

Fig. 7 is the frame construction drawing that plate module is controlled in fluorescence detector of the present invention.

Fig. 8 is the mechanical structure schematic diagram of reagent memory block of the present invention.

Fig. 8 (a) is the reagent the storage box tomograph of reagent memory block.

Fig. 8 (b) is the reagent storage box cover tomograph of reagent memory block.

Fig. 9 is composition schematic diagram inside suitcase of the present invention.

Figure 10 is overhaul flow chart of the invention.

In figure: control circuit board 1, the second light-conductive optic fibre fixed disk 2, the second fiber laser arrays head mounting base 3, light-conductive optic fibre 4, Instrument running LED 5, tool housing 6, shell shading cover 7, fluorescence excitation light source fixed disk 8, fluorescence excitation light source 9, Sample container 10, the first fiber laser arrays head mounting base 11, the first light-conductive optic fibre fixed disk 12, aluminum sample pond 13, heating element Fixing seat 14, upper lower casing fixation fastener 15；Temperature sensor location hole 16, fluorescent emission optical filter 17, the first guide-lighting light Fine fixed disk positioning groove 18, fluorescence excitation light source fixed disk positioning spiro pit 19, fluorescence excitation light source positioning round orifice 20, reagent The storage box 21, flat thin magnet 22 are centrifuged pore 23, and reagent the storage box water filling port 24, reagent the storage box water level limit hole 25, reagent is deposited Storage box lid 26, suitcase 27, liquid-transfering gun the storage box 28, consumptive material the storage box 29, battery case 30, fluorescence detector 31.

Specific embodiment

With reference to the accompanying drawings of the specification, the present invention will be further described, but the invention is not limited to following embodiments.

As shown in figure 9, present invention specific implementation includes fluorescence detector 31, reagent memory block, liquid-transfering gun the storage box 28, consumption Material the storage box 29, battery case 30, above five part are placed in suitcase 27；As shown in Fig. 8 (a) and Fig. 8 (b), wherein reagent Memory block is made of reagent the storage box 21 and reagent storage box cover 26, and the two is adsorbed by flat thin magnet 22.In reagent the storage box 21 It is provided with multiple centrifugation pores 23, in Fig. 8 by taking three holes as an example, for placing centrifuge tube, is held in centrifuge tube and low temperature is needed to store Reagent, pore size can be adjusted according to the specification of centrifuge tube；22 top of reagent the storage box is provided with reagent the storage box water filling port 24, side wall is provided with reagent the storage box water level limit hole 25, is more than maximum injecting quantity Shi Shuicong reagent the storage box water level limit hole 25 Outflow, using water as refrigerant, convenient pollution-free and can Reusability, refrigeration effect is 5-7 hours sustainable.

As shown in Figure 6 and Figure 7, the specific implementation of fluorescence detector 31 of the present invention includes tool housing 6 and is installed on outside instrument Sample frame module, control circuit module, fluorescence detection module, heating module and upper computer module in shell 6, sample frame module, Fluorescence detection module, heating module and control circuit module form monitor station device, and 6 top surface of tool housing is provided with gap slot, lack Sample frame module is installed, sample frame module upper end is in gap slot, and setting shell shading cover 7 covers on gap slot, sample at mouth slot Product frame module connects fluorescence detection module, and fluorescence detection module, heating module are electrically connected with control circuit module；Host computer Module uses PC machine or industrial control all-in-one machine, but not limited to this.

As shown in Figure 1 and Figure 5, sample frame module includes aluminum sample pond 13 and sample container 10；Aluminum sample pond 13 is solid Lower section, aluminum sample pond 13 are fixed on shell 6 by fastener at gap slot, and 13 top surface of aluminum sample pond is equipped with multiple Along the sample port of linear interval arrangement, each sample port holds for placing sample container 10,10 upper end opening of sample container, sample Sample to be tested is filled in device 10, aluminum sample pond 13 carries out heat preservation and shading to sample container 10 (eight connecting leg of PCR)；Each sample Product mouth is the tubaeform bellmouth that diameter reduces from top to bottom, and 13 bottom sides of aluminum sample pond are provided with multiple along linear interval The light-conductive optic fibre hole of arrangement, light-conductive optic fibre hole number is identical with sample port quantity, each light-conductive optic fibre hole respectively with each sample Mouth, which is correspondingly connected with, to be communicated, and light-conductive optic fibre hole horizontal is deeply conducting in itself corresponding sample port bottom.

As depicted in figs. 1 and 2, heating module includes heating element fixing seat 14, heating element and temperature sensor, heating 14 built-in heating element of element fixing seat, heating element geometric center are overlapped with 14 geometric center of heating element fixing seat, heating Element fixing seat 14 is connect with 13 underrun fastener of aluminum sample pond；It is fixed that temperature sensor is additionally provided on aluminum sample pond 13 Position hole 16, mounting temperature sensor at temperature sensor location hole 16, temperature sensor detect the temperature in aluminum sample pond 13 into And control 10 temperature of sample container of heating element heats adjustment sample port.

As shown in figures 1 and 3, fluorescence detection module includes fluorescence excitation light source 9, fluorescent emission optical filter, light-conductive optic fibre 4 And fluorescence detector；Fluorescence excitation light source fixed disk 8 is fixed on 7 bottom surface of shell shading cover, fluorescence excitation light source by fastener 8 bottom surface of fixed disk is equipped with multiple fluorescence excitation light sources 9 along linear interval arrangement, 9 quantity of fluorescence excitation light source and sample port Quantity is identical, and each fluorescence excitation light source 9 is corresponding with each sample port respectively, and fluorescence excitation light source 9 is located at itself corresponding sample Right above product mouth；One end of a light-conductive optic fibre 4 is connected at each light-conductive optic fibre hole by the first fiber laser arrays head mounting base 11, The first fiber laser arrays head mounting base 11 at each light-conductive optic fibre hole is fixed on aluminum sample by the first light-conductive optic fibre fixed disk 12 13 side of product pond；Fluorescent emission optical filter (17) are installed in light-conductive optic fibre hole, transmitting filter center wavelength value is according to fluorescence point Analyse method choice；Control circuit board 1, each 4 other end of light-conductive optic fibre and control circuit board are fixedly mounted in 6 bottom of tool housing The second light-conductive optic fibre fixed disk 2 connection on 1, light-conductive optic fibre 4 are connected to the second guide-lighting light through the second fiber laser arrays head mounting base 3 Fine fixed disk 2 installs fluorescence detector in the control circuit board 1 in second light-conductive optic fibre fixed disk 2, and 4 end of light-conductive optic fibre is logical It crosses the second fiber laser arrays head mounting base 3 fixation for being embedded in the second light-conductive optic fibre fixed disk 2 and is directed at fluorescence detector photosurface.

When the lid of shell shading cover 7 is located at gap slot, the fluorescence excitation light source 9 in fluorescence excitation light source fixed disk 8 is just To the sample port in aluminum sample pond 13, eight sample ports are arranged for placing eight connecting leg of PCR in specific implementation.

As shown in Figure 3 and Figure 4, two threaded holes are additionally provided on aluminum sample pond 13, fastener will by two threaded holes Gap slot is fixed in the aluminum sample pond 13；It is fixed that fluorescence excitation light source fixed disk is additionally provided in fluorescence excitation light source fixed disk 8 Position threaded hole 19, fluorescence excitation light source fixed disk positioning spiro pit 19 is for fluorescence excitation light source fixed disk and aluminum sample pond 13 It is fixed to use.

13 bottom sides of aluminum sample pond open up the first light-conductive optic fibre fixed disk positioning groove 18, and the first light-conductive optic fibre is fixed Disk 12 is in the first light-conductive optic fibre fixed disk positioning groove 18.

Fluorescence excitation light source fixed disk 8 is equipped with multiple fluorescence excitation light source positioning round orifice 20 along linear interval arrangement, Fluorescence excitation light source 9 is installed at each fluorescence excitation light source positioning round orifice 20；9 peak wavelength of fluorescence excitation light source is according to fluorescence point Method choice is analysed, watt level can suitably be adjusted according to measured object concentration by selection driving current by operator.

Tool housing 6 includes upper shell and lower casing, is connected between upper shell and lower casing by shell fixation fastener 15 Fixed is in one.Shell fixation fastener 15 can connection bolt between internal lobes and its internal lobes.

Fluorescence detector uses photodiode, and each fluorescence detector detects each in corresponding aluminum sample pond 13 respectively There is individual detector in a sample port channel, i.e., single channel, and different sample port channels are different according to detection wavelength of fluorescence selection Fluorescence detector, or the fluorescence detector of the wide detection wave band of selection.

Control circuit module includes power module, main control module, fluorescence excitation light source drive module, fluorescence intensity signals tune Manage module, temperature control modules, overheating protection module and communication module；Power module and main control module, fluorescence excitation light source drive Dynamic model block, fluorescence intensity signals conditioning module, temperature control modules, overheating protection module and communication module electrical connection are supplied Electricity, main control module respectively with fluorescence excitation light source drive module, fluorescence intensity signals conditioning module, temperature control modules, overheat Protective module, communication module electrical connection, fluorescence excitation light source drive module are electrically connected fluorescence excitation light source 9, fluorescence intensity signals Conditioning module is electrically connected fluorescence detector, and temperature control modules are electrically connected temperature sensor and heating element, overheating protection module Heating element, communication module and upper computer module is installed on to connect by the modes such as bluetooth module, USB, UART.

Temperature control modules in main control module pass through control algolithm such as PID control, dahlin algorithm, Smith Prediction Control Deng output pwm pulse to heating element and then the heated condition of control heating element, to adjust 13 temperature of aluminum sample pond.

Overheating protection module is made of self-recovery fuse and several electronic components, is pressed in heating element and sample cell 13 back sides guarantee that detection device will not be excessively high due to temperature and there are safety problems.

Fluorescence intensity signals conditioning module includes sequentially connected signal acquisition unit, signal filter unit, signal amplification Unit, data A/D converting unit.

Main control module further includes instrument running LED 5, and instrument running LED 5 is installed on tool housing 6 Outer surface is used for display working condition.When device is powered and instrument running LED 5 shows orange when not running, work as dress Instrument running LED 5 shows green when setting energization and running, and is not run or is sent out by host computer order when device is powered Instrument running LED 5 is displayed in red when raw failure.

Main software component part of the upper computer module as entire detection system, passes through bluetooth module, USB, UART etc. Mode is communicated with control circuit module, and then controls the operation of detection device, and real-time reception detects obtained fluorescence Strength signal.

Upper computer module realizes the regulation of heating block temperature by the temperature control modules in control main control module, upper Machine module controls fluorescence detection module simultaneously, including fluorescence excitation light source drives channel selecting, fluorescence excitation light source driving current Then selection, acquisition fluorescence intensity signals receive the data obtained and the analyses processing such as are filtered, normalizes, show that detection is tied Fruit real-time display simultaneously generates testing result output.Wherein noise is eliminated in normalizing analysis, and the elimination of different channel noises is using system Detection gained background signal when the signal that one standard collects in real time cuts no specimen, but different channels do not use same number Value is eliminated.

Upper computer module of the present invention can also realize following two function by controlling heating module:

1) extraction of sample segment nucleic acid at high temperature, such as e. coli dna are realized, field quick detection is conducive to；

2) solubility curve is generated by gradually increasing temperature real-time monitoring fluorescence signal, the reaction different to determination produces Object, including nonspecific products improve detection accuracy.

When it is implemented, reagent memory block can be placed in the reagent for partially needing low temperature to store, it will be used for what reagent pipetted Liquid-transfering gun is placed in liquid-transfering gun the storage box 28, will test required consumptive material (liquid transfer gun head, centrifuge tube, sample container 10, but unlimited In this) be placed in consumptive material the storage box 29, by powering device battery (lithium battery, but not limited to this) be placed in battery area 30 and glimmering Optical detector 31 is placed in suitcase 27, is used for field quick detection.

Before detection, by sample container 10 (eight connecting leg of PCR, but not limited to this) be put into aluminum sample pond 13, it is ensured that sample 10 bottom centre of container is overlapped with the center of fluorescence excitation light source positioning round orifice 20, needs in detection process shell shading cover 7 is complete The influence to reduce external environmental light to testing result is closed entirely.The selection of the fluorescence analysis according to used by reality is suitable glimmering Light emitting optical filter and fluorescence excitation light source 9, such as fluorescent emission optical filter can choose the peak wavelength of west wind Optical Co., Ltd The LED that peak wavelength is 470nm can be used in the optical filter for being 20nm for 520nm, half-wave width, fluorescence excitation light source 9, but unlimited In this；Transmitting optical filtering disk is coaxial fixed with fluorescent emission optical filter fixation hole 17, and fluorescence excitation light source 9 is excited by fluorescence Positioning spiro pit 19 in light source fixed disk 8 is fixed on the bottom surface in aluminum sample pond 13.Added according to what actual installation was met the requirements Temperature sensor and heating element in thermal modules, it is solid that temperature sensor is placed in 13 bottom of sample cell in the sample frame module Determine in temperature sensor location hole 16, the temperature of the temperature control reaction sample by detecting aluminum sample pond 13, so that Sample temperature meets the temperature requirement of nucleic acid isothermal amplification technology, and the ceramics that Jiangsu Milky Way electronics corporation can be used in heating element add Backing (220V, 30W), but not limited to this.

When detection, software section of the upper computer module as entire detection system, as laboratory technician to entire detection system Row control and interpretation of result human-computer interaction, be connected with control circuit board, to control heating module and fluorescence detection mould Block, hardware components complete entire testing process under the control of software section, and real-time reception detects obtained fluorescence intensity Signal.When in control circuit board communication module and PC machine connected by the modes such as bluetooth module, USB, UART after, receive it is upper The order that machine is sent, the temperature control modules in main control module export pwm pulse to heating element switch pipe and then control heating The heated condition of element adjusts to obtain set temperature by pid algorithm, while according to temperature sensor Real-time Feedback sample cell 13 Actual temperature, and then adjust 13 temperature of sample cell.Overheating protection module is made of self-recovery fuse and several electronic components, It is pressed in heating element and 13 back side of sample cell, guarantees that detection device will not be excessively high due to temperature and there are safety problems. After starting detection, the fluorescence that sample issues is received by light-conductive optic fibre 4 by fluorescence detection device, is improved by fluorescence intensity signals Upper computer module is sent to after resume module, in graph form real-time display；Fluorescence intensity signals conditioning module includes signal Acquisition unit, signal filter unit, signal amplification unit, data A/D converting unit.Main control module includes that instrument operating status refers to Show lamp 5, when device is powered and instrument running LED 5 shows orange, the instrument when device is powered and runs when not running Running LED 5 shows green, and when device is powered and is not run or broken down by host computer order, instrument runs shape State indicator light 5 is displayed in red.

In specific implementation, upper computer module uses computer, provides the control analytic function when detecting to whole system, It is communicated by the interface modes control circuit board such as USB or UART.It is used as a personal-machine interactive interface simultaneously, makes laboratory technician The running that detection system hardware components can be directly controlled is finally completed entire testing process.When beginning, need logical to instrument News, the selection in fluorescence excitation light source driving channel, reaction temperature, detection time carry out initial setting up.To institute in upper computer module Raw florescent intensity signal data the analyses such as is filtered, normalize and handles, obtain testing result real-time display and generate inspection Survey result output.To fluorescence intensity signals value, the measurement of 13 real time temperature of sample cell after gained raw florescent intensity signal value, processing The real-time display of value and last testing result, and can be stored in all setting parameters being related in entire detection process, Current detection environment and testing result.Upper computer module can also control detection device by control heating module and expand sample Increase production object and carries out solubility curve analysis.

As shown in Figure 10, fluorescent dye SYTO9 is used with fluorescent material in fluorescence analysis, using nucleic acid isothermal amplification skill For loop-mediated isothermal amplification technique (LAMP) detection Citrus Huanglongbing pathogen in art, the specific implementation course of work of the invention is such as Under:

1) prepare liquid prepares: reagent needed for taking out from reagent memory block dissolves after shaking up according to conventional nucleic acid augmentation detection Method pipettes required reagent by liquid-transfering gun and configures prepare liquid.

1. configuring determinand solution；

2. configuring the fluorescent dye SYTO9 solution (in the present embodiment for 4 μM) of suitable concentration, guaranteeing that detection is sensitive Fluorescent dye is reduced to the greatest extent to the inhibiting effect of nucleic acid amplification under the premise of degree and accuracy；

3. taking appropriate prepare liquid to mix with suitable SYTO9 solution according to testing requirements and being allowed to reaction bonded；

2) according to detection demand, fluorescence detector 31 installs suitable transmitting optical filter and 9 (this implementation of fluorescence excitation light source Fluorescent emission optical filter selects the optical filter that central wavelength is 20nm for 520nm, half-wave width in example, and fluorescence excitation light source 9 uses Peak wavelength is the LED of 470nm), sample container 10 selects suitable eight connecting leg of PCR, is put into aluminum sample pond 13, eight connecting legs Channel 1 in the corresponding host computer of upper label 1, after placing sample container 10, closes shell shading cover 7, after determining no light leakage, Connect battery area 30 battery and fluorescence detector 31, open the power supply of hardware components, make the components such as fluorescence excitation light source into Enter working condition, instrument running LED 5 shows orange；

3) selection, the reaction temperature in fluorescence excitation light source driving channel system initialization: are provided in upper computer module The testing conditions parameter such as degree, detection time, LED drive current makes hardware components be returned to original state (this reality after confirming parameter Apply selector channel 4 in example, reaction temperature be set as 65 DEG C, detection time 45min, LED drive current be set as 20mA)；

4) it expands and detects: detecting heating to test sample for position to being currently at, while detecting fluorescence signal intensity, Connection ceramic heating element heating sample cell 13 makes the reaction solution in sample container 10 reach 65 DEG C of LAMP reaction temperature, opens Fluorescence excitation light source 9 irradiates this solution, and excitation fluorescent dye generates fluorescence, and fluorescent acceptor receives the fluorescence intensity letter generated Number, amplified reaction data are obtained, instrument running LED 5 shows green；

5) amplified reaction data acquisition and display: when heating element is heated to setting reaction temperature from room temperature, fluorescence reception Device sends received fluorescence intensity signals to host computer (this reality by the modes such as bluetooth module, USB, UART by certain frequency Apply in example and transmitted using bluetooth module), upper computer module is by received original amplification data real-time display；

6) solubility curve is analyzed: part test experience needs to carry out molten to verify whether amplified production is target product Solution curve analysis, after the completion of amplified reaction, sets 95 DEG C for reaction temperature in upper computer module, heating time is set as 5min puts out a fire to enzyme in reaction solution, should not acquire fluorescence intensity signals in the process, then weight when instrument is cooled to 50 DEG C Newly be warming up to 95 DEG C, this stage fluorescent acceptor by certain frequency by received fluorescence intensity signals by bluetooth module, USB, The modes such as UART are sent to host computer (in the present embodiment using bluetooth module transmit), and upper computer module is by received original amplification Data real-time display；

7) data processing and save: upper computer module is filtered the fluorescence intensity signals data collected, normalizing The processing such as change, and testing result is calculated according to related algorithm, it then shows, save:

1. received original amplification data is filtered using digital averaging filtering method, 5 sampled values is continuously taken to carry out Arithmetic average operation excludes random interfering signal；

2. filtered data are normalized --- background signal is rejected, first to the data of background phase into The fitting of row least square method, then cuts the ambient noise estimated out from filtered data；

3. reaching the proliferation time that programmed threshold values are passed through according to the fluorescence intensity signals after normalized, judge Whether contain object in prepare liquid；

8) check data, rehabilitation, measurement terminates: upper computer module executes stop signal, heating module and fluorescence inspection It surveys module and be in run-stopping status, instrument running LED 5 shows orange, then opens monitor station, takes out sample appearance Device 10 discards used eight connecting leg of disposable PCR, closes power supply.

Disregard prepare liquid setup time, entire wheel detection is expanded including sample to be tested and detected total by taking octal as an example Time can be controlled within 30 minutes, realized high-throughput and quickly detection；Simultaneously changeable type transmitting optical filter with The excitation light source driving current of fluorescence excitation light source 9 and optional size, is suitable for different fluorescence analysis, and detection function is rich It is rich；And will test device and required auxiliary tool is integrated in a suitcase, it is convenient for on-site test, it is seen that skill of the invention Art significant effect is prominent.

Above-mentioned specific embodiment rather than is limited the invention for illustrating the present invention, in the present invention Spirit and scope of protection of the claims in, to the present invention any modification and correcting made, both fall within protection of the invention Range.

Claims (9)

1. a kind of top illuminated nucleic acid isothermal amplification detects portable instrument, it is characterised in that: deposited including fluorescence detector (31), reagent Storage area, liquid-transfering gun the storage box (28), consumptive material the storage box (29) and battery case (30), above-mentioned five part are placed in suitcase (27) In；Wherein the reagent memory block is made of reagent the storage box (21) and reagent storage box cover (26) connection, reagent the storage box (21) it is adsorbed between reagent storage box cover (26) by the flat thin magnet (22) that two sides are installed；It is opened in the reagent the storage box (21) There are multiple centrifugation pores (23), for placing the centrifuge tube for holding low temperature storing reagent, pore size can basis for centrifugation pore (23) The specification of centrifuge tube is adjusted；Reagent the storage box (21) top is provided with reagent the storage box water filling port (24), and side wall is provided with Reagent the storage box water level limit hole (25), using water as refrigerant.

2. a kind of top illuminated nucleic acid isothermal amplification according to claim 1 detects portable instrument, it is characterised in that: described glimmering Optical detector (32) includes tool housing (6) and the sample frame module being installed in tool housing (6), control circuit module, glimmering Light detection module, heating module and upper computer module, tool housing (6) top surface are provided with gap slot, install specimen holder at gap slot Module, sample frame module upper end are in gap slot, and setting shell shading cover (7) covers on gap slot, and the connection of sample frame module is glimmering Light detection module, fluorescence detection module, heating module are electrically connected with control circuit module；

The sample frame module includes aluminum sample pond (13) and sample container (10)；Gap slot is fixed in aluminum sample pond (13) Place lower section, aluminum sample pond (13) top surface are equipped with multiple sample ports along linear interval arrangement, and each sample port is for placing sample Product container (10), sample container (10) upper end opening, sample container (10) is interior to fill sample to be tested；Each sample port is from upper The bellmouth reduced to lower diameter, aluminum sample pond (13) bottom sides are provided with multiple light-conductive optic fibres along linear interval arrangement Hole, each light-conductive optic fibre hole is correspondingly connected with each sample port respectively to be communicated, and it is right that light-conductive optic fibre hole horizontal is deeply conducting to itself In the sample port bottom answered；

The heating module includes heating element fixing seat (14), heating element and temperature sensor, heating element fixing seat (14) built-in heating element, heating element geometric center are overlapped with heating element fixing seat (14) geometric center, and heating element is solid Reservation (14) is connect with aluminum sample pond (13) underrun fastener；It is fixed that temperature sensor is additionally provided on aluminum sample pond (13) Position hole (16), mounting temperature sensor at temperature sensor location hole (16), temperature sensor detect aluminum sample pond (13) Temperature and then sample container (10) temperature for controlling heating element heats adjustment sample port；

The fluorescence detection module includes fluorescence excitation light source (9), fluorescent emission optical filter (17), light-conductive optic fibre (4) and glimmering Photodetector；Fluorescence excitation light source fixed disk (8) is fixed on shell shading cover (7) bottom surface, fluorescence excitation light source by fastener Fixed disk (8) bottom surface is equipped with multiple fluorescence excitation light sources (9) along linear interval arrangement, each fluorescence excitation light source (9) point Not corresponding with each sample port, fluorescence excitation light source (9) is located at right above itself corresponding sample port；At each light-conductive optic fibre hole One end that a light-conductive optic fibre (4) is connected by the first fiber laser arrays head mounting base (11), first at each light-conductive optic fibre hole Fiber laser arrays head mounting base (11) is fixed on aluminum sample pond (13) side by the first light-conductive optic fibre fixed disk (12)；Guide-lighting light Fluorescent emission optical filter (17) are installed in fine hole, transmitting filter center wavelength value is selected according to fluorescence analysis method；Outside instrument Control circuit board (1) is fixedly mounted in shell (6) bottom, second on each light-conductive optic fibre (4) other end and control circuit board (1) Light-conductive optic fibre fixed disk (2) connects, and installs fluorescence detector in the control circuit board (1) in the second light-conductive optic fibre fixed disk (2), Light-conductive optic fibre (4) end is fixed simultaneously by the second fiber laser arrays head mounting base (3) for being embedded in the second light-conductive optic fibre fixed disk (2) It is directed at fluorescence detector photosurface.

3. a kind of top illuminated nucleic acid isothermal amplification according to claim 2 detects portable instrument, it is characterised in that: described Two threaded holes are additionally provided on aluminum sample pond (13), fastener is fixed by the aluminum sample pond (13) by two threaded holes In gap slot；It is additionally provided with fluorescence excitation light source fixed disk positioning spiro pit (19) in the fluorescence excitation light source fixed disk (8), Fluorescence excitation light source fixed disk positioning spiro pit (19) is for fluorescence excitation light source fixed disk and the aluminum sample pond (13) It is fixed to use.

4. a kind of top illuminated nucleic acid isothermal amplification according to claim 2 detects portable instrument, it is characterised in that: described Aluminum sample pond (13) bottom sides open up the first light-conductive optic fibre fixed disk positioning groove (18), the first light-conductive optic fibre fixed disk (12) in the first light-conductive optic fibre fixed disk positioning groove (18).

5. a kind of top illuminated nucleic acid isothermal amplification according to claim 2 detects portable instrument, it is characterised in that: described Fluorescence excitation light source fixed disk (8) is equipped with multiple fluorescence excitation light source positioning round orifice (20) along linear interval arrangement, each Fluorescence excitation light source (9) are installed at fluorescence excitation light source positioning round orifice (20).

6. a kind of top illuminated nucleic acid isothermal amplification according to claim 2 detects portable instrument, it is characterised in that: described Tool housing (6) includes upper shell and lower casing, is connected and fixed between upper shell and lower casing by shell fixation fastener (15) In one.

7. a kind of top illuminated nucleic acid isothermal amplification according to claim 2 detects portable instrument, it is characterised in that: described glimmering Photodetector uses photodiode, and each fluorescence detector detects each sample in the corresponding aluminum sample pond (13) respectively Product mouth channel, different sample port channels select different fluorescence detectors according to detection wavelength of fluorescence.

8. a kind of top illuminated nucleic acid isothermal amplification according to claim 2 detects portable instrument, it is characterised in that: the control Circuit module processed includes power module, main control module, fluorescence excitation light source drive module, fluorescence intensity signals conditioning module, temperature Spend control module, overheating protection module and communication module；It is power module and main control module, fluorescence excitation light source drive module, glimmering Light intensity signal conditioning module, temperature control modules, overheating protection module and communication module electrical connection are powered, master control mould Block respectively with fluorescence excitation light source drive module, fluorescence intensity signals conditioning module, temperature control modules, overheating protection module, Communication module electrical connection, fluorescence excitation light source drive module are electrically connected fluorescence excitation light source (9), fluorescence intensity signals conditioning module It is electrically connected fluorescence detector, temperature control modules are electrically connected temperature sensor and heating element, and overheating protection module, which is installed on, to be added Thermal element, communication module are connect with upper computer module.

9. a kind of top illuminated nucleic acid isothermal amplification according to claim 2 detects portable instrument, it is characterised in that: the master Controlling module further includes instrument running LED (5), and instrument running LED (5) is installed on tool housing (6) appearance Face is used for display working condition.