CN107421935A - A kind of fluorescence micro RNA detection means and detection method - Google Patents
A kind of fluorescence micro RNA detection means and detection method Download PDFInfo
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- CN107421935A CN107421935A CN201710690115.1A CN201710690115A CN107421935A CN 107421935 A CN107421935 A CN 107421935A CN 201710690115 A CN201710690115 A CN 201710690115A CN 107421935 A CN107421935 A CN 107421935A
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6486—Measuring fluorescence of biological material, e.g. DNA, RNA, cells
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Abstract
A kind of fluorescence micro RNA detection means and method, belong to technical field of biological, it is characterized in that：Excitation source A is located on the left of sample cell, and excitation source B is located on the right side of sample cell, is respectively welded on circuit boards, in transmission line, for exciting the fluorescent material in sample；Photo detecting unit A is excited between excitation source A and sample cell, photo detecting unit B is excited between excitation source B and sample cell, photo detecting unit B is excited to be below excitation source B with sample groove height, photo detecting unit B is excited to be welded on circuit boards with sample cell, in transmission line, guarantee excites luminous energy to be irradiated to sample in sample cell；Beneficial effect is：Using bandpass filter and photodiode composition fluorescence intensity detection unit, the switching between two kinds of exciting lights is carried out；Increase transmission line module, whole Systems for optical inspection can be isolated and protected, improve accuracy and the sensitivity of experiment.
Description
Technical field
The invention belongs to technical field of biological.
Background technology
Gene (DNA or RNA) is the carrier of hereditary information, in individual growth, development, breeding, heredity and the variation of biology
Etc. playing particularly important effect in life process.The conventional RNA quantitative approach of medical science mainly includes uv-spectrophotometric at present
(260nm), fluorescent quantitative PCR technique etc..But ultraviolet spectrophotometry is frequently subjected to the interference of DNA, albumen, specific quantification compared with
Difference, the detection of sensitivity relative fluorescence is relatively low, it is necessary to which the sample size of consumption is big etc., it is impossible to realizes that RNA's is special quantitative.Fluorescence is determined
It is new technique developed in recent years to measure round pcr, has the characteristics that quantitative rapid, high specificity, high sensitivity, but its
Consume costly, quantitative result is easily influenceed by sample rna quality, objectively limits miniaturization and the portability of instrument.
The content of the invention
The purpose of the present invention is：A kind of fluorescence micro RNA detection means and method are provided, it is using fluorescent dye and spy
The RNA target molecule of the opposite sex is combined, and when low concentration, fluorescence signal can be also launched when fluorescent dye is combined with target molecule.
The technical scheme is that：
The present apparatus includes excitation source A, excitation source B, excites photo detecting unit A, excites photo detecting unit B, fluorescence to visit
Survey unit A, fluorescence detecting unit B, optical filter A, optical filter B, optical filter C, optical filter D, sample cell, transmission line, signal
Detection unit, main control unit, excite light control unit, liquid crystal display, circuit board, housing, sample groove lid, radome.
Excitation source A (1) is located on the left of sample cell (11), and excitation source B (2) is located on the right side of sample cell (11), welds respectively
It is connected on circuit board (17), in transmission line (12), for exciting the fluorescent material in sample.
Excite photo detecting unit A (3) to be located between excitation source A (1) and sample cell (11), excite photo detecting unit B (4)
Between excitation source B (2) and sample cell (11), photo detecting unit B (4) is excited highly to be below exciting with sample cell (11)
Light source B (2), photo detecting unit B (4) is excited to be welded on sample cell (11) on circuit board (17), embedded in transmission line (12)
It is interior, ensure that exciting light can be irradiated to sample cell (11) interior sample, monitored in real time for the state to exciting light, confirm to swash
It is luminous that whether normal work, luminous intensity are stablized.
Fluorescence detecting unit A (5), fluorescence detecting unit B (6) are respectively perpendicular positioned at sample cell (11) and exciting light light path
Both sides, it is welded on circuit board (17), in transmission line (12).Receive and detect by sample slot (11) interior sample to be tested
The fluorescence signal intensity sent, using high accuracy, highly sensitive photodiode, fluoroscopic examination precision can be improved, is measured
Scope is 300-1,000nm.Fluorescence signal can just be launched when these fluorescent dyes are only combined with these target molecules, even in dense
When degree is very low, the inaccurate repeated work for measuring and bringing is avoided.Transmission channel：- the 580nm of the green glow 510, -720nm of feux rouges 665.Choosing
When selecting blue light and exciting, the fluorescence of green or far infrared passage is read.When exciting light is feux rouges, the glimmering of far infrared passage is only read
Light.
Transmission line (12) is closed transmission line, and bottom is fixed on circuit board (17) by expansion bolt,
The each component of light path has been subjected to effectively protection, excitation source A (1), excitation source B (2) transmitting light has been collected, passes through respectively
Optical filter A (7), optical filter B (8) reach sample cell (11), and collect the fluorescence signal excited, by optical filter C (9), filter
Piece D (10) reaches fluorescence detecting unit A (5), fluorescence detecting unit B (6).The interference of external stray light can be effectively eliminated, and
Pollution of the dust to optical path component can be prevented, makes optical transport cleaner, substantially increases accuracy and the sensitivity of detection.
Optical filter A (7), optical filter B (8) respectively positioned at excitation source A (1), excitation source B (2) and sample cell (11) it
Between, in transmission line (12), for filtering out the veiling glare outside exciting light；The optical filter C (9), optical filter D (10)
Respectively between sample cell (11) and fluorescence detecting unit A (5), fluorescence detecting unit B (6), for filtering in addition to fluorescence
Veiling glare.
Detecting signal unit (13) is welded on circuit board (17), for by fluorescence detecting unit A (5), fluorescence detection list
The electric signal of first B (6) conversion, carries out preposition amplification, demodulation, filtering, collection.
Main control unit (14) is welded on circuit board (17), the dual core processor of use, rapidly and accurately quantitative in 5 seconds
DNA, RNA and albumen.Result data is shown to LCDs (16), and stored.
Light control unit (15) is excited to be welded on circuit board (17), for controlling excitation source A (1), excitation source B
(2) output light intensity.
Liquid crystal display (16) is embedded in housing (18) upper surface, is connected by communication cable with circuit board (17), uses
In man-machine interaction, function selection, data storage.
Sample groove lid (19) is located on sample cell (11), is made using light-proof material, is covered tightly during detection, is used for
Reduce influence of the veiling glare in environment to experimental result.
Radome (20) cover using thin aluminum, disturbs on transmission line (12) for shielding electromagnetic signal,
Signal to noise ratio can be effectively improved.
Detection method is：
Using PikoGreen dsDNA quantification kits (Broad Range), it has, and linear dsDNA specificity is good, line
The features such as property scope is wide, easy to operate.
PikoGreen dsDNA quantification kit components：
A:1 × quantify buffer solution 250mL
B:100 × reaction solution 5mL
C:100 × increased response liquid 5mL
D:9 × 0.5mL of dsDNA standard items (0,2,6.25,12.5,25,50,100,150,200ng/uL)
Step 1：Before use, product is recovered to room temperature by being taken out under storage condition.Each component fully shaking or whirlpool
Rotation mixes, centrifugation, to avoid unnecessary reagent loss.
Step 2：Double stranded DNA Quantitation reagent (PikoGreen) working solution is prepared, according to 1:199 proportioning, draws 2 respectively
× TE cushioning liquid and aseptic deionized water (quantitative buffer A), are mixed.It should newly be used with working solution within an hour；
Step 3：Standard items are made, take two EP pipes, each tube is according to 1:19 proportioning adds the dsDNA of various concentrations
Standard specimen and working solution, mix, cumulative volume is 200 μ L, obtains standard items 1 and standard items 2.
Step 4：Standard curve is made, two standard items are sequentially placed into fluorometer sample groove, is incubated at room temperature two points
Clock, is 350nm using excitation wavelength, and launch wavelength is 460 conditioned measurement fluorescent values, it may be determined that go out standard curve (DNA concentration-
Fluorescence intensity)；
Step 5：The DNA concentration of sample is detected, according to 1:199 and 1：Proportioning between 9, take testing sample slow with dilution
Fliud flushing mixes, and cumulative volume is 200 μ L.It is put into fluorometer sample groove, is incubated at room temperature two minutes, reads and sample fluorescence intensity phase
Corresponding DNA concentration.
All reagents are under room temperature, and sample blending, which is put into sample cell, needs incubation at room temperature two minutes.
Obtained calibration value can store, and be used after convenient；Detection data can store, and can at most store 1,000 sample
As a result, and can be exported by USB flash disk.In addition, the expanded range in sample or the detection range more than kit, in instrument screen
Have figure shows.
The beneficial effects of the invention are as follows：Using bidifly light emitting source, the complexity of equipment is simplified, while reduces detection
The volume of unit.In technical scheme, fluorescence intensity detection unit is formed using bandpass filter and photodiode,
By operating in a key interface, the switching between two kinds of exciting lights is carried out；Increase transmission line module, can be by whole optical detection
System is isolated and protected so that the transmitting procedure of whole light is cleaner, improves accuracy and the sensitivity of experiment.We
Method is combined using particular dye by embedded RNA single strand and by the attraction of electric charge with RNA, so as to send fluorescence.With the present invention
Apparatus and method detect its fluorescence intensity, and the standard curve (RNA concentration-fluorescence intensity) drawn with RNA standard items compares,
Read the RNA concentration corresponding with sample fluorescence intensity.Whole detection process is convenient and swift, is detected suitable for laboratory and medical treatment
Aspect.
Brief description of the drawings
The detection principle diagram of Fig. 1 apparatus of the present invention；
Schematic diagram cuts open in the overall structure office of Fig. 2 apparatus of the present invention；
Fluorescence intensity-RNA the concentration standard curves that Fig. 3 present invention measures.
Embodiment
Patent of the present invention is described further with reference to the accompanying drawings and examples：
As illustrated, 1 it is excitation source A, 2 is excitation source B, 3 is that to excite photo detecting unit A, 4 be to excite optical detection list
First B, 5 be fluorescence detecting unit A, 6 be fluorescence detecting unit B, 7 be optical filter A, 8 be optical filter B, 9 be optical filter C, 10 be filter
Mating plate D, 11 be sample cell, 12 transmission lines, 13 be detecting signal unit, 14 be main control unit, 15 be to excite photocontrol list
Member, 16 be liquid crystal display, 17 be circuit board, 18 be housing, 19 be sample groove lid, 20 be radome.
The excitation source A (1) is located on the left of sample cell (11), and excitation source B (2) is located on the right side of sample cell (11), point
It is not welded on circuit board (17), in transmission line (12), for exciting the fluorescent material in sample.
It is described to excite photo detecting unit A (3) to be located between excitation source A (1) and sample cell (11), excite photo detecting unit
B (4) is located between excitation source B (2) and sample cell (11), and both are highly below excitation source, is welded in circuit board (17)
On, in transmission line (12), ensure that exciting light can be irradiated to sample cell (11) interior sample, for the shape to exciting light
State is monitored in real time, confirms whether exciting light whether stablize by normal work, luminous intensity；
The fluorescence detecting unit A (5), fluorescence detecting unit B (6) are located at sample cell (11) for intersection point respectively, with swashing
The vertical both sides of luminous light path, are welded on circuit board (17), in transmission line (12).Receive and detect by sample slot
(11) fluorescence signal intensity that interior sample to be tested is sent, using high accuracy, highly sensitive photodiode, fluorescence can be improved
Accuracy of detection, measurement range 300-1,000nm.Fluorescence can just be launched when these fluorescent dyes are only combined with these target molecules
Signal, when concentration is very low, avoid the inaccurate repeated work for measuring and bringing.Transmission channel：It is-the 580nm of green glow 510, red
- the 720nm of light 665.When selection blue light excites, the fluorescence of green or far infrared passage is read.When exciting light is feux rouges, only read
The fluorescence of far infrared passage.
The transmission line (12), it is closed transmission line, circuit board is fixed in bottom by expansion bolt
(17) on, each component of light path has been subjected to effectively protection, has collected excitation source A (1), excitation source B (2) transmitting light,
Sample cell (11) is reached by optical filter A (7), optical filter B (8) respectively, and collects the fluorescence signal excited, passes through optical filter C
(9), optical filter D (10) reaches fluorescence detecting unit A (5), fluorescence detecting unit B (6).The dry of external stray light can effectively be eliminated
Disturb, and pollution of the dust to optical path component can be prevented, make optical transport cleaner, substantially increase accuracy and the spirit of detection
Sensitivity.
The optical filter A (7), optical filter B (8) are located at excitation source A (1), excitation source B (2) and sample cell respectively
(11) between, in transmission line (12), for filtering out the veiling glare outside exciting light；The optical filter C (9), filter
Piece D (10) is respectively between sample cell (11) and fluorescence detecting unit A (5), fluorescence detecting unit B (6), for filtering out fluorescence
Outside veiling glare.
The detecting signal unit (13) is welded on circuit board (17), for fluorescence detecting unit A (5), fluorescence to be visited
The electric signal of unit B (6) conversion is surveyed, carries out preposition amplification, demodulation, filtering, collection.
The main control unit (14) is welded on circuit board (17), the dual core processor of use, rapidly and accurately fixed in 5 seconds
Measure DNA, RNA and albumen.Result data is shown to LCDs (16), and stored.
It is described to excite light control unit (15) to be welded on circuit board (17), for controlling excitation source A (1), exciting light
Source B (2) output light intensity.
The liquid crystal display (16) is embedded in housing (18) upper surface, is connected by communication cable and circuit board (17)
Connect, for man-machine interaction, function selection, data storage.
The sample groove lid (19) is located on sample cell (11), is made using light-proof material, is covered tightly during detection,
For reducing influence of the veiling glare in environment to experimental result.
The radome (20) is covered on transmission line (12), using thin aluminum, is done for shielding electromagnetic signal
Disturb, signal to noise ratio can be effectively improved.
In this example, bidifly light emitting source includes blue-ray LED and red-light LED, can manually select as needed.When selection is blue
When light excites, the device reads the fluorescence of green or far infrared passage.When exciting light is feux rouges, far infrared passage is only read
Fluorescence.
It is above-mentioned to excite photo detecting unit in this example, the state of exciting light can monitor in real time and defeated by laser
Go out unit to compensate.When not detecting exciting light, then prompting is alarmed on the display screen；When deviation be present in excitating light strength,
Then carry out exciting light compensation.
In this example, each component of light path has been carried out effectively protection by transmission line, and can effectively eliminate the external world
The interference of veiling glare, and pollution of the dust to optical path component can be prevented, make optical transport cleaner, substantially increase detection
Accuracy and sensitivity.
In this example, bandpass filter is directed to two kinds of excitation sources, and holmium glass and didymium glass material has been respectively adopted
Optical filter, improve the degree of accuracy of excitation light wave.
In this example, fluorescence detecting unit improves fluoroscopic examination essence using high accuracy, highly sensitive photodiode
Degree, by detecting signal unit by the electric signal that photodiode change be sent to main control unit carry out data processing, and show and
On display screen, and data can preserve.
In this example, each component of light path has been carried out effectively protection by transmission line, and can effectively eliminate the external world
The interference of veiling glare, and pollution of the dust to optical path component can be prevented, make optical transport cleaner, substantially increase detection
Accuracy and sensitivity.
In this example, fluorescence micro RNA detection means, its application method comprises the following steps：
Step 1：By specific fluorescent reagent and buffer solution with 1:199 mix；
Step 2：Take standard items 1, standard items 2 with buffer solution with 1:19 mix；
Step 3：Standard items 1 and standard items 2 are put into device respectively, are incubated at room temperature two minutes, carry out two-point calibration,
Draw standard curve (RNA concentration-fluorescence intensity)；
Step 4：After obtaining standard curve, testing sample 1-20 μ L are taken to be mixed with 180-199 μ L dilution buffers；
Step 5：It is put into apparatus of the present invention sample cell, is incubated at room temperature two minutes, reads corresponding with sample fluorescence intensity
RNA concentration.
Claims (3)
1. a kind of fluorescence micro RNA detection means, it is characterized in that：
Excitation source A is located on the left of sample cell, and excitation source B is located on the right side of sample cell, is respectively welded on circuit boards, embedded in light
In transmission channel, for exciting the fluorescent material in sample；Photo detecting unit A is excited between excitation source A and sample cell,
Photo detecting unit B is excited to excite photo detecting unit B to be below swashing with sample groove height between excitation source B and sample cell
Light emitting source B, photo detecting unit B is excited to be welded on circuit boards with sample cell, in transmission line, guarantee excites luminous energy to shine
It is mapped to sample in sample cell；
Fluorescence detecting unit A, fluorescence detecting unit B are respectively perpendicular the both sides positioned at sample cell and exciting light light path, are welded on electricity
On the plate of road, in transmission line；
Transmission line is closed transmission line, and bottom is fixed on circuit board by expansion bolt；
Optical filter A, optical filter B are respectively between excitation source A, excitation source B and sample cell, in transmission line；
Detecting signal unit welds on circuit boards, for the electric signal for changing fluorescence detecting unit A, fluorescence detecting unit B,
Carry out preposition amplification, demodulation, filtering, collection；
Main control unit welds on circuit boards；
Light control unit is excited to be welded on circuit board；
Liquid crystal display is embedded in housing upper surface, is connected by communication cable with circuit board.
2. a kind of fluorescence micro RNA detection means as claimed in claim 1, it is characterized in that：
Fluorescence detecting unit A, fluorescence detecting unit B are respectively perpendicular the both sides positioned at sample cell and exciting light light path, are welded in electricity
On the plate of road, in transmission line；Measurement range is 300-1,000nm；Transmission channel is the -580nm of green glow 510, feux rouges
665–720nm；When selection blue light excites, the fluorescence of green or far infrared passage is read；When exciting light is feux rouges, only read remote
The fluorescence of infrared channel.
3. a kind of fluorescence micro RNA detection method, its method are：
Using PikoGreen dsDNA quantification kits (Broad Range),
PikoGreen dsDNA quantification kit components are
A is 1 × quantitative buffer solution 250mL,
B is 100 × reaction solution 5mL,
C is 100 × increased response liquid 5mL,
D is 9 × 0.5mL of dsDNA standard items；
Step 1：Before use, product is recovered mixed to room temperature, each component fully shaking or vortex by being taken out under storage condition
Even, centrifugation, to avoid unnecessary reagent loss；
Step 2：Prepare double stranded DNA Quantitation reagent；Working solution, according to 1:199 proportioning, respectively draw 2 × TE cushioning liquid with
Aseptic deionized water (quantitative buffer A), is mixed；It should newly be used with working solution within an hour；
Step 3：Standard items are made, take two EP pipes, each tube is according to 1:19 proportioning adds the dsDNA standard specimens of various concentrations
With working solution, mix, cumulative volume is 200 μ L, obtains standard items 1 and standard items 2；
Step 4：Standard curve is made, two standard items are sequentially placed into fluorometer sample groove, is incubated at room temperature two minutes, makes
It is 350nm with excitation wavelength, launch wavelength is 460 conditioned measurement fluorescent values, it may be determined that goes out standard curve；
Step 5：The DNA concentration of sample is detected, according to 1:199 and 1：Proportioning between 9, takes testing sample and dilution buffer
Mix, cumulative volume is 200 μ L；It is put into fluorometer sample groove, is incubated at room temperature two minutes, reads corresponding with sample fluorescence intensity
DNA concentration；
All reagents are under room temperature, and sample blending, which is put into sample cell, needs incubation at room temperature two minutes；
Obtained calibration value can store, and be used after convenient；Detection data can store, and can at most store 1,000 sample result,
And it can be exported by USB flash disk；In addition, the expanded range in sample or the detection range more than kit, can in instrument screen
There are figure shows.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107389644A (en) * | 2017-08-11 | 2017-11-24 | 长春理工大学 | A kind of rapid fluorescence proportioning device |
| CN107576802A (en) * | 2017-08-28 | 2018-01-12 | 长春理工大学 | A kind of detection means and detection method of fluorescence micro albumen |
| CN114280025A (en) * | 2021-12-28 | 2022-04-05 | 核工业北京地质研究院 | Device and method for measuring uranium content in solution |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107389644A (en) * | 2017-08-11 | 2017-11-24 | 长春理工大学 | A kind of rapid fluorescence proportioning device |
| CN107576802A (en) * | 2017-08-28 | 2018-01-12 | 长春理工大学 | A kind of detection means and detection method of fluorescence micro albumen |
| CN114280025A (en) * | 2021-12-28 | 2022-04-05 | 核工业北京地质研究院 | Device and method for measuring uranium content in solution |
| CN114280025B (en) * | 2021-12-28 | 2024-05-03 | 核工业北京地质研究院 | Device and method for measuring uranium content in solution |
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