CN105861675A - Primers, probe, kit and method for microchimerism detection and individual recognition - Google Patents
Primers, probe, kit and method for microchimerism detection and individual recognition Download PDFInfo
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Abstract
The invention belongs to the technical field of gene engineering, and discloses a primer-probe combination for microchimerism detection and individual recognition, a kit containing the primer-probe combination, and a method for performing microchimerism detection and individual recognition by using the primer-probe combination or kit. 15 high-individual-recognition-rate InDel sites for Asian people are screened on the basis of the latest InDel site database by using the Asian people as the reference, and the specific primer-probe combination is further designed for each InDel site. Compared with the prior art, the method disclosed by the invention has the advantages of high polymorphism, high specificity, high sensitivity, high convenience and high speed, and provides a very important detection means for microchimerism detection and individual recognition for Asian people.
Description
Technical field
The present invention relates to gene engineering technology field, be particularly used for micro chimerism detection and the primer of individual identification, spy
Pin, test kit and method.
Background technology
Modern genetics man think, gene is the specific nucleoside on DNA (DNA (deoxyribonucleic acid)) molecule with hereditary effect
The general name of acid sequence, is the DNA molecular fragment with hereditary effect.Gene passes to hereditary information not only by replicating
Of future generation, it is also possible to make hereditary information be expressed.This hereditary information lie in the skeleton of people, hair, blood etc. everyone
In soma or organ.By the difference of human genome pleomorphism site, carry out gene test to identify Different Individual, at present
Legal medical expert Yu and clinical trial it are widely used.
Recently multiple genetic marker has been developed for individuality identification.Wherein, STR (Short Tandem
Repeats, STR) owing to detection method is easy, quick, accuracy is high, amplified fragments is of moderate size it is considered to be forensic dna reflects
Standard, the most authoritative approaches and methods in Ding, it is possible to solve most practical problem.But, owing to STR exists height sudden change
Rate (generally 10-2～10-4), the fragment of detection is longer (100～400bp), is difficult to realize the DNA typing to degraded sample, with
Time be fitted together to rate detection sensitivity about 5%, and the when of carrying out micro-transplanting or micro-treatment clinically, general chimeric rate needs
Maintain 1～less than 2%, it is impossible to meet requirement.
Chimera refers to the internal cell chimerism phenomenon that there is different hereditary character of same individuality, i.e. contains two sets or two sets
The most genomic state.Wherein, micro chimerism refers to that the cell component that Different Individual is originated is less than the phenomenon of 1%.Micro chimerism
The more highly sensitive detection method of testing requirement.Therefore, STR can not carry out micro chimerism analysis.
Along with the progress of genome research, pleomorphism site is the most of great interest.Wherein, mononucleotide polymorphic
Property (Single Nucleotide Polymorphism, SNP) has relatively low spontaneous mutation rate (10-8)；And it is single
SNP site amplified production can control at below 200bp, is conducive to the typing of degraded sample.But difference between SNP allele
Only one of which base, therefore, it is difficult to design highly sensitive detection scheme, detection sensitivity can only achieve 1%, the most inapplicable
Detect in micro chimerism.And SNP quantitative approach is complicated, experimental apparatus is expensive, and use cost is high, complex operation, and flux is little, and
Be not suitable for promoting at conventional prudence laboratory.
The pleomorphism site of a new generation inserts or deletion polymorphism (Insertion or Deletion in recent years
Polymorphisms, InDel or DIP), i.e. genome inserts or lacks dissimilar and size DNA fragmentation (4～
Polymorphic markers 22bp) formed, has extensively in terms of forensic qualification, paternity test and other medical tests
Use.From the perspective of forensic medical examination of material evidence, InDel labelling has the following characteristics that (1) content in human genome is rich
Richness, next in number only to SNP；(2) there is close natural mutation rate (～2.3 × 10 with SNP-9)；(3) InDel amplified fragments is more
Short (less than 160bp), is more suitable for detection height degraded and the DNA profiling of low copy, increases the success of DNA degradation sample typing
Rate；(4) there is significant crowd and race difference in InDel site；(5) InDel inspection can be carried out by fluorescent PCR amplification technique
Surveying, technology platform general applicability is strong；(6) based on InDel site, use double color fluorescent quantitative PCR method, determine differentiation donor
After the specific position of receptor, carrying out quantitative PCR, the detection of minimum chimeric rate can reach 0.01～0.05%.Therefore, more closely
The concern of the most many legal medical expert scholars is day by day caused over Nian.But, by the end of so far, based on InDel genetic marker
The test kit for micro chimerism detection and individual identification of commercial comparative maturity is the most rare, only Dutch
Investigator DIP test kit is the commercially produced product of comparative maturity.Simultaneously as there is crowd and race in genetic marker
Difference, Investigator DIP test kit is for asian population, and the suitability is not the strongest.Therefore, research and development one
Set is applicable to just seeming for micro chimerism detection and the individual InDel genetic marker test kit identified of asian population and very must
Want.
Summary of the invention
The present invention is directed to drawbacks described above present in prior art, based on up-to-date InDel site data base, and with Asia
On the basis of crowd, redesign the primer and probe and detection method identified for micro chimerism detection and individuality.The present invention
Detection method polymorphism strong, specificity good, highly sensitive and simple and efficient.
To this end, one aspect of the present invention provides a kind of primer identified for micro chimerism detection and individuality and probe combinations,
It is made up of the primer shown in SEQ ID NO.1-75 and probe.
In a preferred embodiment of the present invention, this primer and probe combinations are further by 1-15 group primer and probe groups
Becoming, wherein the 1st group is made up of the primer shown in SEQ ID NO.1-4 and the probe shown in SEQ ID NO.5, and the 2nd group by SEQ
Primer shown in ID NO.6-9 and the composition of the probe shown in SEQ ID NO.10, the 3rd group by shown in SEQ ID NO.11-14
Probe composition shown in primer and SEQ ID NO.15, the 4th group by the primer shown in SEQ ID NO.16-19 and SEQ ID
Probe composition shown in NO.20, the 5th group by the primer shown in SEQ ID NO.21-24 and the probe shown in SEQ ID NO.25
Composition, the 6th group is made up of the primer shown in SEQ ID NO.26-29 and the probe shown in SEQ ID NO.30, and the 7th group by SEQ
Primer shown in ID NO.31-34 and the composition of the probe shown in SEQ ID NO.35, the 8th group by shown in SEQ ID NO.36-39
Primer and SEQ ID NO.40 shown in probe composition, the 9th group by the primer shown in SEQ ID NO.41-44 and SEQ ID
Probe composition shown in NO.45, the 10th group by the primer shown in SEQ ID NO.46-49 and the probe shown in SEQ ID NO.50
Composition, the 11st group is made up of the primer shown in SEQ ID NO.51-54 and the probe shown in SEQ ID NO.55, the 12nd group by
Primer shown in SEQ ID NO.56-59 and the composition of the probe shown in SEQ ID NO.60, the 13rd group by SEQ ID NO.61-64
Probe composition shown in shown primer and SEQ ID NO.65, the 14th group by the primer shown in SEQ ID NO.66-69 and SEQ
Probe composition shown in ID NO.70, the 15th group by shown in the primer shown in SEQ ID NO.71-74 and SEQ ID NO.75
Probe forms.
The present invention, based on up-to-date InDel site data base, on the basis of asian population, therefrom filters out for Aisan
15 InDel sites of the high individual discrimination of group, they are respectively as follows: N1-1 (rs2308010), N1-2 (rs34855933), N1-
3(rs4646006)、N2-1(rs3042783)、N5-1(rs1610937)、N5-4(rs1610932)、N7-1(rs16458)、
N9-1(rs2308112)、N11-1(rs2307696)、N11-2(rs140790)、N13-1(rs3038530)、N13-2
(rs3049448), N14-1 (rs56024302), N16-2 (rs2067204) and N21-1 (rs16663), the rs table in bracket
Show this site numbering dbSNP data base.
The present invention is further directed to each InDel site above-mentioned, separately design a shared forward primer (F), under
Trip primer (R), insert specific primer F (+) and lack specific primer F (-), wherein expanding fragment length (Amplified
Fragment Length, AFL) it is that the amplified fragments obtained by this primer matches with corresponding forward primer or downstream primer is long
Degree, such as F (+) it is used for specific amplification Insert Fragment with R, R (-) it being used for specific amplification deletion fragment with F, F with R is for expanding
Increase non-specific fragment (wherein: Lycoperdon polymorphum Vitt background color represents the specific base of insertion/deletion).
For 15 InDel sites of above-mentioned asian population height individuality discrimination, the concrete primer of present invention design and spy
Pin situation is as shown in table 1.
Table 1, the primer of the present invention and probe conditions table
Another aspect of the present invention provides a kind of test kit identified for micro chimerism detection and individuality, and it comprises this
Bright described primer and probe combinations.This test kit is stored in-20 DEG C, and is made up of following reagent.
1, Whole Blood Genomic DNA extracts reagent, is purchased from QIAamp DNA Blood Mini kit test kit；
2、RNase-Free ddH2O；
3,2 × Super Real PreMix reactant liquor, is purchased from TIANGEN Biotech (Beijing) Co., Ltd., article No.:
FP206；
4,50 × ROX Reference Dye, is purchased from TIANGEN Biotech (Beijing) Co., Ltd., article No.: FP206；
5, the primer in corresponding numbering centrifuge tube and probe combinations it are divided in respectively.
In a preferred embodiment of the present invention, in test kit, the concentration of every kind of primer is 6 μMs, and the concentration of every kind of probe is
4μM。
Another aspect of the present invention provides primer of the present invention and probe combinations and is preparing micro chimerism detection and individuality
Application in identification agent box.
Further aspect of the present invention provides primer of the present invention and probe combinations, or test kit of the present invention
Application in micro chimerism detection and individual identification.
Last aspect of the present invention provides a kind of micro chimerism detection and individual knowledge method for distinguishing, and it comprises the steps of
1, the DNA of testing sample is obtained.
DNA extraction can use QIAamp DNA Blood Mini kit test kit.
2, use primer of the present invention and probe combinations, or use test kit of the present invention, so that step 1 to obtain
The DNA taken is template, carries out fluorescent PCR amplification, and collects fluorescence signal.
The system of fluorescent PCR amplification is 20uL:10uL 2 × Super Real PreMix reactant liquor (sky root biochemical technology
(Beijing) company limited, article No.: FP206)；(sky root biochemical technology (Beijing) is limited for 0.2uL 50 × ROX Reference Dye
Company, article No.: FP206)；1uL 6uM primers F (+)/F (-)；1uL 6uM primer R；1uL 4uM probe；1uL 20ng/uL
DNA；Use RNase-Free ddH2O supplies reaction system.
The reaction condition of fluorescent PCR is: 95 DEG C of denaturations 15min, and then 60 DEG C of annealing of 95 DEG C of degeneration 3s extend 32s,
Carry out 40 circulations altogether, at 60 DEG C, collect fluorescence signal.
3, judge testing sample polymorphic differences on 15 InDel sites according to Ct value, thus testing sample is entered
The detection of row micro chimerism and individual identification.
The Ct value interval range of each genetic marker positive sample and negative sample, positive sample Ct is judged by Ct value
Value falls between 24-29, and negative sample Ct value is more than 38, and positive sample and negative sample Ct value difference more than 12, expression is drawn
The specificity of thing and probe is good.
In a preferred embodiment of the present invention, the primer of employing and probe are for asian population height individuality discrimination
15 InDel site designs.
In further preferred embodiment of the present invention, the concentration of every kind of primer is 6 μMs, and the concentration of every kind of probe is 4 μ
M。
Seen from the above description, compared with prior art, the present invention possesses following advantage.
1, polymorphism is strong: 15 the InDel sites screened by the present invention, and 94 samples randomly selected are carried out gene
Frequency test, result shows this 15 InDel alleles distributing equilibriums, and polymorphism is strong.According to these 15 InDel
The polymorphism information in site, it is possible to carry out micro chimerism detection and individual identification.
2, specificity is good: 15 the InDel sites screened by the present invention, examines positive sample and negative sample
Surveying, result shows that specificity is good, stability is strong, reproducible, and genotyping result is accurately (comparing on the basis of sequencing result).
3, highly sensitive: when carrying out analysis of leukocyte subsets chimerism, utilize 15 InDel sites that the present invention screens, carry out fluorescence
PCR expands, and when template amount is only 140ng, can carry out accurate quantitative analysis, and minimum chimeric rate detects up to 0.025%.
4, the detection method of the present invention uses double fluorescence labeling amplification technique, and this technology is easy, quick, only needs one
Applied Biosystems 7500 Real Time PCR System, result automatically analyzes, it is possible to ensure not
Accuracy with laboratory data.
Accompanying drawing explanation
Fig. 1: the present invention is used for detection primer and the PCR amplification figure of probe specificity.
Fig. 2: test kit of the present invention is for the PCR amplification figure of individual recognition detection.
Fig. 3: genetic marker N13-1 of the present invention (+) testing result figure.
Fig. 4: genetic marker N13-1 of the present invention (+) standard substance (100%, 90%, 50%, 10%, 1%, 0.1%,
0.05%, 0.025%) amplification curve diagram.
Detailed description of the invention
Below by embodiment, the present invention is described in further detail, it is intended to is used for illustrating rather than restriction
The present invention.It should be pointed out that, to those skilled in the art, under the premise without departing from the principles of the invention, it is also possible to this
Invention carries out some improvement and modification, and these improve and modify and fall under the scope of the present invention too.
Embodiment 1: the design of primer and synthesis
(1) InDel site screening
Use following principle that InDel site is screened:
1, InDel allele fragment length difference (insert or lack base number) is less than 30bp more than 3bp；
2, it is positioned on 22 autosomes of the mankind；
3, the InDel site spacing on same chromosome is more than 5Mb；
4, minimum gene frequency (Minimum allele frequency, MAF) between 0.30 to 0.50 (with
On the basis of the crowd of East Asia)；
5, total number of sites is no less than 30, and cumulative individual discrimination reaches more than 0.9999；
6, being formed without chain on same chromosome, the distribution in crowd meets Hardy-Weinberg Hardy Weinberg equilibrium；
7, the disease phenotype avoiding InDel site and some special is associated, and pleomorphism site is preferably located at intron district
Territory.
Use mentioned above principle, respectively by http://www.ensembl.org/index.html and http: //
Network address shown in www.ncbi.nlm.nih.gov/projects/SNP/, is carried out tentatively the InDel site that polymorphism is stronger
Screening, finally have selected 15 InDel sites, is respectively as follows: N1-1 (rs2308010), N1-2 (rs34855933), N1-3
(rs4646006)、N2-1(rs3042783)、N5-1(rs1610937)、N5-4(rs1610932)、N7-1(rs16458)、N9-
1(rs2308112)、N11-1(rs2307696)、N11-2(rs140790)、N13-1(rs3038530)、N13-2
(rs3049448), N14-1 (rs56024302), N16-2 (rs2067204), N21-1 (rs16663), the rs table in bracket
Show this site numbering dbSNP data base.
(2) samples selection, gene frequency test and primer sequence amendment
Randomly select 94 Chinese population DNA samples, carry out expanding and gene sequencing.Use preliminary selected primer to 94
Individual Healthy People genomic DNA sample detects, in order to assess amplification efficiency and the gene frequency in each site of primer.
1, DNA extraction
DNA extraction is carried out according to QIAamp DNA Blood Mini kit test kit, uses RNase-Free subsequently
ddH2It is standby that O is diluted to 20ng/uL.
2, PCR amplification system
The PCR amplification system of employing 25uL: 2.5uL 10 × buffer reactant liquor；0.75uL 10mM dNTP；1uL 5uM
Primers F；1.8uL 5uM primer R；1.4uL 5uM primers F (+)/F (-)；2uL DNA；0.2uL Hot start Taq；1uL
Mg2+；0.1uL DMSO, uses RNase-Free ddH2O supplies reaction system.
3, PCR reaction condition
Use following PCR reaction condition: 95 DEG C of denaturations 30s, then 58 DEG C of annealing 30s 68 of 95 DEG C of degeneration 30s
DEG C extend 30s, carry out 30 circulations altogether, last 68 DEG C of extension 5min.
4, electrophoresis detection and primer sequence amendment
PCR primer detects after 2% sepharose electrophoresis 30min, and the specific primer of checking design is the most suitable.With
Generation sequencing result is as the criterion, and for electrophoresis false positive and false-negative situation, revises primer sequence, re-starts detection.
After detection and amendment, the primer in 15 the InDel sites finally determined and probe sequence are as shown in table 1,
Shanghai Ying Jun Bioisystech Co., Ltd is entrusted to synthesize above-mentioned primer and probe.
94 samples are detected by primer and probe with 15 InDel sites respectively, and result shows allele frequency
Equilibrium is compared in rate distribution, and concrete polymorphism information is as shown in table 2.Wherein: "+" represent and insert polymorphism；"-" represents deletion polymorphism
Property；"+/+" represent and insert homozygote polymorphism；" +/-" represents insertion/deletion heterozygote polymorphism；"-/-" represent disappearance and isozygoty
Sub-polymorphism.
The polymorphism information (sequencing result) in 2 15 InDel sites of table
Embodiment 2: primer and probe specificity test
Under the sequencing result of embodiment 1, choose 5 respectively and insert homozygote, deletion homozygote and heterozygote, carry out glimmering
Light PCR, for detection primer and the specificity of probe.
1, fluorescent PCR amplification system
Use the fluorescent PCR amplification system of 20uL: 10uL 2 × Super Real PreMix reactant liquor (sky root biochemistry section
Skill (Beijing) company limited, article No.: FP206)；(sky root biochemical technology (Beijing) has 0.2uL 50 × ROX Reference Dye
Limit company, article No.: FP206)；1uL 6uM primers F (+)/F (-)；1uL 6uM primer R；1uL 4uM probe；1uL 20ng/uL
DNA；Use RNase-Free ddH2O supplies reaction system.
2, Fluorescence PCR condition
Use following Fluorescence PCR condition: 95 DEG C of denaturations 15min, then 60 DEG C of annealing of 95 DEG C of degeneration 3s are prolonged
Stretch 32s, carry out 40 circulations altogether, at 60 DEG C, collect fluorescence signal.
3, primer and probe specificity judge
The Ct value interval range of each genetic marker positive sample and negative sample, positive sample Ct is judged by Ct value
Value falls between 24-29, and negative sample Ct value is more than 38, and positive sample and negative sample Ct value difference more than 12, expression is drawn
The specificity of thing and probe is good.
By N1-2 (+) insert as a example by genetic marker, the Ct value of positive sample (inserting homozygote and heterozygote) falls in scope
In 26-28 (seeing Figure of description 1a), the Ct value of negative sample (deletion homozygote) can't detect (i.e. at 40 PCR cycle
Under several, being not detected by fluorescence signal, Ct value is more than 40) (seeing Figure of description 1b), show the specificity of primer and probe
Well.
Embodiment 3: the composition of test kit of the present invention
The present invention, for micro chimerism detection and the individual InDel genetic marker test kit identified, is stored in-20 DEG C, by with
Lower reagent forms.
1, Whole Blood Genomic DNA extracts reagent: be purchased from QIAamp DNA Blood Mini kit test kit.
2、RNase-Free ddH2O。
3,2 × Super Real PreMix reactant liquor, is purchased from TIANGEN Biotech (Beijing) Co., Ltd., article No.:
FP206。
4,50 × ROX Reference Dye, is purchased from TIANGEN Biotech (Beijing) Co., Ltd., article No.: FP206.
5, being divided in the primer in corresponding numbering centrifuge tube and probe combinations respectively, the concentration of every kind of primer is 6uM, every kind
Concentration and probe concentration is 4uM.
Embodiment 4: the detection that test kit of the present invention identifies for individuality
Randomly select the DNA sample (being respectively designated as A and B) of two separate sources, with the InDel in test kit of the present invention
Genetic marker carries out polymorphic detection.
Fluorescent PCR amplification system and reaction condition are with embodiment 2.After pcr amplification reaction, for each genetic marker,
Four kinds of results it are likely to occur, referring specifically to Figure of description 2 after amplification.In Fig. 2 a, sample A and B is the positive；In Fig. 2 b, sample
This A and B is feminine gender；In Fig. 2 c, sample A is positive, and sample B is negative；In Fig. 2 d, sample A is negative, and sample B is positive.
Fig. 2 a and 2b does not has polymorphic differences, Fig. 2 c and 2d to have polymorphic differences, can judge that sample is according to polymorphic differences
No derive from Different Individual.
Judging according to Ct value, in 15 InDel sites, sample A and sample B have 14 genetic marker (N1-1
(+)、N1-2(-)、N1-3(-)、N2-1(+)、N2-1(-)、N5-4(+)、N7-1(+)、N9-1(+)、N11-1(-)、N11-2
(-), N13-1 (+), N13-1 (-), N14-1 (+), N16-2 (-)) there is polymorphic differences.Therefore, can determine whether sample A and sample
B derives from different individualities.
Embodiment 5: primer and probe accuracy and susceptiveness test
Choose 40 pairs of DNA samples and carry out InDel genetic marker qualitative detection, many to obtain the InDel site of every pair of sample
State property information.
For every pair of sample, (available genetic marker need to meet following characteristics: Ct < 28 sun to select available genetic marker
Property sample be set to receptor, Ct > 38 negative sample is set to donor), mix in varing proportions, obtain the standard substance of different proportion, logical
Cross fluorescent PCR and carry out sensitivity and accuracy detection.Meanwhile, in order to reach 10-5Detection sensitivity, the amount of initial DNA profiling
At least 140ng.
Selecting as a example by one pair of which sample (named C and D of sample), specific implementation method is as follows.
1, sample C and sample D is carried out InDel loci polymorphism test
Fluorescence PCR system and condition are with embodiment 2.With sample C as receptor, sample D for supplying, big according to Ct value
Little (C:Ct < 28 positive sample；D:Ct > 38 negative sample), select available genetic marker.
2, standard substance build
For sample C and sample D, in varing proportions mixing (receptor C/ donor D:90%, 50%, 10%, 1%,
0.1%, 0.05%, 0.025%) standard substance that concentration is 20ng/uL of different proportion, are obtained.
3, by the method detection primer of quantitative analysis and the accuracy of probe and susceptiveness
The chimeric ratio of preparation is respectively 100%, 90%, 50%, 10%, 1%, 0.1%, 0.05%, 0.025%
Standard substance are as the template of quantitative analysis, simultaneously with RNase-Free ddH2O is that template is as blank.In order to reach
140ng DNA original template (10-5Detection sensitivity), the standard substance of 7uL 20ng/uL need to be added, other Fluorescence PCR
System and condition are with embodiment 2.
4, quantitative analysis
Pass through fluorescent PCR, it is thus achieved that the genetic marker of the standard substance detection gained of different proportion and the Ct value of reference gene, and
Calculate the difference (Δ Ct) of both gained Ct values.
Figure of description 3 be genetic marker N13-1 (+) testing result.See Fig. 3 it can be seen that genetic marker N13-1
(+) and reference gene amplification curve form are good, and PCR primer specificity is high, without non-specific amplification, and comparison simultaneously is blank organizes nothing
Amplification.
Figure of description 4 be genetic marker N13-1 (+) standard substance amplification curve.Figure shows, standard substance amplification curve
Form is good.Sample before using C as operation transplantation, CD is as the sample after transplanting, according to Δ Ct's front with transplanting after transplanting
Difference (Δ Δ Ct), can calculate acquisition CD and be fitted together to rate %=2-ΔΔCt× 100%, wherein Δ Δ Ct=Δ CtAfter transplanting-ΔCtBefore transplanting
=(CtAfter marker gene is transplanted-CtAfter reference gene is transplanted)-(CtBefore marker gene is transplanted-CtBefore reference gene is transplanted).Calculate obtain by comparing theoretical value and fluorescent PCR
Test value, can be used to judge the sensitivity of each genetic marker and accuracy.
For N13-1 (+) genetic marker, test value is respectively 93.34% (theoretical 90%), 65.19% (theoretical 50%),
13.25% (theoretical 10%), 0.9452% (theoretical 1%), 0.1053% (theoretical 0.1%), 0.0598% (theoretical 0.05%)
And 0.0282% (theoretical 0.025%), showing that primer and probe test accuracy are good, sensitivity can reach 0.025%
Left and right.
Claims (10)
1. the primer identified for micro chimerism detection and individuality and a probe combinations, it is by drawing shown in SEQ ID NO.1-75
Thing and probe composition.
Primer the most according to claim 1 and probe combinations, it is made up of 1-15 group primer and probe, wherein further
1st group is made up of the primer shown in SEQ ID NO.1-4 and the probe shown in SEQ ID NO.5, and the 2nd group by SEQ ID NO.6-
Primer shown in 9 and the composition of the probe shown in SEQ ID NO.10, the 3rd group by the primer shown in SEQ ID NO.11-14 and SEQ
Probe composition shown in ID NO.15, the 4th group by the primer shown in SEQ ID NO.16-19 and the spy shown in SEQ ID NO.20
Pin form, the 5th group is made up of the primer shown in SEQ ID NO.21-24 and the probe shown in SEQ ID NO.25, the 6th group by
Primer shown in SEQ ID NO.26-29 and the composition of the probe shown in SEQ ID NO.30, the 7th group by SEQ ID NO.31-34
Probe composition shown in shown primer and SEQ ID NO.35, the 8th group by the primer shown in SEQ ID NO.36-39 and SEQ
Probe composition shown in ID NO.40, the 9th group by the primer shown in SEQ ID NO.41-44 and the spy shown in SEQ ID NO.45
Pin form, the 10th group is made up of the primer shown in SEQ ID NO.46-49 and the probe shown in SEQ ID NO.50, the 11st group by
Primer shown in SEQ ID NO.51-54 and the composition of the probe shown in SEQ ID NO.55, the 12nd group by SEQ ID NO.56-59
Probe composition shown in shown primer and SEQ ID NO.60, the 13rd group by the primer shown in SEQ ID NO.61-64 and SEQ
Probe composition shown in ID NO.65, the 14th group by shown in the primer shown in SEQ ID NO.66-69 and SEQ ID NO.70
Probe forms, and the 15th group is made up of the primer shown in SEQ ID NO.71-74 and the probe shown in SEQ ID NO.75.
Primer the most according to claim 1 and 2 and probe combinations, it is 15 for asian population height individuality discrimination
The design of InDel site.
4., for micro chimerism detection and the individual test kit identified, it comprises drawing according to any one of claim 1-3
Thing and probe combinations.
Test kit the most according to claim 4, wherein the concentration of every kind of primer is 6 μMs, and the concentration of every kind of probe is 4 μMs.
6. primer and probe combinations according to any one of claim 1-3 are preparing micro chimerism detection and individual identification agent box
In application.
7. the primer according to any one of claim 1-3 and probe combinations, or the test kit described in claim 4 or 5 exists
Application in micro chimerism detection and individual identification.
8. micro chimerism detection and an individual discrimination method, it comprises the steps of
(1) testing sample DNA is obtained；
(2) use the primer according to any one of claim 1-3 and probe combinations, or use the examination described in claim 4 or 5
Agent box, as template with the DNA of acquisition in step (1), carries out fluorescent PCR amplification, and collects fluorescence signal；
(3) judge testing sample polymorphic differences on 15 InDel sites according to Ct value, thus testing sample is carried out micro-
Chimeric detection and individual identification.
Method the most according to claim 8, the primer wherein used and probe are for asian population height individuality discrimination
15 InDel sites designs.
The most according to claim 8 or claim 9, method, wherein the concentration of every kind of primer is 6 μMs, and the concentration of every kind of probe is 4 μ
M。
Priority Applications (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610268840.5A CN105861675A (en) | 2016-04-27 | 2016-04-27 | Primers, probe, kit and method for microchimerism detection and individual recognition |
| PCT/CN2016/109157 WO2017185758A1 (en) | 2016-04-27 | 2016-12-09 | Primer, probe, kit, and method for microchimerism assay and individual recognition |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610268840.5A CN105861675A (en) | 2016-04-27 | 2016-04-27 | Primers, probe, kit and method for microchimerism detection and individual recognition |
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| Publication Number | Publication Date |
|---|---|
| CN105861675A true CN105861675A (en) | 2016-08-17 |
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ID=56629313
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201610268840.5A Pending CN105861675A (en) | 2016-04-27 | 2016-04-27 | Primers, probe, kit and method for microchimerism detection and individual recognition |
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| Country | Link |
|---|---|
| CN (1) | CN105861675A (en) |
| WO (1) | WO2017185758A1 (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2017185758A1 (en) * | 2016-04-27 | 2017-11-02 | 上海荻硕贝肯生物科技有限公司 | Primer, probe, kit, and method for microchimerism assay and individual recognition |
| CN113265474A (en) * | 2021-07-19 | 2021-08-17 | 深圳荻硕贝肯精准医学有限公司 | Detection primer group and probe group for digital PCR (polymerase chain reaction) chimerism rate and application thereof |
| CN116103409A (en) * | 2022-11-16 | 2023-05-12 | 汕头大学医学院 | Method for synchronously detecting bone marrow transplantation chimeric rate of genetically modified mice by utilizing qPCR |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114574595B (en) * | 2022-03-08 | 2022-12-02 | 中国人民解放军军事科学院军事医学研究院 | Application of human chromosome InDel gene locus, primer group, product thereof and individual identification method of test material |
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| CN101575643A (en) * | 2009-05-31 | 2009-11-11 | 中国人民解放军第二军医大学 | Method for detecting the chimerism rate of recipients after allogeneic hematopoietic stem cell transplantation |
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| CN101956005B (en) * | 2010-08-13 | 2012-07-25 | 司法部司法鉴定科学技术研究所 | Fluorescently-labeled insertion/deletion (InDel) genetic polymorphism locus composite amplification system and application thereof |
| CN104131067B (en) * | 2013-05-03 | 2016-03-02 | 司法部司法鉴定科学技术研究所 | A kind of fluorescently-labeled X-InDel site composite amplification system and application thereof |
| CN103468800B (en) * | 2013-08-23 | 2014-12-10 | 四川大学 | Forensic medicine composite detection kit based on 20 multiple insertion/delection genetic markers |
| CN105861675A (en) * | 2016-04-27 | 2016-08-17 | 上海荻硕贝肯生物科技有限公司 | Primers, probe, kit and method for microchimerism detection and individual recognition |
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2016
- 2016-04-27 CN CN201610268840.5A patent/CN105861675A/en active Pending
- 2016-12-09 WO PCT/CN2016/109157 patent/WO2017185758A1/en active Application Filing
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| CN101575643A (en) * | 2009-05-31 | 2009-11-11 | 中国人民解放军第二军医大学 | Method for detecting the chimerism rate of recipients after allogeneic hematopoietic stem cell transplantation |
| CN101671736A (en) * | 2009-10-15 | 2010-03-17 | 北京市道培医院 | Gene detection kit used for detecting cell chimerism or individual recognition |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2017185758A1 (en) * | 2016-04-27 | 2017-11-02 | 上海荻硕贝肯生物科技有限公司 | Primer, probe, kit, and method for microchimerism assay and individual recognition |
| CN113265474A (en) * | 2021-07-19 | 2021-08-17 | 深圳荻硕贝肯精准医学有限公司 | Detection primer group and probe group for digital PCR (polymerase chain reaction) chimerism rate and application thereof |
| CN113265474B (en) * | 2021-07-19 | 2021-11-02 | 深圳荻硕贝肯精准医学有限公司 | Detection primer group and probe group for digital PCR (polymerase chain reaction) chimerism rate and application thereof |
| CN116103409A (en) * | 2022-11-16 | 2023-05-12 | 汕头大学医学院 | Method for synchronously detecting bone marrow transplantation chimeric rate of genetically modified mice by utilizing qPCR |
Also Published As
| Publication number | Publication date |
|---|---|
| WO2017185758A1 (en) | 2017-11-02 |
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