CN101509040A - Reagent kit for inosculating status analysis after hemopoietic stem cell transplantation and uses thereof - Google Patents
Reagent kit for inosculating status analysis after hemopoietic stem cell transplantation and uses thereof Download PDFInfo
- Publication number
- CN101509040A CN101509040A CNA2009100478385A CN200910047838A CN101509040A CN 101509040 A CN101509040 A CN 101509040A CN A2009100478385 A CNA2009100478385 A CN A2009100478385A CN 200910047838 A CN200910047838 A CN 200910047838A CN 101509040 A CN101509040 A CN 101509040A
- Authority
- CN
- China
- Prior art keywords
- peak
- donor
- 1min
- site
- receptor
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Images
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention relates to a kit for analyzing chimerism after hemopoietic stem cell is transplanted and the application thereof. The kit contains specific primers containing the following STR loci: D3S3045, D4S2366, D4S2639, D5S818, D13S317, D18S1002, D20S481 and D22S689. The kit adopts primer pairs which are not possessed by the commercialized kit, and the primer pairs provide reagent with higher informedness among recipients, so that the kit is suitable for testing the assembling single information locus and applied for quantitative trace in chimerism.
Description
Technical field
The present invention relates to biological technical field, be specifically related to utilize STR to detect the diagnostic kit and the application thereof of the chimerism after the hematopoietic stem cell transplantation.
Background technology
Hemopoietic stem cell is one of blood ingredient, is the initiator cell that generates various hemocytes, claims hematopoietic pluripotential stem cell again, is present in marrow, embryonic liver, peripheral blood and the Cord blood.It had both had height self ability, had the ability of each system's progenitor cell of further differentiation again.Modern age, blood transfusion just utilized this two kinds of abilities, made its immunity system be subjected to press down back input blood donor's hemopoietic stem cell to the blood recipient with radiation or heavy dose of chemicals, allowed it retain in blood recipient's marrow and differentiation and proliferation, and this promptly is a hematopoietic stem cell transplantation.Hematopoietic stem cell transplantation now is used for the treatment of leukemia, many malignant tumours and some heredopathia just increasingly extensively, because hemopoietic stem cell can obtain from marrow, peripheral blood, bleeding of the umbilicus, tire liver, therefore bone marrow transplantation, autologous peripheral blood stemcell transplant, cord blood cells and fetal liver transplantation have appearred clinically, and according to the source of hemopoietic stem cell can be divided into from body, allosome is homogenic (as twins) and recessive allele (as the donor of father and mother, compatriot or consanguinity-less relation) is transplanted.Perfect along with development of transplanting theory and technology and marrow storehouse, the application clinically of recessive allele hematopoietic stem cell transplantation more and more widely.
For judging that transplanting recurrence situation whether successful, monitor patients also implements immunosuppressant therapy, prognosis etc. in time, need detect and observe its variation tendency in the intravital situation of receptor to donor's cells after transplanting, the chimerism of therefore transplanting back detection of dynamic hemocyte has great importance to judging transplantation effect, the clinical early intervention treatment of enforcement.
From genetics, heterogenic donor-recipient's blood chimerism is the mixing of two different genotype individual cells in essence.Therefore, utilize the genetic marker on the karyomit(e) can distinguish donor-recipient's cell and donor-recipient's cell proportion is carried out quantitative analysis.Traditional mosaic analysis means comprises RFLP-PCR and the VNTR/STR-PCR technology based on the first-generation and the detection of s-generation genetic marker, and the FISH technology of analyzing based on chromosome segment, because FISH only can be used for the donor-recipient that sex does not conform to, so its application limitation is bigger.STR (short tandem repeat, STR) be some simple repeated sequences on the genome karyomit(e), STR has height polymorphism and somatocyte stability, heredity stably in family, STR-PCR has now become the strong tool of differentiating Different Individual DNA, is used for paternity test as far back as forensic science.Because this method good reproducibility is responsive efficient, the detection that has therefore become present IBMTR recommendation is for the gold standard that is subjected to chimerism.
Because the special mosaic assay kit of Shang Weiyou adopts the paternity test test kit to realize the application of STR-PCR technology in mosaic is analyzed at present usually.
Below be foremost in the world paternity test test kit manufacturer-PE Applied Biotechnology company limited product the test kit title and simultaneously the amplification the site:
AmpFlSTR?
?PCR?Amplification?Kit：
D3S1358，D16S539，TH01，TPOX，CSF1PO，D7S820，Amelogenin；
CSF1PO，D2S1338，D3S1358，D5S818，D7S820，D8S1179，D13S317，D16S539，D18S51，D19S433，D21S11，FGA，TH01，TPOX，vWA，Amelogenin；
?MiniFiler
TM?PCR?Amplification?Kit：
D8S1179，D3S1358，TH01，D19S433，vWA，D5S818，TPOX；
?Profiler?
?ID?PCR?Amplification?Kit：
D3S1358，vWA，FGA，D8S1179，D21S11，D18S51，D5S818，D13S317，D7S820，Amelogenin.
?Profiler?
?PCR?Ampl?ification?Kit：
D3S1358，D5S818，D7S820，D8S1179，D13S317，D18S51，D21S11，FGA，and?vWA.；
CSF1PO，D3S1358，D5S818，D7S820，D13S317，FGA，TH01，TPOX，and?vWA
D2S1338，D3S1358，D8S1179，D16S539，D18S51，D19S433，D21S11，SE-33，FGA，vWA
D2S1338，D3S1358，D8S1179，D16S539，D18S51，D19S433，D21S11，TH01，FGA，vWA
D8S1179，D21S11，D7S820，CSF1PO，D3S1358，D5S818，D13S317，D16S539，D2S1338，D19S433，vWA，D12S391，D18S51，Amelogenin，D6S1043，FGA
Some and chimeric rate be can contain in these test kits and irrelevant detection site such as sex site detected, and, because parent-offspring's judgement is the qualitative analysis to a plurality of sites, require quantitatively different with chimeric rate analysis, when therefore choosing in the STR site, it also is discrepant to informational requirement, and so-called informedness is meant that the donor-recipient side is had unique band that the opposing party do not have or is called the per-cent that the segmental case of allelotrope accounts for all cases, and unique band needs differ 2 tumor-necrosis factor glycoproteinss at least with other band.The high expression explanation of informedness can clearly quantitatively pick out the ability of Different Individual, the ability that the low then expression of informedness clearly quantitatively picks out Different Individual a little less than.Desirable information site is exactly to have band or the fragment that can clearly differentiate on this site between the donor-recipient, and all bands that have between the donor-recipient can not influence each other, thereby produce peak height or the peak area corresponding with fluorescent signal behind the needed raw value-electrophoresis of chimeric rate and exert an influence calculating.Combination in our experience is existing viewpoint in the world, we think that general desirable information site need meet 4 requirements: 1, this site is the repetition of 4 Nucleotide, 2, the allelotrope fragment that has a uniqueness between the donor-recipient at least, 3, Du Te allelotrope satisfies the condition that differs 2 tumor-necrosis factor glycoproteinss with other any allelic sequence lengths of both sides at least, any allelotrope sequence length of 4, one side all must and the opposing party's allelotrope sequence length can not only differ a tumor-necrosis factor glycoproteins.Can comprise not high enough site of some informednesses and sex site etc. in parent-offspring's detection kit, and behind the electrophoretic migration like the position class the segmental peak height of different fluorescently-labeled allelotrope or peak area can influence each other, the quantitative result in these sites is subject to disturb.If adopt the paternity test test kit to obtain chimeric rate information, it is many to primer to increase simultaneously, and can not be only to carrying out trace detection in some information site, and because the not high enough site of informedness also participates in experiment simultaneously, wasted manpower and financial resources, and its quantitative accuracy is not high enough.In the time can not obtaining the information site, also can't increase the site that other can provide desirable information flexibly.
In recent years, along with the widespread use of mosaic analysis in leukemia relapse and the little remaining kitchen range monitoring of leukemia, clinical practice is also had higher requirement to the detection sensitivity and the quantitative accuracy of mosaic analytical technology.So that the doctor earlier takes the medical intervention measure to obtain more good therapeutic action.
Summary of the invention
The diagnostic kit and the detection method thereof that the purpose of this invention is to provide the chimerism after the hematopoietic stem cell transplantation solve the available reagent box and can only increase manyly to primer simultaneously, can not only carry out trace detection to the information site after transplanting; And adopting the unexistent primer of present commercialization reagent right, these primers are to providing informedness higher reagent between the donor-recipient, thereby the detection in the single information of suitable assembly unit site; Can be used for the quantitative tracking of chimerism.
The invention discloses a kind of reagent kit for inosculating status analysis after hemopoietic stem cell transplantation, the Auele Specific Primer that contains following STR site in the test kit: D3S3045, D4S2366, D4S2639, D5S818, D13S317, D18S1002, D20S481 and D22S689, the fragment that these sites are 4bp repeats.
The present invention has carried out research screening to known STR site, selects to have higher informedness and most ofly be the unexistent STR of other paternity test test kit site, and it is 4bp that repeated fragment is all satisfied in these STR sites.Studies show that, meet the STR site of these conditions, during as the quantitative analysis of chimeric rate, disturb little, data precision height, and suitable asian population.
The Auele Specific Primer in described STR site is through fluorescently-labeled Auele Specific Primer.
Preferably, the Auele Specific Primer of above-mentioned STR site correspondence is SEQ ID NO.1-SEQID NO.16.
The corresponding relation such as the following table in each Auele Specific Primer and STR site are listed:
Behind the information site of determining between the donor-recipient, only the information site is followed the tracks of for convenience, in the test kit of the present invention, the independent packaging (as being divided in the different small packagess) respectively of the Auele Specific Primer of each STR site correspondence.
Except that primer, also can comprise dNTP, Mg in the test kit
2+Or PCR common agents such as Taq-Gold enzyme, test kit also can only comprise primer, and other PCR common agents of purchasing with other are used in use.
The present invention also further discloses the using method of above-mentioned reagent kit for inosculating status analysis after hemopoietic stem cell transplantation, comprises the following steps:
1. be template with donor before the bone marrow transplantation and receptor's hemopoietic stem cell DNA respectively,,, determine allelotrope according to electrophoresis result with going up the capillary electrophoresis of genetic analyzer after the pcr amplification product sex change with the pcr amplifications respectively of all primers in the test kit; According to the donor-recipient's who obtains allelotrope, determine desirable information site.
2. be template with the hemopoietic stem cell DNA that transplants the back receptor, with the primer PCR amplification of the definite desirable information site correspondence of step 1;
3. after the pcr amplification product sex change that step 2 is obtained, the capillary electrophoresis of last genetic analyzer is obtained the peak height or the peak area data of amplified production correspondence, and is calculated chimeric rate in view of the above.
Desirable information site in the above-mentioned steps 1 is meant the allelotrope fragment that meets 1, has a uniqueness between the donor-recipient at least, 2, Du Te allelotrope satisfies the condition that differs 2 tumor-necrosis factor glycoproteinss with other any allelic sequence length of both sides at least, 3, except that unique allelotrope fragment, any allelotrope sequence length of any allelic sequence length of the side and the opposing party differs 2 tumor-necrosis factor glycoproteinss or identical at least.All primers in the test kit are being distinguished pcr amplifications, and with after going up the capillary electrophoresis of genetic analyzer after the pcr amplification product sex change, the product fragment of corresponding same information site amplification, the different peak situations that go out can appear in donor and receptor, demonstrating different and peak that correspond to the molecular weight that differs from 1 repeating unit of looking younger, the position at peak during capillary electrophoresis is different peaks, otherwise then is identical peak.If the product fragment of corresponding same information site amplification, different peaks corresponds to look younger and differs from the molecular weight of 2 repeating units, condition 1 and 2 is before satisfied in the site that then produces these peaks, can be considered the information site, if except these peaks, other peak that donor or receptor produce in this site, all do not have only to differ a repeating unit with any peak of the opposing party, then this information site also satisfies condition 3, desirable information site can be further be regarded as, the trace detection of the chimeric rate in back can be used to transplant separately.
As donor on certain site is 16,18 two allelotrope, the receptor is 13,19 two allelotrope, then satisfy condition 1 and 2, the allelotrope 13,16 that two uniquenesses are arranged, but because 18,19 differ a repeated fragment size, and belong to Different Individual, just undesirable 3, this site does not just meet the requirement in desirable information site.
But it should be noted that, judgement for condition 3, at least the requirement that differs 2 tumor-necrosis factor glycoproteinss proposes at difference side, 2 allelotrope that only differ a repeated fragment size all belong to same side, and all differ 2 fragments at least with the opposing party's fragment, then still meet the site requirement of desirable information.As donor on certain site is 16,17 two allelotrope, the receptor is 13,19 two allelotrope, then satisfy condition 1 and 2,13,19 is unique allelotrope, 16,17 two allelotrope can not be can be regarded as unique allelotrope, but all differ 2 fragments at least with the opposing party's fragment, thus 3 the requirement of also satisfying condition, so this site meets the site requirement of desirable information.
The condition of pcr amplification is in above-mentioned steps 1 and the step 2:
95℃，8min；
94 ℃, 1min, 65 ℃ of 1min, 72 ℃, 1min, 2 circulations;
94 ℃, 1min, 61 ℃ of 1min, 72 ℃, 1min, 2 circulations;
94 ℃, 1min, 58 ℃ of 1min, 72 ℃, 1min, 25 circulations;
94 ℃, 1min, 55 ℃ of 1min, 72 ℃, 1min, 5 circulations;
60℃，10min。
In the above-mentioned steps 2, transplant the back sample desirable information site of only need increasing, only need to add the primer in desirable information site.
Derive from the shared ratio of donor hemopoietic stem cell in receptor's peripheral blood cells after above-mentioned chimeric rate promptly refers to transplant.Receptor source sexual cell proportion is called residual rate, chimeric rate+residual rate=1.
Because human chromosome is paired, corresponding STR site, on a pair of karyomit(e), article two, chromosomal sequence may be identical, also may be different, therefore, the amplified production in a corresponding STR site, one or two peak may appear during capillary electrophoresis, the molecular weight standard thing comparison that adds simultaneously according to capillary electrophoresis the time, the allelotrope that can know detected individuality is the fragment of the molecular weight size of corresponding how many tumor-necrosis factor glycoproteinss, and according to the power of fluorescence, obtains to occur the area and the peak height at the peak at fragment place.
Corresponding different situations, the calculation formula of chimeric rate is as follows respectively:
When respectively there are two peaks in the corresponding information site with the receptor of donor, and have only a peak difference between the donor-recipient, when another peak was identical, chimeric rate calculation formula was: (dA)/(rA+dA); In identical peak is not counted in when calculating
When donor has two peak dA1, dA2, receptor have two peak rA1, rA2, each peak all not simultaneously, chimeric rate calculation formula is: (dA1+dA2)/(rA1+rA2+dA1+dA2);
When donor has two peak dA1, dA2, receptor have only a peak rA1, each peak all not simultaneously, chimeric rate calculation formula is: 1-((2 * rA1)/(2 * rA1+dA1+dA2)) or (dA1+dA2)/(2 * rA1+dA1+dA2);
When the receptor has two peak rA1, rA2, donor have a peak dA1, each peak all not simultaneously, chimeric rate calculation formula is: (2 * dA1)/(2 * dA1+rA1+rA2);
When the corresponding information site with the receptor of donor all had only a peak, chimeric rate calculation formula was: dA1/ (rA1+dA1).
In the above-mentioned formula, r represents the receptor, and d represents donor, A: expression peak height or peak area.Different peaks refers to the product fragment of the corresponding same information of donor and receptor site amplification, demonstrates the position difference at peak during its capillary electrophoresis, and corresponds to the peak of the molecular weight that differs from 1 tumor-necrosis factor glycoproteins of looking younger.Otherwise then be identical peak.
The used most of site of this patent all is to be short of in the product of existing paternity test, but these site assembly units also can detect separately, and informedness is also than higher, can be used for the tracking in information site and replenishing flexibly as other products, solved can only the increase disadvantage in whole sites of present product, can only need the desirable information of amplification site, be used to follow the trail of the variation of chimeric rate, thereby it is more economical, the result detects more simple and clear, avoided the interfering with each other of different fluorescence, so be more suitable for quantitative analysis in block polymer.And can be used as the additional reagent of difficult paternity test, or the additional test kit during judicial expertise.
Description of drawings
Fig. 1: the different concns biased sample detected blending ratio typical curve of D22S689STR between the donor-recipient
That X-coordinate is represented is the receptor who sneaks in the test sample: donor DNA ratio, ordinate zou is the detected receptor who sneaks into of this test kit: donor DNA ratio data, Raw data represents the original receptor that not do not obtain through overcorrection: donor DNA ratio (residual rate), because the apparatus measures dna content has deviation, raw data in the time of need sneaking into according to 50%, carrying out mathematical model proofreaies and correct, Calibrated represents that this test kit is detected through the gauged receptor of mathematical model: the DNA ratio of donor, the receptor that expection should obtain on the Expected representation theory: the DNA ratio of donor.Can find that Calibrated overlaps substantially with Expected, result and the expected results basically identical measured are described.
Fig. 2: D4S2366 points out in the site chimeric rate of the routine patient to change the prompting recurrence
A: donor general layout, B: patient transplants back 30 days general layout C: patient transplant back 170 days general layout as seen from the figure 30 days the time patient's general layout with the general layout of donor consistent (124,132), illustrate and implant 91.54%, but during by 170 days patient's self general layout reply for self general layout be main body (116), it is 20.11% that chimeric rate falls to, the prompting recurrence.
Fig. 3: the D22S689 general layout changes prompting patient's recessive allele hemopoietic stem cell and implants
A:7 days, chimeric rate 0%B:14 days, chimeric rate 9.21%C:21 days, chimeric rate 95.1%D:27 days, chimeric rate 95.7%
The result shows that patient has only the general layout (206,210) of oneself in the time of 7 days, and the general layout of donor (194,222) presents gradually in the time of 14 days, and 21 days onset human inner cell major parts are the cell of donor source property, and the cell of donor source property still is main in the time of 27 days, not recurrence.The black triangle is the molecular weight witness marker.
Embodiment
Below enumerate specific embodiment with further elaboration the present invention, should understand embodiment is not to be used to limit protection scope of the present invention.
The screening in embodiment 1 STR site
Asian hemopoietic stem cell DNA is carried out the analysis of STR site information,
Test method:
1, after obtaining patient's informed consent, extract the patient that 16 routine marrow hemopoietic stem cells transplant and the peripheral blood 2ML of donor thereof, wherein 5 examples are that the donor-recipient of non-blood relationship is right, 11 examples are that the donor-recipient between the compatriot is right.According to sky root whole blood DNA extraction agent box (day root company, BeiJing, China) specification sheets, extract genomic dna.After measuring DNA concentration (nucleic acid-protein detector, Eppendorf, Germany), be diluted to 10ug/mL, packing is preserved.
2, respectively with TAMRA, FAM, the synthetic different primers of HEX difference mark (entrusting Shanghai to give birth to the worker), be used for amplification.The amplification site sees Table 1.
3, pcr amplification: amplification system is 20uL, comprises 0.2mM dNTP, 2mM Mg2+, and the 4uM primer, 1 unit of Taq-Gold (applying biological science and technology company-PE company, the U.S.), 10ug/mLDNA1-2uL increases on PE9600 amplification instrument.Each donor or receptor respectively increase 8 the pipe, have only a pair of primer in every pipe.
The amplification parameter is 95 ℃, 8min;
94 ℃, 1min, 65 ℃ of 1min, 72 ℃, 1min, 2 circulations;
94 ℃, 1min, 61 ℃ of 1min, 72 ℃, 1min, 2 circulations;
94 ℃, 1min, 58 ℃ of 1min, 72 ℃, 1min, 25 circulations;
94 ℃, 1min, 55 ℃ of 1min, 72 ℃, 1min, 5 circulations;
60℃，10min。
4, amplification finishes the back in 1.5% agarose voltage 100V, and 30 minutes electrophoresis after confirming to increase successfully, enter next step analysis.
5, after above-mentioned each site amplified production equivalent is merged into 1 pipe, get 1uL, (having the fluorescein-labeled double chain DNA fragment of ROX by 16 forms to add the interior mark of 1uL methane amide denaturing agent and ROX-500,70,80,100,120,140,160,180,200,240,280,320,360,400,450,490,500bp molecular weight is respectively:, can be used as the molecular weight standard thing) after, 95 ℃ after 2 minutes in 4 ℃ 5 minutes, go up electrophoresis in ABI3100 genetic analyzer (ABI, the U.S.).
6, determine the amplified fragments position according to fluorescent signal,, determine fragment molecular weight size, thereby confirm the allelotrope in this site with the comparison of molecular weight marker thing.Experimental data Genescan genotype software collection analysis.
7, sum up the allelotrope of different loci between every couple of donor-recipient, computing information, donor informedness, receptor's informedness, ideal bit dot information.
Informedness is calculated as the donor-recipient an a certain side is had the allelotrope that a opposing party does not have at least in certain site, and differ 2 repeated fragments between this allelotrope and other allelotrope at least, promptly have unique allelotrope case and account for the ratio that all detect case;
The donor informedness has the allelotrope that a opposing party does not have for the donor-recipient at least to a donor, and the case that differs 2 repeated fragments between this equipotential gene and other allelotrope at least accounts for the ratio that all detect case;
Receptor's informedness is then calculated the donor-recipient receptor is had the ratio that a unique allelotrope case accounts for all detection cases at least.The informedness in desirable site is calculated the per-cent that the case that meets desirable information site accounts for all cases, desirable information site is promptly on the basis that meets informedness calculating, except having unique allelotrope, other any allelotrope fragment and another individual fragment can not only differ 1 repeated fragment on sequence length, but 2 fragments of same individuality can only differ 1 repeated fragment.
8, according to experimental procedure 7,16 examples that statistical computation is obtained with PE company paternity test test kit by Shanghai judicial expertise institute are over the years transplanted the informedness in each site of donor-recipient, are listed in table 2.Wherein 2 pairs is the compatriot, and 14 pairs is that the donor-recipient of non-blood relationship is right.
Test-results: 1, the summary of STR site information such as the table 1 of this research configuration.
The analytical results of the test kit STR site information of this trial production of table 1
2, sum up informedness that usefulness existing P E company test kit in the past obtains in table 2.
Table 2, PE company test kit are analyzed the site information that transplant patient obtains
| The site | Informedness | The donor informedness | Receptor's informedness | The ideal bit dot information |
| D8S1179 | 0.47 | 0.47 | 0.47 | 0.20 |
| D21S11 | 0.13 | 0.13 | 0.13 | 0 |
| D7S820 | 0.63 | 0.38 | 0.63 | 0.5 |
| CSF1P0 | 0.19 | 0.19 | 0.13 | 0.13 |
| D3S1358 | 0.31 | 0.25 | 0.19 | 0.19 |
| TH01 | 0.50 | 0.25 | 0.42 | 0.42 |
| D16S539 | 0.44 | 0.38 | 0.38 | 0.25 |
| D2S1338 | 0.63 | 0.50 | 0.63 | 0.38 |
| D19S433 | 0.13 | 0.13 | 0.13 | 0 |
| vWA | 0.47 | 0.33 | 0.47 | 0.33 |
| TPOX | 0.46 | 0.23 | 0.31 | 0.38 |
| D18S51 | 0.56 | 0.50 | 0.56 | 0.38 |
| FGA | 0.56 | 0.56 | 0.50 | 0.38 |
Listed site is most of in the table 2 is the most authoritative medical jurisprudence individual recognition detection kit at present
PCR Amplification Kit is had. because Amelogenin is the sex site, can not provide informedness, so exclude in the table 2
3, comparison sheet 1, table 2, informedness in the table 1 generally is higher than table 2, and table 1, table 2 average ideal site information are respectively 0.28,0.27, there is not obvious gap. may be that similarly compatriot's transplanting donor-recipient is right because the object of this test kit has more hereditary feature, and that the research object of PE test kit majority is the donor-recipient of consanguinity-less relation is right, caused this kit information property height, but desirable informedness is compared with the information letter of this test kit much lower.But illustrate that at least the site information that this research is selected for use is not less than the most authoritative existing selected site of commercialization paternity test test kit.
The assembling of embodiment 2 test kits
The primer that synthesizes SEQID NO.1-SEQ ID NO.16 respectively.
All forward primers that are numbered odd number are all adopted 5 ' fluorescent mark TAMRA or FAM or HEX, specifically see Table 1.
The different primers that mark is good are respectively charged in the different 0.5mL plastics tubings, and the test kit of packing into after the sealing lucifuge is finished the assembling of test kit.
Embodiment 3 test kits detect the accuracy rate assessment of chimeric rate
Test method:
1. select for use an example for being subjected to right sample on the experiment basis of example 1 at random, determine that desirable information site is the D22S689STR site, the receptor has 16,17 allelotrope, and donor has 13,20 allelotrope.
DNA, the donor DNA of receptor before transplanting all be positioned over 37 ℃ 30 minutes, DNA concentration is adjusted into 10ug/mL in advance.
3. gradient sample prepares equal-volume and mixes above-mentioned DNA, prepare 50% donor: receptor DNA, and, mixes the gradient sample of the chimeric rate of formation 75%, 87.5%, 93.75%, 96.8%, 98.4%, 99.2% respectively successively as the initial product of gradient sample with the DNA of donor; 50% donor: receptor DNA biased sample is transplanted preceding DNA with the receptor successively respectively and is mixed the gradient sample of the chimeric rate of formation 25%, 12.5%, 6.25%, 3.13%, 1.56%, 0.8% as initial sample.75% chimeric specimen preparation is that equal-volume mixes 50% sample and donor DNA, and 62.5% sample is to mix the receptor with 75% sample equal-volume to transplant preceding DNA, and 37.5% sample is that equal-volume mixes 50% sample and the receptor transplants preceding DNA.Whole gradient sample R/D DNA comprises 0% (receptor DNA before promptly transplanting), 0.8%, 1.56%, 3.13%, 6.25%, 12.5%, 25%, 37.5%, 50%, 62.5%, 75%, 87.5%, 93.75%, 96.8%, 98.4%, 99.2%, 100% (being donor DNA).
4, the pcr amplification amplification system is 20uL, comprises 0.2mM dNTP, 2mM Mg2+, and 4uM SEQ ID NO.15 and SEQ ID NO.16,1 unit of Taq-Gold (applying biological science and technology company, the U.S.), DNA1-2uL increases on PE9600 amplification instrument.
5, the amplification parameter is 95 ℃, 8min;
94 ℃, 1min, 65 ℃ of 1min, 72 ℃, 1min, 2 circulations;
94 ℃, 1min, 61 ℃ of 1min, 72 ℃, 1min, 2 circulations;
94 ℃, 1min, 58 ℃ of 1min, 72 ℃, 1min, 25 circulations;
94 ℃, 1min, 55 ℃ of 1min, 72 ℃, 1min, 5 circulations;
60℃，10min。
6, get above-mentioned amplified production 1uL, add in 1uL methane amide denaturing agent and the ROX-500 back 95 ℃ of marks (molecular weight marker thing) after 2 minutes in 4 ℃ 5 minutes, go up electrophoresis in ABI3100 genetic analyzer (ABI, the U.S.).
7, determine the amplified fragments position according to fluorescent signal, compare with the molecular weight marker thing, determine fragment molecular weight size, thereby confirm the allelotrope in this site, and with example 1 in this donor-recipient's the result of D22S689 contrast, confirm that gradient sample is 13, band appears in the clip size place of 16,17,20 allelotrope correspondences, interpret sample is errorless, continues following experiment.
8, experimental data is collected with Genescan genotype software, obtains peak height and peak area.
9, calculate chimeric rate: selecting for use when donor has two peak dA1, and dA2, receptor have two peak rA1, rA2, each peak all not simultaneously, chimeric rate calculation formula is (dA1+dA2)/(rA1+rA2+dA1+dA2); Calculate with peak area and peak height respectively, this moment dA1, dA2 correspondence 13,20 allelic peaks, rA1, rA2 correspondence 16,17 allelic peaks.Obtain the chimeric rate of different gradient sample correspondences.
10, the correction of chimeric rate: because some difference is understood in the DNA concentration determination on different instruments, these differences may make detected concentration and actual concentrations that skew is arranged, and this research is attempted with mathematical model these skews being proofreaied and correct.
If DNA concentration correction coefficient is X,, r represents the receptor, and d represents donor, A: expression peak height or peak area:
(dA1+dA2) * X/ (rA1+rA2+ (dA1+dA2) * X)=0.5, i.e. X=(rA1+rA2)/(dA1+dA2), this moment rA and dA correspondence be the peak height or the peak area at each peak during 50% blended gradient sample in theory, calculate DNA concentration correction coefficient X;
1-(dA1+dA2) * X/ (rA1+rA2+ (dA1+dA2) * X) is the residual rate after the correction,
(dA1+dA2) * (rA1+rA2+ (dA1+dA2) * X) is the chimeric rate after proofreading and correct to X/;
Donor in the compute gradient sample during other each concentration or receptor's peak height or peak area are inserted above-mentioned formula, can obtain the correction residual rate of each concentration correspondence in the gradient sample.
Experimental result:
1, calculates the correction residual rate according to step 10, draw typical curve Fig. 1 of gradient sample.
2, calculate chimeric result respectively according to peak height and honeybee area, list in table 3.
It is very approaching to can be observed the chimeric rate that peak height and honeybee area obtain respectively from table 3, calculate both correlation coefficient r=0.999979 with the raw data that obtains, in other sample, also obtain similar results, explanation can be calculated with peak height or peak area, do not influence result's interpretation, some investigator tends to calculate with peak area, but in our data, do not observe both notable difference is arranged, value that the gauged typical curve of process obtains and expected value gap are substantially less than 0.015, and prompting detects specific inaccuracy less than 1.5%.Every group the variation coefficient (CV) is also all less than 5%, and gauged peak height group and peak area group are respectively 0.77% and 1.704%.Illustrate that this test kit detects chimeric rate and has higher accuracy and accuracy.
The result that table 3 obtains according to peak height and calculated by peak area relatively
Known proportion R/D DNA: according to the artificial mixing donor-recipient's of test method step 2 preparation dna ladder degree sample
The practical application 1 of embodiment 4 test kits
This test kit can effectively detect the chimeric variation after the transplanting of transplant patient, thereby the prompting graft is actually for implanting or repelling.
1. the acquisition of donor and receptor's peripheral blood DNA:
After obtaining informed consent, extract transplant patient 1 and transplant preceding, transplanting back 7,14,21,27,30,170 days and donor peripheral blood 2mL,
According to sky root whole blood DNA extraction agent box (day root company, BeiJing, China) specification sheets, extract genomic dna.After measuring DNA concentration (nucleic acid-protein detector, Eppendorf, Germany), be diluted to 10ug/mL, packing is preserved.
2. be template with donor before transplanting and receptor's DNA respectively, get in the test kit that embodiment 2 makes each, distinguish pcr amplification primer, amplification system: 0.2mM dNTP, 2mM Mg2+, 4uM primer, 1 unit of Taq-Gold enzyme and 10ugDNA, final volume is 20uL.
The PCR reaction conditions: 95 ℃, 8min;
94 ℃, 1min, 65 ℃ of 1min, 72 ℃, 1min, 2 circulations;
94 ℃, 1min, 61 ℃ of 1min, 72 ℃, 1min, 2 circulations;
94 ℃, 1min, 58 ℃ of 1min, 72 ℃, 1min, 25 circulations;
94 ℃, 1min, 55 ℃ of 1min, 72 ℃, 1min, 5 circulations;
60℃，10min。
3. will respectively get 1uL from all PCR products of same donor mixes, (having the fluorescein-labeled double chain DNA fragment of ROX by 16 forms to get the interior mark of mix products 1uL adding 1uL methane amide denaturing agent and ROX-500, molecular weight is respectively: 70,80,100,120,140,160,180,200,240,280,320,360,400,450,490,500bp, can be used as the molecular weight standard thing) back 95 ℃ after 2 minutes in 4 ℃ 5 minutes, in ABI3100 genetic analyzer (ABI, the U.S.) go up electrophoresis, obtain the amplification general layout of donor, obtain receptor's amplification general layout with quadrat method, donor and receptor's amplification general layout is compared, in the amplification general layout prompting D4S2366 site, patient has a peak: 116, donor has two peaks: 124,132, meet the feature in desirable information site.
4. transplant the back sample information site of only need increasing, only need the primer in adding information site.Therefore after transplanting, only it is transplanted the back sample and follow the trail of D4S2366.DNA with receptor after transplanting is a template, primer amplification with the D4S2366 correspondence, get the above-mentioned PCR product of 1uL add in 1uL methane amide denaturing agent and the ROX-500 back 95 ℃ of marks after 2 minutes in 4 ℃ 5 minutes, electrophoresis on the ABI3100 genetic analyzer is obtained peak height and peak area.The amplification general layout as shown in Figure 2
5. the calculating of chimeric rate adopts following formula to calculate chimeric rate: (124 peak areas+132 peak areas)/(2 * 116 peak areas+124 peak areas+132 peak areas).
As seen from Figure 2 30 days the time patient's general layout with the general layout consistent (124,132) of donor, implanted 91.54%, but patient's self general layout is replied to self general layout is main body (116) during by 170 days, it is 20.11% that chimeric rate falls to, prompting is recurred.
The practical application 2 of embodiment 5 test kits
Adopt the test kit of embodiment 2, adopt the method identical, detect another donor and receptor with embodiment 4.
Donor and receptor's amplification general layout is compared, and in the amplification general layout prompting D22S689 site, patient has 2 peaks: 206,210, and donor has 2 peaks: 194,222, meet the feature in information site.
After transplanting, it is transplanted the back sample follow the trail of D22S689.Peripheral blood cells DNA with receptor after transplanting is a template, primer amplification with the D22S689 correspondence, get the above-mentioned PCR product of 1uL add in 1uL methane amide denaturing agent and the ROX-500 back 95 ℃ of marks after 2 minutes in 4 ℃ 5 minutes, electrophoresis on the ABI3100 genetic analyzer is obtained peak height and peak area.The amplification general layout as shown in Figure 3.
The calculating of chimeric rate adopts following formula to calculate chimeric rate: (194 peak areas+222 peak areas)/(206 peak areas+210 peak areas+194 peak areas+222 peak areas).
Fig. 3 result shows that patient has only the general layout (206,210) of oneself in the time of 7 days, and the general layout of donor (194,222) presents gradually in the time of 14 days, and this moment, chimeric rate was 9.2%; And 21 days onset human inner cell major parts are the cell of donor source property, and this moment, chimeric rate was 95.1%; The cell of donor source property still is main in the time of 27 days, and chimeric rate is 95.8%, illustrates that implantation success does not have recurrence at present from peripheral blood cells increase gradually in recipient's body of the hemopoietic stem cell of donor.
Embodiment 5-19
Adopt the test kit of embodiment 2, adopt the method identical with embodiment 4, to the trace detection block polymer after 15 routine patients' transplanting, the result is as follows:
The successful utilization of the test kit of the present invention of The above results explanation.
Sequence table
＜110〉Shanghai City Blood Center
＜120〉reagent kit for inosculating status analysis after hemopoietic stem cell transplantation and application thereof
<130>PCNXY090121
<160>16
<170>PatentIn?version?3.1
<210>1
<211>20
<212>DNA
<213>artificial?sequence
<220>
＜223〉primer
<400>1
<210>2
<211>20
<212>DNA
<213>artificial?sequence
<220>
＜223〉primer
<400>2
<210>3
<211>20
<212>DNA
<213>artificial?sequence
<220>
＜223〉primer
<400>3
<210>4
<211>25
<212>DNA
<213>artificial?sequence
<220>
＜223〉primer
<400>4
<210>5
<211>20
<212>DNA
<213>artificial?sequence
<220>
＜223〉primer
<400>5
<210>6
<211>23
<212>DNA
<213>artificial?sequence
<220>
＜223〉primer
<400>6
<210>7
<211>20
<212>DNA
<213>artificial?sequence
<220>
＜223〉primer
<400>7
<210>8
<211>20
<212>DNA
<213>artificial?sequence
<220>
＜223〉primer
<400>8
<210>9
<211>20
<212>DNA
<213>artificial?sequence
<220>
＜223〉primer
<400>9
<210>10
<211>20
<212>DNA
<213>artificial?sequence
<220>
＜223〉primer
<400>10
<210>11
<211>26
<212>DNA
<213>artificial?sequence
<220>
＜223〉primer
<400>11
<210>12
<211>26
<212>DNA
<213>artificial?sequence
<220>
＜223〉primer
<400>12
<210>13
<211>20
<212>DNA
<213>artificial?sequence
<220>
＜223〉primer
<400>13
<210>14
<211>21
<212>DNA
<213>artificial?sequence
<220>
＜223〉primer
<400>14
<210>15
<211>22
<212>DNA
<213>artificial?sequence
<220>
＜223〉primer
<400>15
<210>16
<211>20
<212>DNA
<213>artificial?sequence
<220>
＜223〉primer
<400>16
Claims (9)
1. reagent kit for inosculating status analysis after hemopoietic stem cell transplantation contains the Auele Specific Primer in following STR site: D3S3045, D4S2366, D4S2639, D5S818, D13S317, D18S1002, D20S481 and D22S689 in the test kit.
2. reagent kit for inosculating status analysis after hemopoietic stem cell transplantation according to claim 1 is characterized in that the Auele Specific Primer in described STR site is selected from SEQ ID NO.1-SEQ ID NO.16.
3. reagent kit for inosculating status analysis after hemopoietic stem cell transplantation according to claim 1 is characterized in that the Auele Specific Primer in described STR site is through fluorescent mark.
4. reagent kit for inosculating status analysis after hemopoietic stem cell transplantation according to claim 1 is characterized in that, the independent packaging respectively of the Auele Specific Primer of described each STR site correspondence.
5. as reagent kit for inosculating status analysis after hemopoietic stem cell transplantation as described in arbitrary claim among the claim 1-4, it is characterized in that, also comprise dNTP, the Mg of independent packaging in the described test kit
2+Or one or more reagent in the Taq-Gold enzyme.
6. as the using method of reagent kit for inosculating status analysis after hemopoietic stem cell transplantation as described in arbitrary claim among the claim 1-5, comprise the following steps:
A. be template with donor before the bone marrow transplantation and receptor's peripheral blood cells DNA respectively,,, determine allelotrope according to electrophoresis result with going up the capillary electrophoresis of genetic analyzer after the pcr amplification product sex change with the pcr amplifications respectively of all primers in the test kit; According to the donor-recipient's who obtains allelotrope, determine desirable information site;
B. be template with the peripheral blood cells DNA that transplants the back receptor, with the primer PCR amplification of the definite desirable information site correspondence of step a;
C. after the pcr amplification product sex change that step b is obtained, the capillary electrophoresis of last genetic analyzer is obtained the peak height or the peak area data of amplified production correspondence, and is calculated chimeric rate in view of the above.
7. as the using method of reagent kit for inosculating status analysis after hemopoietic stem cell transplantation as described in the claim 6, it is characterized in that described desirable information digit point meets following condition: the allelotrope fragment that has a uniqueness between the donor-recipient at least; Unique allelotrope satisfies the condition that differs 2 tumor-necrosis factor glycoproteinss with other any allelic sequence length of both sides at least; Any allelotrope sequence length of one side's any allelic sequence length and the opposing party differs the STR site of 2 tumor-necrosis factor glycoproteinss at least.
8. as the using method of reagent kit for inosculating status analysis after hemopoietic stem cell transplantation as described in the claim 6, it is characterized in that the condition of pcr amplification is among step a and the step b:
95℃，8min；
94 ℃, 1min, 65 ℃ of 1min, 72 ℃, 1min, 2 circulations;
94 ℃, 1min, 61 ℃ of 1min, 72 ℃, 1min, 2 circulations;
94 ℃, 1min, 58 ℃ of 1min, 72 ℃, 1min, 25 circulations;
94 ℃, 1min, 55 ℃ of 1min, 72 ℃, 1min, 5 circulations;
60℃，10min。
9. as the using method of reagent kit for inosculating status analysis after hemopoietic stem cell transplantation as described in the claim 6, it is characterized in that, the situation that step c is corresponding different, the calculation formula of chimeric rate is as follows respectively:
When respectively there are two peaks in the corresponding information site with the receptor of donor, and have only a peak difference between the donor-recipient, when another peak was identical, identical peak was not considered, and chimeric rate calculation formula is: (dA)/(rA+dA);
When donor has two peak dA1, dA2, receptor have two peak rA1, rA2, each peak all not simultaneously, chimeric rate calculation formula is: (dA1+dA2)/(rA1+rA2+dA1+dA2);
When donor has two peak dA1, dA2, receptor have only a peak rA1, each peak all not simultaneously, chimeric rate calculation formula is: 1-((2 * rA1)/(2 * rA1+dA1+dA2)) or (dA1+dA2)/(2 * rA1+dA1+dA2);
When the receptor has two peak rA1, rA2, donor have a peak dA1, each peak all not simultaneously, chimeric rate calculation formula is: (2 * dA1)/(2 * dA1+rA1+rA2);
When the corresponding information site with the receptor of donor, when all having only a peak dA1 and rA1, chimeric rate calculation formula is: dA1/ (rA1+dA1);
In the above-mentioned formula, r represents the receptor, and d represents donor, A: expression peak height or peak area.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2009100478385A CN101509040B (en) | 2009-03-19 | 2009-03-19 | Reagent kit for inosculating status analysis after hemopoietic stem cell transplantation and uses thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2009100478385A CN101509040B (en) | 2009-03-19 | 2009-03-19 | Reagent kit for inosculating status analysis after hemopoietic stem cell transplantation and uses thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN101509040A true CN101509040A (en) | 2009-08-19 |
| CN101509040B CN101509040B (en) | 2011-11-23 |
Family
ID=41001572
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN2009100478385A Active CN101509040B (en) | 2009-03-19 | 2009-03-19 | Reagent kit for inosculating status analysis after hemopoietic stem cell transplantation and uses thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN101509040B (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107674920A (en) * | 2016-12-19 | 2018-02-09 | 苏州大学附属第医院 | Chimera multiple PCR primer composition and detection method |
| CN107960107A (en) * | 2015-06-15 | 2018-04-24 | 默多克儿童研究所 | The method for measuring chimerism |
| 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 |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| GB0104690D0 (en) * | 2001-02-26 | 2001-04-11 | Cytogenetic Dna Services Ltd | Diagnostic test |
-
2009
- 2009-03-19 CN CN2009100478385A patent/CN101509040B/en active Active
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107960107A (en) * | 2015-06-15 | 2018-04-24 | 默多克儿童研究所 | The method for measuring chimerism |
| CN107674920A (en) * | 2016-12-19 | 2018-02-09 | 苏州大学附属第医院 | Chimera multiple PCR primer composition and detection method |
| CN107674920B (en) * | 2016-12-19 | 2021-04-13 | 苏州大学附属第一医院 | Chimeric multiplex PCR primer composition and detection method |
| 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 |
Also Published As
| Publication number | Publication date |
|---|---|
| CN101509040B (en) | 2011-11-23 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN103906848B (en) | For detecting composition and the method for allelic variant | |
| Zubakov et al. | Towards simultaneous individual and tissue identification: A proof-of-principle study on parallel sequencing of STRs, amelogenin, and mRNAs with the Ion Torrent PGM | |
| JP2007509613A (en) | QRT-PCR assay system for gene expression profiling | |
| EP2937423A1 (en) | Se33 mutations impacting genotype concordance | |
| CN111020031A (en) | Method for detecting tumor gene mutation by combining sequence specific blocker with specific PCR (polymerase chain reaction) program | |
| CN104131072A (en) | Method and system for individual recognition and paternity identification of unknown sample | |
| US20110306505A1 (en) | X-STR multiplex PCR amplification system | |
| KR20210112350A (en) | Quantitative amplicon sequencing for detection of multiple copy number variations and quantification of allele ratios | |
| de Lima et al. | PCR amplicons identify widespread copy number variation in human centromeric arrays and instability in cancer | |
| CN101509040B (en) | Reagent kit for inosculating status analysis after hemopoietic stem cell transplantation and uses thereof | |
| CN110295218B (en) | Method for quantifying mutant allele burden of target gene | |
| CN113416769B (en) | Method, composition and use for detecting microsatellite instability of non-control sample based on next generation sequencing technology | |
| CN110628920A (en) | Fluorescence labeling multiplex amplification kit for 35 STR loci of human Y chromosome and application thereof | |
| CN113151419B (en) | Spinal muscular atrophy detection method | |
| US20170051332A1 (en) | Methods and compositions for detecting expression of target genes | |
| US9528161B2 (en) | Materials and methods for quality-controlled two-color RT-QPCR diagnostic testing of formalin fixed embedded and/or fresh-frozen samples | |
| CN112522375A (en) | Detection kit and detection method for gene mutation of folate metabolism related molecular marker | |
| CN103352070A (en) | ROS1 fusion gene screening method | |
| Whitlam et al. | Use of ubiquitous, highly heterozygous copy number variants and digital droplet polymerase chain reaction to monitor chimerism after allogeneic haematopoietic stem cell transplantation | |
| Petkova et al. | Measuring Telomere Length—From Ends to Means | |
| CN111440876A (en) | Kit and method for quantitatively detecting methylation degree of human MGMT gene | |
| Sel et al. | Can novel methods replace the gold standard chimerism method after allogeneic hematopoietic stem cell transplantation? | |
| CN112029841B (en) | Method for quantifying telomere length and genomic motifs | |
| CN116445596B (en) | Product and method for human genotyping and application thereof | |
| CN112226528B (en) | Quality inspection method for detecting bacterial contamination by biological tissue sample |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant |