CN101671736A - Gene detection kit used for detecting cell chimerism or individual recognition - Google Patents
Gene detection kit used for detecting cell chimerism or individual recognition Download PDFInfo
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Abstract
The invention discloses a gene detection kit which is used for detecting cell chimerism or individual recognition, in particular relating to an oligonucleotide array sequence, a kit and a method usedfor detecting cell chimerism or individual recognition. The kit utilizes gene insertion/deletion polymorphism (INDELs) and site-specific fluorescent quantitative PCR to quantitatively detect that different individuals of human beings have chimerism or microchimerism of foreign body cells, which can be used for the detection of mother and son/daughter cell microchimerism, cell chimerism of donor/receptor after the transplantation of hematopoietic stem cells or other organs and residual diseases after the transplantation of hematopoietic stem cells of leukemia, and for the forensic individual identification, paternity test and the like. The kit in the invention has good specificity and high detection sensitivity, can accurately and quantitatively detect with convenient operation and with norequirement of special devices; and when adopting the method in the invention to carry out individual identification detection, only agaroseelectrophoresis is needed to distinguish the results.
Description
Technical field
The present invention relates to a kind of gene detecting kit and nucleotide sequence and using method thereof that is used to detect cell chimerism state or individual recognition, belong to biological technical field.
Background technology
Utilize the gene information of different biology individual uniqueness separately to distinguish the individuality source of different individualities or cell, can be used for detecting the childbirth stepmother daughter cell long-standing microchimerism relevant with autoimmune disease, donor after hemopoietic stem cell or other organ transplantations/donee's cells chimerism, minimal residual disease etc. is having a wide range of applications aspect forensic evaluation, paternity test and other medical tests after the leukemia hematopoietic stem cell transplantation.Genome is the fragment that is comprising all genetic information of people, has with living, and remains unchanged all the life.This genetic information lies in everyone body tissue such as bone, hair, blood of people or the organ.Since polytype pleomorphism site of One's name is legion is arranged in the human genome, often all variant on a lot of sites between the Different Individual, and carry out gene test and only need can carry out by micro-sample.Utilize gene pleiomorphism to carry out individual recognition and become legal medical expert and the widely used method of clinical detection.
Develop multiple genetic marker in recent years and be used for individual recognition.Wherein STR (Short TandemRepeat is called for short STR) has developed into the most frequently used individual recognition certification mark in each medical jurisprudence laboratory at present because detection method is easy, quick, accuracy is high, amplified fragments is of moderate size.But because clip size difference seldom (often being 3～5 base length) between the different microsatellite polymorphism fragments, therefore need high-resolution electrophoresis method (polyacrylamide gel electrophoresis or use the sequenator analysis) just can detect.Because the polyacrylamide gel electrophoresis complicated operation, poor stability is tending towards superseded gradually; And the cost costliness of sequenator has limited this broad application.
The definition of mosaic on the genetics (Chimerism) is the cell chimerism of different inherited character in the same individual body or mixes performance.A this body is formed the simultaneous state of different clone by two or more karyomit(e) simultaneously and is called chimerism.Have been found that the long-standing microchimerism of childbirth stepmother's daughter cell, relevant with autoimmune disease; Donor after hemopoietic stem cell or other organ transplantations/donee's cells chimerism is relevant with prognosis, and minimal residual disease chimerism etc. is directly related with treatment after the leukemia hematopoietic stem cell transplantation.Detection for the research of pathogenic mechanism and treatment, conditions of patients is all significant.For example, in the allosome hematopoietic stem cell transplantation, implanted source of human stem cell is in not belonging to the donor of same individuality with the patient, after these stem cells are implanted in patient's body, though can in patient's body, grow oneself, but the gene genetic information that it carries still is the information that belongs to donor, has therefore just formed chimerism.Therefore the monitoring of donor-recipient's cell chimerism state is the important indicator of hematopoietic stem cell transplantation success or not, at present implanting states that adopt after the STR method detects hematopoietic stem cell transplantation more.
Micro chimerism phenomenon (Microchimerism) is meant that the small amounts of cells of a certain individuality transfers to another intravital phenomenon.It is micro chimerism that the general composition that claims that a kind of individuality is originated in the blended cell is lower than at 1% o'clock, and the component proportions in all sources was a chimerism greater than 1% o'clock all.The cell of oneself may be transferred to fetus as far back as the fifties in last century with regard to having been found that mother, and after fetus birth, the cell of maternal source also can exist in child's body even many decades for a long time.Also found afterwards, also may carry a spot of child's of deriving from cell for a long time in childbirth back mother's the body.The chimeric phenomenon of the known this mothers and sons of present research is relevant with some autoimmune disorders, but also has the more mechanism person of requiring study to explore.In recent years, along with the application of organ transplantation and going deep into of research, the investigator also finds also extensively to exist the micro chimerism phenomenon in organ transplantation.Be present in the peripheral blood of patients as in patient's body of heart transplantation or renal transplantation, detecting the cell long-period that derives from donor on a small quantity, the repulsion of this micro chimerism phenomenon and graft with tolerate relevant.
Different with individual recognition is that individual recognition is to be used for detecting two samples whether to derive from same individuality respectively, perhaps whether has the genetic connection on the genetics; And the chimerism analysis is in a sample, and how many cellular constituents in Different Individual source respectively accounts for.Therefore, individual recognition is that the polymorphic position dot information according to different specimens compares analysis, to assess whether belonging to same individuality on its genetics or not having genetic connection; And the analysis of chimeric rate is to be clue according to the known polymorphism information that belongs to two or more Different Individual respectively, comes the shared ratio of two or more Different Individual cells in the quantitative analysis compound sample.
Therefore the detection that the detection requirement of chimerism can quantitative analysis, especially microchimerism requires more highly sensitive detection method.And the at present conventional STR method of using is carried out chimerism when detecting, because the restriction of detection method and instrument, detection sensitivity can only reach 1%, therefore can not carry out the analysis of micro chimerism.When carrying out micro chimerism research at present, adopt the method that in women's sample, detects the Y chromosome specific gene more.It is good that this method has specificity, the characteristics that detection sensitivity is high.But because Y chromosome only is present in male sex's cell, so this method can only be used for detecting the cell in male sex source in women's sample, and versatility is poor, causes most samples to detect and to analyze.Another kind of method commonly used is the polymorphism according to the HLA site, according to the difference of two individual HLA loci polymorphisms, increases detection sensitivity in conjunction with nido locus specificity PCR method.But, often need each is all wanted special contrived experiment scheme to sample, and can not carry out quantitative analysis because the HLA loci polymorphism is very strong.
Along with the progress of genome research, the pleomorphism site of a new generation is found in succession and uses.(Single Nucleotide Polymorphisms, SNP) detection scheme of individual recognition is set in the site also to have the investigator to adopt single nucleotide polymorphism.But because the allelotrope differences of single nucleotide polymorphism has only a base, therefore be difficult to set highly sensitive detection scheme, detection sensitivity often can only reach 1%, still can not be used for the detection of micro chimerism.And SNP quantivative approach complexity, poor practicability.
INDELs (insertions and deletions promptly inserts and lacks) polymorphism is the pleomorphism site of a new generation of discovered in recent years, is meant to insert or lacked dissimilar and big or small small pieces segment DNA in the genome.With respect to the STR polymorphism,, therefore can't improve detection sensitivity by the method for setting locus specificity PCR primer because the STR site is the tumor-necrosis factor glycoproteins of different numbers; And the INDELs polymorphism is the disappearance or the variation of inserting, and disappearance or the fragment inserted can be severally not wait to a hundreds of base, therefore can come the allelotype of insertion/disappearance is distinguished by the method that designs locus specificity PCR primer.With respect to the SNP polymorphism, because SNP is the replacement sudden change of single base, sequence difference is not less between isoallele, and it is specific therefore having only 3 ' terminal base according to the locus specificity primer of SNP site design, is difficult to improve detection sensitivity; But the difference between the disappearance of INDELs polymorphism or the allelotype of insertion can be several even a hundreds of base, therefore between the Auele Specific Primer of different loci very big difference can be arranged, therefore can improve the specificity and the detection sensitivity of primer greatly.In addition, determine that some individual gene INDELs polymorphism features are having a wide range of applications aspect forensic evaluation, paternity test and other medical tests.
Summary of the invention
The technical problem to be solved in the present invention provides one group in order to variant cell chimerism, microchimerism in the human body or Different Individual is carried out the oligonucleotide of gene recognition.
Another technical problem that the present invention will solve provide a kind of special, sensitive, be used to detect the gene detecting kit of cell chimerism state or individual recognition efficiently.This test kit can be used for mother and sons' cell micro chimerism, donor after hemopoietic stem cell or other organ transplantations/donee's cells chimerism, the detection of residual disease after the leukemia hematopoietic stem cell transplantation, legal medical expert's individual recognition and paternity test etc.
For solving the problems of the technologies described above, the present invention by the following technical solutions:
The invention provides one group of oligonucleotide that is used to detect cell chimerism state or individual recognition, be the oligonucleotide of sequence table SEQ IDNo.1 to base sequence shown in the sequence table SEQ ID No.32;
Wherein, SEQ ID No.1 is for detecting the rs2308010 pleomorphism site Auele Specific Primer that inserts polymorphism, SEQID No.2 is for detecting the rs2308010 pleomorphism site Auele Specific Primer of deletion polymorphism, and SEQ ID No.3 is that upstream primer, the SEQ ID No.4 of rs2308010 pleomorphism site is the downstream primer of rs2308010 pleomorphism site;
SEQ ID No.5 is for detecting the rs4150017 pleomorphism site Auele Specific Primer that inserts polymorphism, SEQ IDNo.6 is for detecting the rs4150017 pleomorphism site Auele Specific Primer of deletion polymorphism, and SEQ ID No.7 is that upstream primer, the SEQ ID No.8 of rs4150017 pleomorphism site is the downstream primer of rs4150017 pleomorphism site;
SEQ ID No.9 is for detecting the rs2307553 pleomorphism site Auele Specific Primer that inserts polymorphism, SEQ IDNo.10 is for detecting the rs2307553 pleomorphism site Auele Specific Primer of deletion polymorphism, and SEQ ID No.11 is that upstream primer, the SEQ ID No.12 of rs2307553 pleomorphism site is the downstream primer of rs2307553 pleomorphism site;
SEQ ID No.13 is for detecting the rs2308144 pleomorphism site Auele Specific Primer that inserts polymorphism, SEQ IDNo.14 is for detecting the rs2308144 pleomorphism site Auele Specific Primer of deletion polymorphism, and SEQ ID No.15 is that upstream primer, the SEQ ID No.16 of rs2308144 pleomorphism site is the downstream primer of rs2308144 pleomorphism site;
SEQ ID No.17 is for detecting the rs4646006 pleomorphism site Auele Specific Primer that inserts polymorphism, SEQ IDNo.18 is for detecting the rs4646006 pleomorphism site Auele Specific Primer of deletion polymorphism, and SEQ ID No.19 is that upstream primer, the SEQ ID No.20 of rs4646006 pleomorphism site is the downstream primer of rs4646006 pleomorphism site;
SEQ ID No.21 is for detecting the rs34132048 pleomorphism site Auele Specific Primer that inserts polymorphism, SEQ IDNo.22 is for detecting the rs34132048 pleomorphism site Auele Specific Primer of deletion polymorphism, and SEQ ID No.23 is that upstream primer, the SEQ ID No.24 of rs34132048 pleomorphism site is the downstream primer of rs34132048 pleomorphism site;
SEQ ID No.25 is for detecting the rs1610932 pleomorphism site Auele Specific Primer that inserts polymorphism, SEQ IDNo.26 is for detecting the rs1610932 pleomorphism site Auele Specific Primer of deletion polymorphism, and SEQ ID No.27 is that upstream primer, the SEQ ID No.28 of rs1610932 pleomorphism site is the downstream primer of rs1610932 pleomorphism site;
SEQ ID No.29 is for detecting the rs56024302 pleomorphism site Auele Specific Primer that inserts polymorphism, SEQ IDNo.30 is for detecting the rs56024302 pleomorphism site Auele Specific Primer of deletion polymorphism, and SEQ ID No.31 is that upstream primer, the SEQ ID No.32 of rs56024302 pleomorphism site is the downstream primer of rs56024302 pleomorphism site.
The sequence of various primers sees table 1 for details, and wherein expanding fragment length is the expanding fragment length of this primer and downstream primer (R) pairing gained.
Table 1. primer sequence
The present invention also provides a kind of gene detecting kit that is used to detect cell chimerism state or individual recognition, and the 10tests/ box is stored in-20 ℃, is made up of following reagent:
1) extracting genome DNA reagent (20 person-times) is available from liking to pursue progress biotechnology (Hangzhou) company limited, article No.: AP-MN-BLGDNA-50;
2) ultrapure water (preparation of MilliporeMILLI-Q PF PLUS water purification machine, resistivity＞18.2M Ω) 5ml;
3) 2 * PCR reaction solution 2ml, available from TIANGEN Biotech (Beijing) Co., Ltd., article No.: KT201;
4) 2 * SYBRGreen PCR reaction solution 2ml, available from u.s.a. applied biosystem company limited, article No.: 4367659;
5) detect employed each 50ul of various primers (various primers are as shown in table 1, and its sequence is synthetic by American I ntegratedDNA Technologies company, are 2pmol/ul concentration with 1 * TE dilution), be divided in respectively in the 0.2ml centrifuge tube of corresponding numbering;
6) genomic dna reference substance 1 (50ng concentration, 50ul);
7) genomic dna reference substance 2 (50ng concentration, 50ul);
Rs2308010, the rs4150017 of wherein said genomic dna reference substance 1 and reference substance 2, rs2307553, rs2308144, rs4646006, rs34132048, rs1610932 and rs56024302 loci polymorphism have carried out gene sequencing.The loci polymorphism information of genomic dna reference substance 1 provided by the invention and reference substance 2 sees Table 2 (wherein polymorphism is inserted in "+" representative, and "-" represents deletion polymorphism).
The polymorphism information in 8 INDELs sites of table 2. test kit reference substance
Advantage of the present invention is:
1, the present invention only adopts regular-PCR instrument and agarose electrophoresis method can carry out the polymorphism qualitative analysis in INDELs site, and carries out individual recognition or sibship analysis according to the polymorphism in each INDELs site.With respect to the STR method that extensively adopts at present, the present invention does not need expensive instruments such as sequenator, is easy to promote and use.
2, carry out chimeric or microchimerism when analyzing, with respect to the detection method that adopts the Y chromosome specific sequence, it is restricted that present method does not have sex.The sample of any sex all can be analyzed, and makes analysis no longer be subjected to the restriction of sample sex, has better generality.
3, carry out chimeric or microchimerism when analyzing, with respect to the method that adopts HLA locus specificity primer, present method versatility is better, and can carry out accurate quantitative analysis.
4, carry out chimeric or during the microchimerism quantitative analysis, because present method combines the characteristics and the nest-type PRC technology of INDELs polymorphism.When increasing the original template amount, guaranteed detection by quantitative result's accuracy again.When adopting the Y chromosome specific sequence to detect micro chimerism, because initial dna profiling quantitative limitation, detection sensitivity can only reach about 10
-4But when adopting present method, because the increase of original template amount, detection sensitivity can bring up to 10
-5, further improved detection sensitivity.
The invention will be further described below in conjunction with specification drawings and specific embodiments, all any this areas of doing according to the disclosure of invention be equal to replacement, all belong to protection scope of the present invention.
Description of drawings
Fig. 1 is a design of primers synoptic diagram of the present invention, and wherein is a gene order,
For inserting polymorphic sequence, F is a upstream primer, and R is a downstream primer ,+be insert type locus specificity primer ,-be absence type locus specificity primer;
Fig. 2 detects electrophoresis result figure for this test kit is used for individual recognition, what "+" and "-" represented this reaction detection respectively is insert type and absence type polymorphism, in 8 INDELs sites of electrophorogram this A of indicating and sample B, there is the polymorphism in 6 sites (rs4150017, rs2307553, rs2308144, rs4646006 rs34132048, rs56024302) variant;
Fig. 3 is used to detect the electrophoresis result figure of chimerism, the classification situation of detected result when the electrophorogram indicating originally is in chimerism and INDELs pleomorphism site for the present invention;
Each loci polymorphism detected electrophoresis result figure, the classification situation of INDELs pleomorphism site among electrophorogram this E of indicating and the sample F when Fig. 4 detected mother and daughter's microchimerism for detection;
Fig. 5 is standard substance amplification gained amplification curve of the present invention and canonical plotting; R: confidential reference items primer amplification curve; A:50% standard substance primer amplified curve; B:5% standard substance primer amplified curve; C:0.5% standard substance primer amplified curve; D:0.05% standard substance primer amplified curve;
Fig. 6 detects the amplification curve and the graphic representation that dissociates for sample of the present invention.
Embodiment
Embodiment 1: primer design is with synthetic
Be chosen in the candidate INDELs site that has strong polymorphism in the Chinese han population: according to the U.S. state-run bioinformation center (The National Center for Biotechnology Information, NCBI) website snp database (http://www.ncbi.nlm.nih.gov/sites/entrez? db=snp) the INDELs polymorphism information in is selected, and just is selected in the INDELs site that this crowd's polymorphism is stronger in the database data.
Design of primers such as Fig. 1, illustrate primer design method of the present invention and principle: according to each INDELs site sequence design of choosing and prepare corresponding primer sequence: for each INDELs site, design a shared upstream primer (F), a shared downstream primer (R) respectively, detect and insert polymorphism (Insersion, I) locus specificity primer (+) and detection deletion polymorphism (Deletsion, locus specificity primer (-) D).3 ' end of every locus specificity primer has the corresponding gene order each INDELs site or deletion polymorphism of 4-6 base specific respectively, to reach stronger locus specificity effect.
Sample is selected and experiment: 100 health adults of Han nationality of picked at random (50 of men, 50 of woman) genomic dna sample increases and the gene sequencing analysis.Finally determine the INDELs site of adopting according to the polymorphism situation in each site of actual detected.Primer with above-mentioned design detects 100 parts of health adult's genomic dna samples, with the polymorphism situation in assessment primer amplification efficient and each site.
The extraction of genomic dna like the to pursue progress description of product of biotechnology (Hangzhou) company limited carries out, and it is standby to 5ng/ul with 1 * TE solution dilution to extract the genomic dna that obtains.The PCR reaction system is 20ul, contain genomic dna 10ng in each reaction system, each 4pmol of primer (inserting polymorphism and deletion polymorphism in order to detect respectively) of being numbered of each INDELs site correspondence " F, R ,+" or " F, R ,-", 2 * PCR reaction solution 10ul supplies reaction system with ultrapure water.The PCR reaction conditions is 95 ℃ of pre-sex change 5 minutes, and 35 circulations were carried out in 95 ℃ of sex change-58 ℃ of annealing in 15 seconds-72 ℃ of extensions in 30 seconds in 45 seconds altogether then, and last 72 ℃ were extended 7 minutes.The PCR product detected through 2% agarose electrophoresis in 30 minutes, judged the polymorphism situation in each site according to the stripe size that occurs.
According to experimental result, finally chosen 8 INDELs sites that have strong polymorphism in the crowd of Han nationality, the numbering in snp database is respectively: rs2308010, rs4150017, rs2307553, rs2308144, rs4646006, rs34132048, rs1610932, rs56024302.The primer sequence sees Table 1, and the polymorphism information in detected 8 sites sees Table 3 in 100 routine samples, wherein "+/+" representative insertion polymorphism of isozygotying; "+/-" representative insertion/disappearance heterozygosis polymorphism; "-/-" represent the homozygous deletion polymorphism.
Above the primer is synthetic by American I ntegrated DNA Technologies company.
The polymorphism detected result in selected 8 the INDELs sites of this test kit in the routine sample of table 3.100
The composition of embodiment 2 test kits of the present invention
The gene detecting kit that is used to detect cell chimerism state or individual recognition of the present invention, the 10tests/ box is stored in-20 ℃, is made up of following reagent:
1) extracting genome DNA reagent (20 person-times) is available from liking to pursue progress biotechnology (Hangzhou) company limited, article No.: AP-MN-BLGDNA-50;
2) ultrapure water (preparation of MilliporeMILLI-Q PF PLUS water purification machine, resistivity＞18.2M Ω) 5ml;
3) 2 * PCR reaction solution 2ml, available from TIANGEN Biotech (Beijing) Co., Ltd., article No.: KT201;
4) 2 * SYBRGreen PCR reaction solution 2ml, available from u.s.a. applied biosystem company limited, article No.: 4367659;
5) detect employed each 50ul of various primers (various primers are as shown in table 1, and its sequence is synthetic by American I ntegratedDNA Technologies company, are 2pmol/ul concentration with 1 * TE dilution), be divided in respectively in the 0.2ml centrifuge tube of corresponding numbering;
6) genomic dna reference substance 1 (50ng concentration, 50ul);
7) genomic dna reference substance 2 (50ng concentration, 50ul);
The preparation of genomic dna reference substance 1 and reference substance 2:
Taking two healthy each 10ml of donor peripheral blood sample, pursue progress after company extracting genome DNA reagent extracts Whole Blood Genomic DNA with above-mentioned love, is to be reference substance after 50ng/ul and the packing with the dilution of 1 * TE damping fluid.
Wherein genomic dna reference substance 1 and reference substance 2 provide as quality control product, and sample derives from two healthy donors.Reference substance 1 and reference substance 2 are united use, and the various insert types in 8 INDELs sites and the genotypic DNA sample of absence type are provided.The loci polymorphism of genomic dna reference substance 1 and reference substance 2 confirms that through gene sequencing concrete polymorphism information sees Table 2 (wherein polymorphism is inserted in "+" representative, and "-" represents deletion polymorphism).
Embodiment 3: this test kit is used for the detection of individual recognition
The genomic dna sample of two Different Individual of picked at random (respectively called after A and B), carry out this test kit respectively in the polymorphism in 8 INDELs sites detect.
The PCR reaction system is 20ul, contain genomic dna 10ng in each reaction system, each 4pmol of primer (inserting polymorphism and deletion polymorphism in order to detect respectively) of being numbered of each INDELs site correspondence " F, R ,+" or " F, R ,-", 2 * PCR reaction solution 10ul supplies reaction system with ultrapure water.The PCR reaction conditions is 95 ℃ of pre-sex change 5 minutes, and 35 circulations were carried out in 95 ℃ of sex change-58 ℃ of annealing in 15 seconds-72 ℃ of extensions in 30 seconds in 45 seconds altogether then, and last 72 ℃ were extended 7 minutes.The PCR product detected through 2% agarose electrophoresis in 30 minutes, judged the polymorphism situation in each site according to the stripe size that occurs.
Electrophoresis result is seen Fig. 2, and what "+" and "-" represented this reaction detection respectively is insert type and absence type polymorphism.Can judge according to electrophoresis result, in 8 INDELs sites of sample A and sample B, there is the polymorphism in 6 sites (rs4150017, rs2307553, rs2308144, rs4646006 rs34132048, rs56024302) variant, therefore can judges that sample A derives from different individualities with sample B.
Embodiment 4: this test kit is used for the detection of chimerism after the bone marrow transplantation
Choosing an example carries out before leukaemic's the transplanting of bone marrow transplantation peripheral blood, donor peripheral blood, donor peripheral blood and transplants back peripheral blood sample (being labeled as C, D and CD respectively) and detect.Transplant back peripheral blood sample and account for 13.12% for patient's derived cell, for donor and patient mix chimerism through STR method detected result with classics.The STR method adopts ABI Identifilier test kit and ABI 310 sequenators to detect.
Identical with the STR method is that when adopting this test kit to carry out the chimerism detection, sample and donor sample detect before need transplanting the patient equally, to obtain donor and patient's INDELs loci polymorphism information separately.But this test kit only needs agarose electrophoresis to detect and the ordinary gel imager can be analyzed chimerism.
Specific implementation method is:
1, respectively C, D and CD sample are carried out the detection in 8 INDELs sites: the PCR reaction system is 20ul, contain genomic dna 10ng in each reaction system, each 4pmol of primer (inserting polymorphism and deletion polymorphism in order to detect respectively) of being numbered of each INDELs site correspondence " F, R ,+" or " F, R ,-", 2 * PCR reaction solution 10ul supplies reaction system with ultrapure water.The PCR reaction conditions is 95 ℃ of pre-sex change 5 minutes, and 35 circulations were carried out in 95 ℃ of sex change-58 ℃ of annealing in 15 seconds-72 ℃ of extensions in 30 seconds in 45 seconds altogether then, and last 72 ℃ were extended 7 minutes.The PCR product detected through 2% agarose electrophoresis in 30 minutes, judged the polymorphism situation in each site according to the stripe size that occurs.This routine electrophoresis result is seen Fig. 3.
2, the classification of pleomorphism site: according to the situation of each loci polymorphism, 8 INDELs sites can be divided into 4 classes: 1, donor and patient's polymorphism are identical is 0 class site (as rs4150017 in this example and rs34132048 site), be the noninformation bit point, this type of site can not be used for carrying out the chimerism analysis; 2, patient (C) is for inserting homozygosity, donor (D) for lacking homozygosity, or patient (C) is I class site for disappearance homozygosity, donor (D) for the site (as rs56024302 site in this example) of inserting homozygosity, and the analysis of various chimerisms can be carried out in this type of site.3, patient (C) is that insertion/disappearance heterozygosity, donor (D) are IIa class site (as the rs2308010 in this example, rs2308144, rs4646006 site) for the site of inserting or lack homozygosity, this type of site is fit to carry out the less relatively chimerism analysis of patient's cell concentration, is carrying out having bigger error when the less relatively chimerism of donor's cells is analyzed.3, donor (D) is that insertion/disappearance heterozygosity, patient (C) are IIb class site (as the rs2307553 in this example, rs1610932 site) for the site of inserting or lack homozygosity, this type of site is fit to carry out the less relatively chimerism analysis of donor's cells's amount, is carrying out having bigger error when the less relatively chimerism of patient's cell is analyzed.
3, the calculating of chimeric ratio:
The calculating of chimeric rate at first will be carried out gray analysis to the insertion and the absence type PCR electrophoresis fragment in I class and II class site in the chimeric sample (being sample CD in this example) with the gel imaging instrument.Used gel imaging instrument is a Tanon GIS-2010 gel imaging instrument in this example, and used gray analysis software is a day energy GIS gel images treatment system V4.0.
A) 0 class site does not participate in the analysis of chimerism, does not calculate.
B) the chimeric rate calculation formula in I class site is:
The chimeric rate of patient's sample=patient-specific band gray-scale value/(total gray-scale value of insertion/disappearance band)
The chimeric rate of donor sample=donor specific band gray-scale value/(total gray-scale value of insertion/disappearance band)
C) IIa class site is mainly used in the chimeric rate calculating of patient's composition, and calculation formula is:
The chimeric rate of patient's sample=patient-specific band gray-scale value/(total gray-scale value of insertion/disappearance band)
D) IIb class site is mainly used in the chimeric rate calculating of donor composition, and calculation formula is:
The chimeric rate of donor sample=donor specific band gray-scale value/(total gray-scale value of insertion/disappearance band)
The rs56024302 site is I class site in this routine sample, and chimeric rate calculation result is 11.80% for patient's cell proportion; Rs2308010, rs2308144, rs4646006 are IIa class site, and calculating patient's cell proportion is 13.51%, 12.12% and 10.17%.Patient's cell chimerism ratio mean value is 12.05%, and is close with the result who detects with the STR method.
Embodiment 5: this test kit is used for the detection of microchimerism
When this test kit is used for the detection (as mother and sons' micro chimerism) of microchimerism, need equally earlier two parts of samples from Different Individual to be detected, to obtain donor and patient's INDELs loci polymorphism information separately, select the information available site again and carry out further micro chimerism detection.Because during microchimerism, the cell proportion of micro chimerism generally very low (＜1%), because the amplified production amount is very low and the restriction of detection method, agarose electrophoresis often detects the amplified production less than micro chimerism when detecting, therefore need combined with fluorescent quantitative PCR to detect.For reaching 10
-5Detection sensitivity, needing at least, the initial gene group dna profiling of 500ng detects.But when the template total amount is too much, can influence the amplification efficiency of PCR and the detection of low copy template, need increase in advance when therefore chimeric or microchimerism being carried out quantitative analysis, i.e. the amplification of the first round of nest-type PRC.The pre-amplification time-division does not adopt the upstream primer (F) in each INDELs site, downstream primer (R) combination to increase.When carrying out fluorescence quantitative PCR detection, also to make up typical curve, to carry out accurate quantitative analysis.
We adopt this test kit that 41 couples of mothers and sons/woman are carried out mother to have carried out the detection of microchimerism for mother's sample of the bone marrow transplantation of donor.The result detects the micro chimerism of son/women cell therein in 10 examples (24%) mother peripheral blood, and the ratio of micro chimerism cell is 10
-5～10
-3Between.This measure wherein an example carry out the explanation (mother's sample called after E, daughter's sample called after F) of application method.
Specific implementation method is:
1, among sample E and the sample F polymorphism in each INDELs site detect and the sorting technique of pleomorphism site with embodiment 3.The loci polymorphism electrophorogram of two parts of samples is seen Fig. 4, because both are mother and daughter's genetic connection, does not therefore have I class site.Can carry out the microchimerism analysis of mother (E) cell in daughter (F) body according to rs2308010, rs4150017 in the II class site, rs2308144 and rs4646006 site.
2, the first round of nest-type PRC amplification: the PCR reaction system is 20ul, contains genomic dna 660ng in each reaction system and (is equivalent to 10 approximately
5The genomic dna of individual cell), each 4pmol of primer that is numbered " F, R " of each INDELs site correspondence, 2 * PCR reaction solution 10ul supplies reaction system with ultrapure water.The PCR reaction conditions is 95 ℃ of pre-sex change 5 minutes, and 20 circulations were carried out in 95 ℃ of sex change-58 ℃ of annealing in 15 seconds-72 ℃ of extensions in 30 seconds in 45 seconds altogether then, and last 72 ℃ were extended 7 minutes.
3, the structure of standard substance: choose known genomic dna sample with other people of relevant polymorphism, the polymorphism sample of the different insertion of isozygotying, homozygous deletion or heterozygosity is mixed in varing proportions (as deletion polymorphism: insert the polymorphism ratio and be respectively 50%, 5%, 0.5%, 0.05%), obtain the standard substance of different ratios.
4, nido second is taken turns the PCR reaction, be the quantitative analysis of microchimerism: the chimeric ratio that will prepare is respectively standard substance or 500 times of templates as quantitative analysis of the resulting product dilution of nido first round pcr amplification of 50%, 5%, 0.5%, 0.05%, is that template is done blank with the ultrapure water simultaneously.Can carry out the INDELs site (being rs2308010, rs4150017, rs2308144 and rs4646006 site in this example) of quantitative analysis for each, adopt the locus specificity primer (+) of detection insertion polymorphism or the locus specificity primer (-) of detection deletion polymorphism to be combined into the nest-type PRC amplification that performing PCR second is taken turns respectively according to corresponding situation, adopt upstream primer (F) and downstream primer (R) to make up simultaneously and carry out the amplification of internal control gene with downstream primer (R).The pcr amplification of this step carries out on quantitative real time PCR Instrument, and adopts the SYBRGreen dye method to carry out the real-time detection of amplified production.This routine fluorescence quantitative PCR detection is carried out on AB 7300 type quantitative real time PCR Instruments, and the PCR reaction system is 20ul, comprising first round PCR product 2ul, SYBRGreen PCR reaction solution 10ul, every each 4pmol of primer after the dilution.Reaction conditions be 50 ℃ made that the UNG enzyme works in 2 minutes, 95 ℃ of pre-sex change in 10 minutes and heat of activation start enzyme, 15 seconds, 60 ℃ of 95 ℃ of sex change were carried out 40 circulations in 1 minute altogether then, did the dissociation curve analysis after the PCR circulation to judge the specificity of PCR product.
5, typical curve analysis: the difference (goal gene CT-internal control gene CT value, i.e. Δ CT value) that detects the CT value of the goal gene of gained and internal control gene according to the standard substance of different concns is done typical curve.Figure 5 shows that the standard substance amplification and the canonical plotting in rs2308010 site in this example, display standard product amplification curve form is good among the figure, typical curve R
2=0.9915, linear dependence is good.
6, the quantitative analysis of microchimerism: insert or the specificity of the dissociation curve assay products of absence type locus specificity primer and confidential reference items primer amplification gained according to each pleomorphism site, and calculate the difference (Δ CT) of both gained CT values.Detect the Δ CT value of gained according to the standard substance of different concns and do typical curve, can calculate the ratio of chimeric or microchimerism in the sample then according to the Δ CT value of the typical curve of gained and sample to be checked.Figure 6 shows that the detected result in rs2308010 site in this example, show among the figure institute's mark in this internal control gene and specificity site (insertion pleomorphism site) Gene Handling tracing pattern good, dissociation curve shows that the PCR product is special, blank does not have amplification.Because the genotype in daughter rs2308010 site is the disappearance homozygosity in this example, and the genotype in this site of mother be an insertion/disappearance heterozygous, so detected insert type gene fragment derives from the Disease in Infants cell of micro chimerism in daughter's genomic dna.The insert type gene micro chimerism rate in rs2308010 site is calculated as 0.036% according to detected Δ CT value and typical curve in the sample in this example, because the genotype in this site is an insertion/disappearance heterozygous in mother's body, so the micro chimerism rate of mother cell should be 0.036% * 2=0.072%.In this example quantitative analysis has been carried out in other three sites (rs4150017, rs2308144 and rs4646006) equally, the mean value that detected result is respectively 0.056%, 0.078%, 0.067%, four pleomorphism site cell micro chimerism is 0.068%.Therefore have the microchimerism of the cell of maternal source in daughter's sample, Disease in Infants cell chimerism ratio is about 0.068%.
Sequence table
＜110〉Beijing Daopei Hospital
＜120〉a kind of gene detecting kit that is used to detect cell chimerism state or individual recognition
<130>
<160>32
<170>PatentIn?version?3.3
<210>1
<211>30
<212>DNA
＜213〉synthetic primer rs2308010+
<400>1
ctgactagca?acaagcgtgg?aattagtcag 30
<210>2
<211>29
<212>DNA
＜213〉synthetic primer rs2308010-
<400>2
agtcctgcaa?caagcgtgga?attaggact 29
<210>3
<211>31
<212>DNA
＜213〉synthetic primer rs2308010 F
<400>3
gaacttatgc?aatattactg?ccatcaagtt?c 31
<210>4
<211>30
<212>DNA
＜213〉synthetic primer rs2308010 R
<400>4
ctcgtttgtg?tgatcatcaa?actcaacgag 30
<210>5
<211>28
<212>DNA
＜213〉synthetic primer rs4150017+
<400>5
agacaggtac?ggtgtgatgc?cactgtct 28
<210>6
<211>28
<212>DNA
＜213〉synthetic primer rs4150017-
<400>6
atcaaggtac?ggtgtgatgc?cacttgat 28
<210>7
<211>29
<212>DNA
＜213〉synthetic primer rs4150017 F
<400>7
acaaggagtg?caacagaacg?agaccttgt 29
<210>8
<211>28
<212>DNA
＜213〉synthetic primer rs4150017 R
<400>8
gtcttgtcct?ttcaacggtt?tccaagac 28
<210>9
<211>28
<212>DNA
＜213〉synthetic primer rs2307553+
<400>9
gtcagtgcaa?agtgggcatt?tgactgac 28
<210>10
<211>28
<212>DNA
＜213〉synthetic primer rs2307553-
<400>10
ctctgtgcaa?agtgggcatt?tgacagag 28
<210>11
<211>33
<212>DNA
＜213〉synthetic primer rs2307553 F
<400>11
gaaagtgaca?tgtgtattag?acctgccact?ttc?33
<210>12
<211>26
<212>DNA
＜213〉synthetic primer rs2307553 R
<400>12
gctcacatga?gtgcacagcc?atgagc 26
<210>13
<211>25
<212>DNA
＜213〉synthetic primer rs2308144+
<400>13
cttcattgtc?ctcgcctcca?tgaag 25
<210>14
<211>26
<212>DNA
＜213〉synthetic primer rs2308144-
<400>14
cttcagcacc?ttgtcctcgc?ctgaag 26
<210>15
<211>30
<212>DNA
＜213〉synthetic primer rs2308144 F
<400>15
ctgcttgatg?tctgtcagcc?ttctaagcag 30
<210>16
<211>27
<212>DNA
＜213〉synthetic primer rs2308144 R
<400>16
ctgattgcgt?caccatttgt?gaatcag 27
<210>17
<211>27
<212>DNA
＜213〉synthetic primer rs4646006+
<400>17
tgagtgctgg?taaatggcac?acactca 27
<210>18
<211>27
<212>DNA
＜213〉synthetic primer rs4646006-
<400>18
tgcacctggt?aaatggcaca?cagtgca 27
<210>19
<211>27
<212>DNA
＜213〉synthetic primer rs4646006 F
<400>19
ctaagtggca?gcattttcag?cacttag 27
<210>20
<211>31
<212>DNA
＜213〉synthetic primer rs4646006 R
<400>20
agctgacgag?tctccagatc?tatgatcagc?t 31
<210>21
<211>29
<212>DNA
＜213〉synthetic primer rs34132048+
<400>21
tgttaggtgc?ccagtgagca?gaactaaca 29
<210>22
<211>26
<212>DNA
＜213〉synthetic primer rs34132048-
<400>22
gactgtgccc?agtgagcaga?acagtc 26
<210>23
<211>32
<212>DNA
＜213〉synthetic primer rs34132048 F
<400>23
ttggtcggag?acagtaggta?gatgttgacc?aa 32
<210>24
<211>31
<212>DNA
＜213〉synthetic primer rs34132048 R
<400>24
cagagacgtg?gttgtcaaat?gaaggtctct?g 31
<210>25
<211>27
<212>DNA
＜213〉synthetic primer rs1610932+
<400>25
agaacccttt?gcaactcacc?aggttct 27
<210>26
<211>25
<212>DNA
＜213〉synthetic primer rs1610932-
<400>26
gcatctttgc?aactcacctg?gatgc 25
<210>27
<211>31
<212>DNA
＜213〉synthetic primer rs1610932 F
<400>27
ctgtctgtat?cacagctgaa?taagcagaca?g 31
<210>28
<211>26
<212>DNA
＜213〉synthetic primer rs1610932 R
<400>28
caacatccag?ggagagcatg?gtgttg 26
<210>29
<211>28
<212>DNA
＜213〉synthetic primer rs56024302+
<400>29
gatagatcca?ttcacccatc?catctatc 28
<210>30
<211>28
<212>DNA
＜213〉synthetic primer rs56024302-
<400>30
actttgccat?tcacccatcc?atcaaagt 28
<210>31
<211>31
<212>DNA
＜213〉synthetic primer rs56024302 F
<400>31
cagctagtcc?attcctgtac?cagagtagct?g 31
<210>32
<211>28
<212>DNA
＜213〉synthetic primer rs56024302 R
<400>32
ctcattagga?gtttggagct?gcaatgag 28
Claims (2)
1. one group of oligonucleotide that is used to detect cell chimerism state or individual recognition is characterized in that: be the oligonucleotide of sequence table SEQ ID No.1 to base sequence shown in the sequence table SEQ ID No.32; Wherein,
SEQ ID No.1 is for detecting the rs2308010 pleomorphism site Auele Specific Primer that inserts polymorphism, SEQ IDNo.2 is for detecting the rs2308010 pleomorphism site Auele Specific Primer of deletion polymorphism, and SEQ ID No.3 is that upstream primer, the SEQ ID No.4 of rs2308010 pleomorphism site is the downstream primer of rs2308010 pleomorphism site;
SEQ ID No.5 is for detecting the rs4150017 pleomorphism site Auele Specific Primer that inserts polymorphism, SEQ IDNo.6 is for detecting the rs4150017 pleomorphism site Auele Specific Primer of deletion polymorphism, and SEQ ID No.7 is that upstream primer, the SEQ ID No.8 of rs4150017 pleomorphism site is the downstream primer of rs4150017 pleomorphism site;
SEQ ID No.9 is for detecting the rs2307553 pleomorphism site Auele Specific Primer that inserts polymorphism, SEQ IDNo.10 is for detecting the rs2307553 pleomorphism site Auele Specific Primer of deletion polymorphism, and SEQ ID No.11 is that upstream primer, the SEQ ID No.12 of rs2307553 pleomorphism site is the downstream primer of rs2307553 pleomorphism site;
SEQ ID No.13 is for detecting the rs2308144 pleomorphism site Auele Specific Primer that inserts polymorphism, SEQ IDNo.14 is for detecting the rs2308144 pleomorphism site Auele Specific Primer of deletion polymorphism, and SEQ ID No.15 is that upstream primer, the SEQ ID No.16 of rs2308144 pleomorphism site is the downstream primer of rs2308144 pleomorphism site;
SEQ ID No.17 is for detecting the rs4646006 pleomorphism site Auele Specific Primer that inserts polymorphism, SEQ IDNo.18 is for detecting the rs4646006 pleomorphism site Auele Specific Primer of deletion polymorphism, and SEQ ID No.19 is that upstream primer, the SEQ ID No.20 of rs4646006 pleomorphism site is the downstream primer of rs4646006 pleomorphism site;
SEQ ID No.21 is for detecting the rs34132048 pleomorphism site Auele Specific Primer that inserts polymorphism, SEQ IDNo.22 is for detecting the rs34132048 pleomorphism site Auele Specific Primer of deletion polymorphism, and SEQ ID No.23 is that upstream primer, the SEQ ID No.24 of rs34132048 pleomorphism site is the downstream primer of rs34132048 pleomorphism site;
SEQ ID No.25 is for detecting the rs1610932 pleomorphism site Auele Specific Primer that inserts polymorphism, SEQ IDNo.26 is for detecting the rs1610932 pleomorphism site Auele Specific Primer of deletion polymorphism, and SEQ ID No.27 is that upstream primer, the SEQ ID No.28 of rs1610932 pleomorphism site is the downstream primer of rs1610932 pleomorphism site;
SEQ ID No.29 is for detecting the rs56024302 pleomorphism site Auele Specific Primer that inserts polymorphism, SEQ IDNo.30 is for detecting the rs56024302 pleomorphism site Auele Specific Primer of deletion polymorphism, and SEQ ID No.31 is that upstream primer, the SEQ ID No.32 of rs56024302 pleomorphism site is the downstream primer of rs56024302 pleomorphism site.
2. gene detecting kit that is used to detect cell chimerism state or individual recognition, the 10tests/ box, form by following reagent:
1) extracting genome DNA reagent;
2) ultrapure water;
3) 2 * PCR reaction solution;
4) 2 * SYBRGreen PCR reaction solution;
5) detect employed primer, promptly sequence table SEQ ID No.1 is divided in respectively in the centrifuge tube to the oligonucleotide of base sequence shown in the sequence table SEQ ID No.32, and the concentration of every kind of primer is 2pmol/ul;
6) the genomic dna reference substance 1;
7) the genomic dna reference substance 2;
Rs2308010, the rs4150017 of wherein said genomic dna reference substance 1 and reference substance 2, rs2307553, rs2308144, rs4646006, rs34132048, rs1610932 and rs56024302 loci polymorphism have carried out gene sequencing.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| CN106480198A (en) * | 2016-11-01 | 2017-03-08 | 公安部物证鉴定中心 | A kind of method and system for carrying out individual identification to unknown sample |
| CN107012209A (en) * | 2017-03-22 | 2017-08-04 | 浙江省血液中心 | A kind of multiple real-time quantitative method agents useful for same for assessing HSCT implanting state |
| CN109988826A (en) * | 2017-12-30 | 2019-07-09 | 安诺优达基因科技(北京)有限公司 | It is a kind of to calculate the method and system that mixing sample is fitted into ratio based on STR |
-
2009
- 2009-10-15 CN CN2009101781854A patent/CN101671736B/en not_active Expired - Fee Related
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| 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 |
| CN106480198A (en) * | 2016-11-01 | 2017-03-08 | 公安部物证鉴定中心 | A kind of method and system for carrying out individual identification to unknown sample |
| CN106480198B (en) * | 2016-11-01 | 2019-09-17 | 公安部物证鉴定中心 | A kind of method and system carrying out individual identification to unknown sample |
| CN107012209A (en) * | 2017-03-22 | 2017-08-04 | 浙江省血液中心 | A kind of multiple real-time quantitative method agents useful for same for assessing HSCT implanting state |
| CN107012209B (en) * | 2017-03-22 | 2018-06-12 | 浙江省血液中心 | A kind of multiple real-time quantitative method agents useful for same for assessing hematopoietic stem cell transplantation implanting state |
| CN109988826A (en) * | 2017-12-30 | 2019-07-09 | 安诺优达基因科技(北京)有限公司 | It is a kind of to calculate the method and system that mixing sample is fitted into ratio based on STR |
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| Publication number | Publication date |
|---|---|
| CN101671736B (en) | 2012-01-04 |
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