CN109355178A - A kind of contiguous volume gradient capillary digital pcr device and its application method - Google Patents
A kind of contiguous volume gradient capillary digital pcr device and its application method Download PDFInfo
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- CN109355178A CN109355178A CN201811248584.9A CN201811248584A CN109355178A CN 109355178 A CN109355178 A CN 109355178A CN 201811248584 A CN201811248584 A CN 201811248584A CN 109355178 A CN109355178 A CN 109355178A
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Abstract
The present invention provides a kind of contiguous volume gradient capillary digital pcr device, which includes: a programmable heat circulating instrument, the capillary of a set of cross section size consecutive variations and an optical signalling acquisition equipment.The present invention can simplify the operating procedure of digital pcr while save the cost, and can reach higher DNA and quantify accuracy.
Description
Technical field
The present invention relates to digital pcr field, in particular to a kind of contiguous volume gradient capillary digital pcr device and its make
Use method.
Background technique
Modern biology research, especially medical research are related to the demand to the quantitative analysis of nucleic acids often.Such as detection blood
Certain dissociative DNAs and its content in liquid can instruct the clinical diagnosis of certain cancers and the therapeutic effect of monitoring cancer.With
Past quantitatively mainly takes quantitative fluorescent PCR to carry out relative quantification, that is, chooses the transcript conduct of a stable gene of expression
Reference, come judge purpose nucleic acid amount height.But interference of this method vulnerable to non-target nucleic acid molecules (background noise), only
Suitable for qualitative or low accuracy detection, it is unable to satisfy the detection of the especially rare target nucleic acid molecules of certain contents.
Digital pcr (dPCR) then can be by large-scale parallel PCR amplification, by faint amplified signal from background noise
It extract, the number for the molecule being amplified is counted with " endpoint signal with or without ".In recent years, as nanometer manufactures skill
" Water-In-Oil PCR " technology that the development of art and micro-fluidic technologies and two generation sequencing technologies grow up, can once generate tens of thousands of
Or even millions of nanoliters even single Water-In-Oil droplet of picoliters rank, in this, as the sample dispersion carrier of dPCR, and produce
Raw droplet type digital pcr (ddPCR) technology, is the digital pcr platform of current relative maturity.
On the one hand, ddPCR needs to realize the generation of uniform droplet by drop generator, and droplet is transferred to 96 orifice plates
After carrying out PCR, and droplet analyzer is needed to carry out signal-obtaining.The instrument price that the process is related to is higher, and common lab is difficult
To bear.
On the other hand, every in digital pcr since digital pcr is according to positive drop number come statistical mask copy number
In a drop the copy number of template cannot it is too high can not be too low, too high on the low side, the too low and meeting that will cause final statistics copy number
Cause statistical error excessive, is statistically most perfectly 1 or 0.For pilot scale study template consumption, mainstream number
PCR (including ddPCR) requires to do preliminary experiment to determine experiment pattern dosage；For some very precious nucleic acid materials, do
The cost of preliminary experiment will be very high, or even not enough do a preliminary experiment at all.
Summary of the invention
In order to solve the problems, such as at high cost in the prior art and need to do preliminary experiment, the present invention provides a kind of contiguous volumes
Gradient capillary digital pcr device.
The present invention provides a kind of capillary, the descending consecutive variations of internal diameter, it is characterised in that: the capillary is thin
Holding internal diameter is 10~50 μm, butt end internal diameter < 1mm, and capillary pipe length is 0.8~4cm.
The present invention also provides purposes of the aforementioned capillary in digital pcr.
The present invention also provides a kind of contiguous volume gradient capillary digital pcr devices, which is characterized in that the device packet
It includes:
One programmable heat circulating instrument；
Capillary as previously described；
One optical signalling acquires equipment.
Further, the programmable heat circulating instrument is PCR instrument；And/or aforementioned fluorescent signal collecting device is that fluorescence is aobvious
Micro mirror.
The present invention also provides the application methods of aforementioned device, which is characterized in that comprises the steps of:
(1) by the PCR reaction system containing fluorophor and with water isodensity oil mutually according to 1:(2~4) ratio shake it is mixed
It is even, it is placed in the capillary of the descending consecutive variations of internal diameter, closes capillary both ends；
(2) heating makes oily mutually with water phase along the distribution of capillary radially alternating；
(3) PCR reaction is carried out, optical signalling acquires the droplet that positive 30%~50% region of droplet accounting is counted under equipment
Several and droplet radius calculates pcr template copy number.
Method above-mentioned, which is characterized in that step (2) heating condition is to maintain 10min at 95 DEG C.
Method above-mentioned, which is characterized in that the PCR reaction system and be 1: 3 (v/ with the ratio of water isodensity oil phase
v)。
Method above-mentioned, which is characterized in that the oil is mutually that castor oil and paraffin oil are mixed in 3: 1 (v/v) ratios, then are used
The mixture prepare cholesterol saturated solution to get.
Method above-mentioned, which is characterized in that the oil mutually also contains surfactant.
Method above-mentioned, which is characterized in that the fluorophor of the PCR reaction system is located on probe, or is located at double
On chain luminescent dye molecule.
Method above-mentioned, which is characterized in that the positive drop accounting in the region for counting droplet number and droplet radius is
30%~50%.
By the capillary and high-temperature process of the descending consecutive variations of the internal diameter of apparatus of the present invention, make drop in non-individual body
Integral is distributed in capillary；Drop increases with the probability of target molecule with the increase of droplet size.After PCR, positive liquid
The distribution situation of drop is that big drop positive ratio is high, and each drop contains multiple initial copies, and counting positive drop can underestimate just
Beginning copy number；Droplet positive ratio is low, and statistical error can be very big；The positive drop ratio that centre has one section of region is moderate,
Initial copy number is at 1 or so in corresponding drop, applicants experimentally found that in positive 25%~55% area of droplet accounting
When domain, the copy number of pcr template can be calculated by the ratio and droplet size of positive drop, is 30% in positive drop accounting
~50% accuracy rate is higher.After the present invention selects suitable region, directly it can detect and calculate, avoid conventional numerical
The multiple Sample Dilution preliminary experiment that PCR carries out to keep positive drop ratio moderate.
Since the volume of the PCR drop of the invention formed is very small, the DNA copy contained is considerably less, can using the present invention
To realize effect identical with digital pcr, the minim DNA in sample to be examined is detected.
The present invention compares existing digital pcr since consumptive material can use cheap capillary, greatly save ground at
This, and operating procedure is simplified, and detection method is accurate, it has a good application prospect.
The present invention is described in further details below by specific embodiment, but is not to limit of the invention
System, above content according to the present invention, according to the ordinary technical knowledge and customary means of this field, not departing from, the present invention is above-mentioned
Under the premise of basic fundamental thought, the modification, replacement or change of other diversified forms can also be made.
Detailed description of the invention
Fig. 1 is the Water-In-Oil drop of contiguous volume gradient in capillary, that is, capillary
Specific embodiment
Embodiment device uses test
The present embodiment is using artificial synthesized EGFR gene as test template.
1. method
The preparation of 1.1 contiguous volume gradient capillaries
The use of internal diameter is that 1mm capillary makes contiguous volume gradient capillary, under alcolhol burner barbecue, uniformly firmly draws
Capillary modifies capillary tip to being broken, and exposed tip hole is unobstructed.Obtain butt end internal diameter 1mm, 20 μm of taper end internal diameter, long 3cm
Capillary.
1.2 oil are mutually prepared
Density is prepared first close to the oil of water density, and the present embodiment is with castor oil (0.98g/1) and paraffin oil according to 3: 1
Volume ratio matches even and cholesterol is added, and ratio is that 100ml castor oil addition excess cholesterol is allowed to be dissolved as saturation state, is added
0.5% sorbester p17 or 0.1% 60 surfactant of tween, concussion uniformly, make castor oil, paraffin oil and cholesterol mixing
Object mixed stability, homoepitaxial.
The preparation of 1.3PCR oil water mixture
10 μ l of buffer is premixed using conventional 2 × PCR, upstream and downstream primer, probe and template is added, then supplement volume with water
It is PCR aqueous phase system to 20 μ l.In aqueous phase system: primer and probe final concentration is 0.25 μm of ol/l, and template concentrations are
300copies/μl.Primer, probe, template sequence are as follows:
PCR primer:
VEGFR-F:GTGGACAACCCCCACGTGT (SEQ ID NO.1)；
VEGFR-R:CCGAAGGGCATGAGCTTCG (SEQ ID NO.2).
TaqMan probe: FAM-CCTCACCTCCACCGTGCAGCTC (SEQ ID NO.3)-TAMRA.
Template:
GTGGACAACCCCCACGTGTGGCATCTGCCTCACCTCCACCGTGCAGCTCATATGCAGCTCATGCCCTT
CGGCAGCCTCCTGGACTATGTCCGGGGCCAAACTGCTGGGTGC(SEQ ID NO.4)。
20 μ l aqueous phase systems and the aforementioned oily phase of 60 μ l are mixed, fullys shake uniformly, obtains PCR oil water mixture.
1.4 contiguous volume gradient capillary PCR reaction
It is inserted under liquid level with stub end capillary, oil water mixture has been configured using capillarity sucking, by capillary
Tip is placed downward, and oil water mixture is made to be filled to capillary tip using gravity, is sintered tip with flame, is closed capillary
Pipe, stub end are closed using sealing compound, and capillary is put into PCR pipe, and PCR pipe water filling is put into quantitative PCR instruments, is heated to 95
DEG C maintain 10 minutes, will form an oil extraction packet water drop in capillary at this time along capillary arranged radially, liquid-drop diameter is drop
The internal diameter (as shown in Figure 1) of position capillary.Then carry out PCR amplification, i.e., 95 DEG C maintain 10 seconds, and 50 DEG C maintain 30 seconds,
60 DEG C maintain 30 seconds, carry out 35 circulations and complete amplification.
1.5 high intension microscopes calculate drop fluorescence signal acquisition in capillary and statistics
It is put into high intension microscopically observation after capillary is taken out and reads data, calculates for convenience, we choose and connect
Drop at nearly 50% positive drop is as analysis object.Positive drop number is counted, first, region drop half is measured
Diameter R1, a droplet radius R of regional center2, region the last one droplet radius R3.Drop total volume is calculated with following formula:
(4/3×πR1 3+4/3×πR2 3+4/3×πR3 3)/3 × total drop number=drop total volume；And it is corrected with Poisson distribution method
Calculate the copy number of positive template in average each drop, formula: (1- is positive by every drop positive template copy number M=-ln
Probability)；It is the copy number in unit volume with the region template sum divided by the total volume of the region drop, multiplied by adding
Sample volume is the template copy sum in prepared PCR reaction system.
2. result
The positive drop number of selected 50 Water-In-Oil drops amplification is 21, calculates to obtain each drop positive template
Number is about about to contain 27.24 copies (template copy) in 0.5447,50 drops.
Through measuring, R1=1.36 μm, R2=1.25 μm, R3=1.13 μm, it is included in above-mentioned formula calculating and is included in the 50 of statistics
A drop total volume is about 412nL, so template concentrations are 27.24/0.412=66copies/ μ L in PCR amplification system, then
About 20 × 66=1322copies containing template to be measured in 20 μ L reaction systems, with known template concentrations 1500copies/5 μ l base
Originally it is consistent.
To sum up, contiguous volume gradient capillary digital pcr device provided by the invention, can be reached not with lower cost
Wrong DNA quantitative detection precision, and the preliminary experiment without doing sample extension rate, have a good application prospect.
Claims (10)
1. a kind of capillary, the descending consecutive variations of internal diameter, it is characterised in that: the capillary taper end internal diameter is 10~50
μm, butt end internal diameter≤1mm, capillary pipe length is 0.8~4cm.
2. purposes of the capillary described in claim 1 in digital pcr.
3. a kind of contiguous volume gradient capillary digital pcr device, which is characterized in that the device includes:
One programmable heat circulating instrument；
Capillary described in claim 1；
One fluorescence signal acquisition equipment.
4. device as claimed in claim 3, which is characterized in that the programmable heat circulating instrument is PCR instrument；And/or it is described glimmering
Light signal collection equipment is fluorescence microscope.
5. the application method of claim 3 described device, which is characterized in that comprise the steps of:
(1) by the PCR reaction system containing fluorophor and with water isodensity oil mutually according to 1: the ratio of (2~4), which is shaken, to be mixed, and is set
In the capillary for entering the descending consecutive variations of internal diameter, capillary both ends are closed；
(2) heating makes oily mutually with water phase along the distribution of capillary radially alternating；
(3) carry out PCR reaction, optical signalling acquire the droplet number that positive 30%~50% region of droplet accounting is counted under equipment and
Droplet radius calculates pcr template copy number.
6. method as claimed in claim 5, which is characterized in that step (2) heating condition is to maintain 10min at 95 DEG C.
7. method as claimed in claim 5, which is characterized in that the PCR reaction system and the ratio with water isodensity oil phase
It is 1: 3 (v/v).
8. method as claimed in claim 5, which is characterized in that the oil is mutually castor oil and paraffin oil in 3: 1 (v/v) ratios
Mixing, then with the mixture preparation cholesterol saturated solution to get.
9. the method as described in claim 5 or 8, which is characterized in that the oil mutually also contains surfactant.
10. method as claimed in claim 5, which is characterized in that the fluorophor of the PCR reaction system is located on probe,
Or it is located on double-strand luminescent dye molecule.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| WO2024001219A1 (en) * | 2022-06-27 | 2024-01-04 | 北京本立科技有限公司 | Digitizing device and method for liquid sample |
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