CN206940892U - High flux ring mediated isothermal amplification identifies food borne pathogenses body device - Google Patents
High flux ring mediated isothermal amplification identifies food borne pathogenses body device Download PDFInfo
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- CN206940892U CN206940892U CN201621129863.XU CN201621129863U CN206940892U CN 206940892 U CN206940892 U CN 206940892U CN 201621129863 U CN201621129863 U CN 201621129863U CN 206940892 U CN206940892 U CN 206940892U
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- isothermal amplification
- mediated isothermal
- ring mediated
- high flux
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- 238000006243 chemical reaction Methods 0.000 claims abstract description 23
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Landscapes
- Apparatus Associated With Microorganisms And Enzymes (AREA)
- Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The utility model is that a kind of high flux ring mediated isothermal amplification identifies food borne pathogenses body device, including constant temperature mechanism（1）, reaction plate（2）, fluorescence excitation device（3）, image capture mechanism（4）, portable computer（5）, wherein reaction plate（2）It is arranged on constant temperature mechanism（1）Top, fluorescence excitation device（3）It is arranged on reaction plate（2）Top, image capture mechanism（4）It is arranged on fluorescence excitation device（3）Top, portable computer（5）With image capture mechanism（4）Connection.The utility model uses the image capture device being connected with portable computer, and food-borne pathogens are detected by the fluorescence intensity in captured in real-time ring mediated isothermal amplification process, and by analyzing.High flux ring mediated isothermal amplification identification food-borne causal agent device volume of the present utility model is small, with battery powered, can be conducted a field operation under the conditions of without civil power and complete the high-throughput nucleic acid identification of sample.Detection method of the present utility model is simple to operate, convenient and practical.
Description
Technical field
The utility model is a kind of high flux ring mediated isothermal amplification identification food borne pathogenses body device, particularly a kind of to use
Food-borne causal agent is identified in the high flux ring mediated isothermal amplification for the Rapid identification that food-borne causal agent is completed under the conditions of POC
Device, belong to the innovative technology of high flux ring mediated isothermal amplification identification food borne pathogenses body device.
Background technology
Medical diagnosis or nursing, referred to as medical care (POC) are carried out at one's side in patient, are research fields active at present,
And make important progress.It is in recent years to have been reported that more.At present poc testing method is provided with primary care and emergency ward.Some are most normal
The example seen is disposable gestation detection and continuous blood sugar monitoring system (CGMS).However, these equipment are also many other
The application of renewal.For example, based on the method for nucleic acid due to its high specific and selectivity, it is considered to be detection and identification microorganism
With the goldstandard of virus.The POC equipment that one kind can carry out DNA cloning with such as polymerase chain reaction (PCR) technology can show
Writing reduces testing cost, reduces sample volume, and contract in the environment of most standard laboratory equipment in various can not touch
It is short go out result time.
POC systems based on nucleic acid are currently in the starting stage and had a extensive future.In microfluid and micro-/ nano technical field
Latest developments can develop small-sized and sensitive POC " chip lab " diagnostic instrments, quickly be analyzed with providing nucleic acid amplification.
Examination of the portable diagnostic equipment to POC DNA is ideal.Detected in the occasion of such as resource-constrained, and family,
Proposed during including limited professional knowledge and experimental facilities for global health-center.Especially when POC equipment is examined with nucleic acid amplification
When surveying the function of (NA-POC detectors), it is possible in the accurately and quickly diagnostic assistance of infectious agent, infected in prevention
Propagation when play a very important role [4].
The POC equipment of high performance-price ratio can set up cheap screening station with low-level infrastructure needs, can develop
Disease control network and Site Detection infectious pathogen.There is the meaning that several situations demonstrate NA-POC tests.For example, food service industry
Spending a large amount of financial resources, (United States Department of Agriculture specially spends annual 100000000000 dollars, for controlling the cause of disease of food origin disease.POC is examined
Examining system will allow it is all be engaged in food production and commercialized each participant detection pathogen product, recalled with reducing or eliminating
The disease relevant with food.The Viral diagnosis for including being harmful to patient for POC equipment other important applications.It is if all
There is virus checking equipment on airport, and H1N1 flu outbreaks in 2009 should can be avoided or at least controlled.Checkpoint
It is equipped with and checks equipment, you can to detect infected patient, it can be prevented and infected to other countries.Developed 40 years half
The key that PoC systems are implemented as this benefit, portability and quick detection is transplanted in conductor technology research.Fast temperature circulates,
It is the key for entering performing PCR measure within 10 minutes or less time, element of the semiconductor as rapid thermal cycles can be used, has
There is the characteristics of small thermal mass local heater and small size.Semiconductor can also detect DNA cloning product, avoid using with expensive and
Bulky optical system.
The nucleic acid amplification detection of wherein pathogen is to identify the important method of microorganism and virus.At present, conventional nucleic acid expands
It is the method using optical system detection fluorescence or turbidity to increase detection.However, this method needs complicated and expensive equipment (light
Fibre, LED sensor etc.), cheapest real-time PCR analysis instrument is also required to ten tens thousand of yuan, can increase cost and increase POC equipment
The quantity of element.
Utility model content
The purpose of this utility model is consideration above mentioned problem and provides a kind of cheap, small volume, and structure is simpler
High flux ring mediated isothermal amplification identification food borne pathogenses body device.The utility model is designed reasonably, it is convenient and practical.
The technical solution of the utility model is：High flux ring mediated isothermal amplification of the present utility model identifies food borne pathogenses
Body device, include constant temperature mechanism, reaction plate, fluorescence excitation device, image capture mechanism, portable computer, wherein reacting
Plate is arranged on the top of constant temperature mechanism, and fluorescence excitation device is arranged on the top of reaction plate, and image capture mechanism is arranged on fluorescence
The top of excitation apparatus, portable computer are connected with image capture mechanism；Above-mentioned fluorescence excitation device is by that can launch exciting light
LED or quartz burner and filter set into, LED or quartz burner and optical filter are located at the top side of reaction plate, and
It is fixed on constant temperature mechanism.
The utility model using the structure for being used to computer picture method detect amplified production, nucleic acid amplification due to that can be become
Must be less expensive, it is smaller and simpler.The utility model utilizes small-sized image collecting device, for temperature cycles and nucleic acid amplification
Detection is the primary element of successful NA-POC detecting instruments.The utility model is connected using cheap with portable computer
Image capture device, by the fluorescence intensity in captured in real-time ring mediated isothermal amplification process, and pass through software analysis, inspection
Survey food-borne pathogens.The utility model small volume, with battery powered, it can be conducted a field operation under the conditions of without civil power and complete sample
This high-throughput nucleic acid identification.The device design of high flux ring mediated isothermal amplification identification food-borne causal agent of the present utility model
Ingenious, function admirable is convenient and practical.The detection of high flux ring mediated isothermal amplification identification food-borne causal agent of the present utility model
Method is simple to operate, convenient and practical.
Brief description of the drawings
Fig. 1 is schematic diagram of the present utility model；
Fig. 2 is the enlarged drawing for the micropore that microwell plate is provided with.
Embodiment
Embodiment 1：
Schematic diagram of the present utility model is as shown in figure 1, the identification of the utility model high flux ring mediated isothermal amplification is food-borne
The device of pathogen, include constant temperature mechanism 1, reaction plate 2, fluorescence excitation device 3, image capture mechanism 4, portable computer
5, wherein reaction plate 2 is arranged on the top of constant temperature mechanism 1, and fluorescence excitation device 3 is arranged on the top of reaction plate 2, image capture
Mechanism 4 is arranged on the top of fluorescence excitation device 3, and portable computer 5 is connected with image capture mechanism 4 by data wire 6.On
State fluorescence excitation device 3 to be made up of the LED or quartz burner 31 and optical filter 32 that can launch exciting light, LED or ultraviolet lamp
Pipe 31 and optical filter 32 are located at the top side of reaction plate 2, and are fixed on constant temperature mechanism 1.
In the present embodiment, above-mentioned constant temperature mechanism includes moist closet, power supply, heating element heater, radiator valve, TEMP
Device, heating element heater and temperature sensor are arranged in moist closet, and power supply is connected with heating element heater, radiator valve and heating element heater
Connection.
In addition, above-mentioned radiator valve is provided with temperature setting device, temperature setting device is that temperature sets panel or opened
Close.Said temperature setting panel is mainly set to constant temperature room temperature, and temperature setting panel can use touch control liquid crystal panel, and switch can
Set using toggle switch.And confirm to carry out temperature control process after the completion of setting；Above-mentioned moist closet is formed can be according to used
DNA cloning enzyme, preference temperature is set to, thermostat temperature scope control is in the range of ± 0.5 DEG C；Above-mentioned heating element heater resistance
Piece, or electrothermal tube, or semiconductor or low voltage heater, power are 10-100 watts.；Above-mentioned radiator valve is controlled by single-chip microcomputer
Circuit board group is into receiving temperature information from sensor by design temperature, control the switch of heater, complete high-precision temperature control
System；Said temperature sensor is by heating site sensor with specimen locations sensor group into heating site sensor connection is simple
Temperature control power switch device, mainly prevent temperature is too high to cause instrument damage.
In order to ensure heat insulation effect, above-mentioned thermostatic chamber forms a cube cavity by 3-tier architecture, and outer layer is protective layer, by metal
Or engineering plastics are compressing；Intermediate layer is insulation material；Internal layer is metal level, and thermostatic chamber madial wall fixes heating unit by bottom
Part and temperature sensor, there are atoleine or the metal perforated plate agreed with reaction plate in heating chamber, passed with carrying out effective heat
Pass process.
In the present embodiment, above-mentioned power unit includes small battery and regulator rectifier circuit, charging circuit, and can lead to
Conventional alternating current is crossed to be powered or charge.
In the present embodiment, the structure of above-mentioned reaction plate 2 is microwell plate, and microwell plate is provided with micropore 7, is placed with micropore 7
Reaction solution 71, exciting light 72 goes out reflected light 73 by the back reflection of reaction solution 71, as shown in Fig. 2 the material of microwell plate is polyethylene
Or other organic polymer materials, DNA polymerase reaction should not be influenceed.
In the present embodiment, the LED or quartz burner 31 and optical filter 32 of above-mentioned fluorescence excitation device 3 are located at reaction plate 2
Top side, and be fixed on the thermostatic chamber of constant temperature mechanism 1.The ripple of the exciting light of the above-mentioned transmitting of LED or quartz burner 31
A length of nanoscale, SYBR Green I are excited at less than 485nm wavelength.
In the present embodiment, above-mentioned image capture mechanism 4 includes digital camera 41, camera lens 42, fluorescent optical filter 43, shading
Cover 44, wherein camera lens 42 is placed in the lower section of digital camera 41, and fluorescent optical filter 43 is placed in the lower section of camera lens 42, and fluorescent optical filter
43 are placed in the top of light shield 44.The use of digital camera 41 can carry out the camera or camera of fluorescence shooting, and camera lens 42 should meet
Each position fluorescence can photograph when orifice plate is shot and image is undistorted；Fluorescent optical filter 43 is selected only in DNA dye emissions
Wavelength, such as λ=495nm when using Evagreen, the λ=520nm optical filter for having high pass characteristic during SYBR Green I；Shading
Cover 44 can use the black cloth of multi-layer thick or plastic plate to prepare, and prevent ambient light interference fluorescence shooting；Above-mentioned portable computer 5 is adopted
With common notebook computer, installation operation system such as windows, and image capture interface, as USB3 or IEEE1394 with
Carry out high-speed image sampling.Software section should have image capture software and Fluorescence image analysis software.
The utility model is divided using the detection operation of the device of high flux ring mediated isothermal amplification identification food-borne causal agent
For four-stage：
The first step：Prepare ring mediated isothermal amplification reagent and buffer solution：
For each sample, prepared by the preparation ring mediated isothermal amplification reagent and buffer soln for needing cumulative volume 10-50 μ L
Composition, preferably 30 μ L；
Preparing ring mediated isothermal amplification reagent and buffering agents includes following component, and the following content that indicates is final concentration：
Glycine betaine 500-1500mM, triphosphate deoxyribose nucleotide (dNTPs) each 1.5mM of mixture, 1 × isothermal buffer solution (three hydroxyls
Aminomethane hydrochloride (Tris-HCl) 20mM, ammonium sulfate 10mM, Qula lead to (Triton) X-1008mM, potassium chloride
20mM), magnesium sulfate 5-15mM, 0.5-3.5 μM of primer mixture, bacillus stearothermophilus (BST) thermal starting polymerase 0.4-1
Unit/μ L, 20 μM of fluorescent dye；
Second step：Above-mentioned preparation of samples takes the clinical sample of appropriate food, patient's secretion or excrement under sterile working
This, is inoculated in enrichment culture medium, the incubated 12-16 hours at 37 DEG C；The nutrient solution of requirement is taken under aseptic condition, again
It is inoculated in enrichment liquid of the same race, shaken cultivation 4-6 hours；1-2ml bacterium solutions are taken to centrifuge, rotating speed 5000rpm, 5-6 minute obtains
Bacterial sediment；With distilled water washing thalline 2-3 after, add 100 μ L aseptic double-distilled waters and suspend, about 10-12 is boiled at 100 DEG C
Minute, centrifugation, rotating speed 12000rpm, 3-4 minute obtains supernatant, the as rough DNA of bacterium.Positive control standard and negative control
Standard is handled by same procedure；
3rd step：Above-mentioned sample-adding, which is pressed, adds LAMP solution per hole 10-30 μ L volumes, then adds 1 μ L samples respectively, and control
Product, per the μ L paraffin oils of Kong Zaijia 20 after the completion of sample-adding, prevent reaction solution dehydration；
4th step：Above-mentioned real-time fluorescence imaging is per minute once to record using the camera shooting photo for possessing automatic gate
Fluorescence intensity in amplification procedure, fluoroscopic image is shot plus FITC filters, image preserves post analysis successively.
Claims (8)
1. a kind of device of high flux ring mediated isothermal amplification identification food-borne causal agent, it is characterised in that include constant temperature mechanism
（1）, reaction plate（2）, fluorescence excitation device（3）, image capture mechanism（4）, portable computer（5）, wherein reaction plate（2）If
Put in constant temperature mechanism（1）Top, fluorescence excitation device（3）It is arranged on reaction plate（2）Top, image capture mechanism（4）If
Put in fluorescence excitation device（3）Top, portable computer（5）With image capture mechanism（4）Connection；Above-mentioned image capture machine
Structure（4) digital camera (41), camera lens (42), fluorescent optical filter (43), light shield (44) are included, wherein camera lens (42) is placed in number
The lower section of code-phase machine (41), fluorescent optical filter (43) is placed in the lower section of camera lens (42), and fluorescent optical filter (43) is placed in light shield
(44) top, fluorescent optical filter (43) are in DNA dye emission wavelength, using λ during Evagreen=495 nm, SYBR
The nm of λ during Green I=520 optical filter.
2. the device of high flux ring mediated isothermal amplification identification food-borne causal agent according to claim 1, its feature exist
Include thermostatic chamber, power supply, heating element heater, radiator valve, temperature sensor, heating element heater and temperature in above-mentioned constant temperature mechanism
Sensor is arranged in moist closet, and power supply is connected with heating element heater, and radiator valve is connected with heating element heater.
3. the device of high flux ring mediated isothermal amplification identification food-borne causal agent according to claim 2, its feature exist
Temperature setting device is provided with above-mentioned radiator valve, temperature setting device is that temperature sets panel or switch.
4. the device of high flux ring mediated isothermal amplification identification food-borne causal agent according to claim 2, its feature exist
A cube cavity is formed by 3-tier architecture in above-mentioned thermostatic chamber, outer layer is protective layer, compressing by metal or engineering plastics；It is middle
Layer is insulation material；Internal layer is metal level, and thermostatic chamber madial wall fixes heating element heater and temperature sensor, heating chamber by bottom
On have atoleine or the metal perforated plate agreed with reaction plate.
5. the device of high flux ring mediated isothermal amplification identification food-borne causal agent according to claim 2, its feature exist
Include small battery and regulator rectifier circuit, charging circuit in above-mentioned power supply, and be powered by conventional alternating current or
Charging.
6. the device of high flux ring mediated isothermal amplification identification food-borne causal agent according to claim 1, its feature exist
In above-mentioned reaction plate（2）Structure be the microwell plate provided with micropore.
7. the device of high flux ring mediated isothermal amplification identification food-borne causal agent according to claim 2, its feature exist
In above-mentioned fluorescence excitation device（3）By the LED or quartz burner that can launch exciting light（31）And optical filter（32）Composition, LED
Or quartz burner（31）And optical filter（32）Positioned at reaction plate（2）Top side, be fixed on thermostatic chamber.
8. the device of high flux ring mediated isothermal amplification identification food-borne causal agent according to claim 7, its feature exist
In above-mentioned LED or quartz burner（31）The wavelength of the exciting light of transmitting is nanoscale, and SYBR Green I are less than 485nm ripples
Strong point is excited.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201621129863.XU CN206940892U (en) | 2016-10-17 | 2016-10-17 | High flux ring mediated isothermal amplification identifies food borne pathogenses body device |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201621129863.XU CN206940892U (en) | 2016-10-17 | 2016-10-17 | High flux ring mediated isothermal amplification identifies food borne pathogenses body device |
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| CN206940892U true CN206940892U (en) | 2018-01-30 |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113755311A (en) * | 2021-11-09 | 2021-12-07 | 北京华牛世纪生物技术研究院 | Gene chip reader |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113755311A (en) * | 2021-11-09 | 2021-12-07 | 北京华牛世纪生物技术研究院 | Gene chip reader |
| CN113755311B (en) * | 2021-11-09 | 2022-02-18 | 北京华牛世纪生物技术研究院 | Gene chip reader |
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