CN102399879A - Hematopoietic chimera real-time quantitative PCR (Polymerase Chain Reaction) detection and specimen collection method - Google Patents
Hematopoietic chimera real-time quantitative PCR (Polymerase Chain Reaction) detection and specimen collection method Download PDFInfo
- Publication number
- CN102399879A CN102399879A CN2011103629365A CN201110362936A CN102399879A CN 102399879 A CN102399879 A CN 102399879A CN 2011103629365 A CN2011103629365 A CN 2011103629365A CN 201110362936 A CN201110362936 A CN 201110362936A CN 102399879 A CN102399879 A CN 102399879A
- Authority
- CN
- China
- Prior art keywords
- real
- time quantitative
- quantitative pcr
- chimera
- pcr
- 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.)
- Pending
Links
Images
Abstract
The invention provides a PCR (Polymerase Chain Reaction) real-time quantitative detection method for detecting a hematopoietic chimera by using fluorescent dye SYBR green. The method comprises the following steps of: collecting a specimen; selecting genetic polymorphism markers to design a primer and a probe; detecting the hematopoietic chimera through real-time quantitative PCR; artificially synthesizing different dilute concentrations of hematopoietic chimera; and performing statistical analysis. The step of specimen collection comprises the specific procedures of clinically collecting blood samples of a donor and a receptor before transplantation and one month after transplantation; and directly extracting genome DNA by using the conventional method. In the invention, 12 specific genetic polymorphism markers capable of being used for Q-PCR to detect the hematopoietic chimera are introduced. Moreover, a melting curve is also used for verifying the specificity of the PCR product in order to overcome the defect that the fluorescent dye SYBR green brought into double-chain DNA is nonspecific. The real-time quantitative PCR method provided by the invention has the advantages of low price, long quality guarantee period and high sensitivity and is a fast, simple, convenient and reliable hematopoietic chimera detection method.
Description
Technical field
The present invention relates to a kind of hemopoietic chimera real-time quantitative PCR (Polymerase Chain Reaction; The polymerase chain reaction) detection method, particularly a kind of PCR that utilizes optical dye SYBR green to detect hemopoietic chimera detects and the specimen collection method.
Background technology
Along with the allosome hemopoietic stem cell is used for treating various pernicious and nonmalignant hematologic diseases, many patients have prolonged total survival time and DFS time.The major cause that causes treating failure is palindromia, and graft is ostracised, graft versus host disease.Therefore the donor mosaic is to be used for diagnosing graft to become to live after the stem cell transplantation of quantitative analysis patient allosome, early detection and treatment palindromia, the important tool of transplant rejection.Chimeric monitoring after the allosome HSCT has become the routine inspection means; Particularly at RIC (Reduced Intensity Conditioning; The reduction strength condition) heteroplastic transplantation; The chimeric result of donor can be used to instruct whether carry out therapeutic intervention after the allosome stem cell transplantation, has played decisive role in the recurrence that changes immunosuppressant therapy and treatment disease especially.
The detection of hemopoietic chimera can utilize acceptor to measure with donor polymorphum genetic marker or the different of its product.The method of widespread use at present comprises cytogenetics, red cell grouping, and (Fluorescence In Situ Hybridization, FISH) technology is popped one's head in through XY karyomit(e) and is measured for restriction fragment length polymorphism analysis and immunofluorescence in situ hybridization.But above method or time-consuming perhaps is not suitable for all patients.Pop one's head in through XY karyomit(e) such as immunofluorescence in situ hybridization (FISH) technology and to measure hemopoietic chimera, therefore only be applicable to that acceptor and donor are the allosome HSCTs of different sexes; And the red cell grouping method only is applicable to the acceptor and the donor of different blood groups.
Use in recent years method that PCR (Polymerase Chain Reaction, polymerase chain reaction) real-time quantitative detects hemopoietic chimera because of its application extensively and highly sensitive be used more and more.Present received molecular biology method is to detect the Microsatellite sequence with PCR method--a kind of weak point and the sequence that repeats not encode.Because in different acceptors and donor, thereby the number of iterations of this sequence difference causes varying in size of PCR product.And then with acrylamide gel electrophoresis and automatic sequence analysis PCR product.This method sensitivity relatively low (1～5%), and time-consuming, and effort costs an arm and a leg.
The real time quantitative PCR method of recently a kind of single nucleic acid polymorphum of new application TaqMan technology for detection (SNP, Single Nucleotide Polymorphisms) is established.SNP is an allelic variation, and wide expression is at human chromosome coding and non-coding region.Approximately per 1000 base pairs just have a SNP to take place, and therefore for chimeric analysis an information flag storehouse widely are provided.The real time quantitative PCR method of this TaqMan technology need be used special TaqMan probe, has to cost an arm and a leg and shortcoming that the quality guaranteed period is short.Comparatively speaking, the optical dye SYBR green that can include double-stranded PCR product in is cheap because of it, highly sensitive and being widely used on the real time quantitative PCR method.
Summary of the invention
The objective of the invention is to set up a kind of PCR that utilizes optical dye SYBR green to detect hemopoietic chimera and detected novel method and specimen collection method thereof, said PCR detection method can improve the sensitivity that PCR method detects, low and cost saving consuming time.
For reaching the object of the invention, the real-time quantitative PCR detection method of detection hemopoietic chimera of the present invention may further comprise the steps:
S1. make a collection of specimens;
S2. select polymorphum genetic marker design primer and probe (probe);
S3. real-time quantitative PCR detects hemopoietic chimera;
S4. the hemopoietic chimera of 12 different weaker concns of synthetic; And
S5. statistical analysis.
The detection key of hemopoietic chimera is to select to distinguish the specificity polymorphum genetic marker of acceptor and donor.Introduce 12 among the present invention and can be used for the specificity polymorphum genetic marker that real-time fluorescence quantitative PCR (Real time Q-PCR) detects hemopoietic chimera.With these 12 polymorphum genetic markers 37 pairs of donors and acceptor are screened, the result shows that the informedness (informativity) of each polymorphism mark is between 3% to 47%.12 polymorphum genetic markers can be distinguished 94% donor and acceptor.Except that a pair of identical twin, all 37 pairs of donors and acceptor can find suitable specificity polymorphism mark further to carry out quantitative PCR and detect hemopoietic chimera.
To include double-stranded DNA in be nonspecific in order to remedy optical dye SYBR green, moves the specificity that melting curve (melting curve) can be used for verifying the PCR product simultaneously.Further use the hemopoietic chimera (from 0.01% to 100%) of 12 different weaker concns of synthetic; Utilize donor dna to dilute receptor dna or receptor dna dilutes donor dna, the amplification curve of standard is made by the increase hemopoietic chimera of above synthetic of the PCR in the allelotrope site of donor or receptor-specific.The negative allelotrope of donor or acceptor can be used as negative control.The per-cent of the donorcells after the transplanting can calculate from the amplification curve of standard.
The contriver of the application's case compares the real-time quantitative PCR of this method and TaqMan probe, and the result shows that its sensitivity and accuracy are the same high with the real time quantitative PCR method of TaqMan probe.Because Sybr green low price, long quality-guarantee period makes it in practical application, more be superior to Taqman PCR.The contriver of the application's case has detected 18 cases with this novel method, and detected result is consistent with result with traditional M icrosatellite/FISH.But present method is widely used, and is applicable to all kinds of allosome stem cell transplantations (comprising with sex and different sexes), and sensitivity is higher than two kinds of methods in addition far away.
In sum, the detection hemopoietic chimera real-time quantitative PCR detection method set up of present patent application is a kind of shortcut and simple and method reliably.
Description of drawings
Through the detailed description below in conjunction with accompanying drawing, aforesaid purpose, the feature and advantage with other of the present invention will become obvious.Wherein:
Shown in Figure 1 is the step synoptic diagram of the real-time quantitative PCR detection method of detection hemopoietic chimera of the present invention;
Two allelotrope that produce for the PCR product shown in Figure 2 demonstrate two visibly different melting curves;
Shown in Figure 3ly make the standard amplification curve for the increase hemopoietic chimera of synthetic of the PCR by the allelotrope site of donor or receptor-specific.
Embodiment
As shown in Figure 1, according to the object of the invention, a kind of novel method of utilizing optical dye SYBR green to detect hemopoietic chimera that one embodiment of the present of invention are set up may further comprise the steps:
S1. make a collection of specimens: before clinical collection donor and acceptor are transplanted with transplant back one month blood sample, therefrom directly extract genomic DNA (genomic dna) with ordinary method, with Nanodrop spectrophotometer measurement DNA concentration, then be stored in 4 ℃ subsequent use.
S2. select polymorphum genetic marker design primer and probe (probe): the short insertion/deletion polymorphism (human biallelic short insertion/deletion polymorphisms) of the human allelotrope of the nucleotide sequence of polymorphum genetic marker obtains from Marshfiled Clinic (http://research.marshfieldclinic.org/genetics/Genetic Research).Wherein, choice criteria is: comprise and contain at least two different polymorphic alleles of above successive base, in general crowd, show the heterozygosity of height.With this Standard Selection 12 polymorphum genetic markers as shown in table 1.Design primer with software Prime 3, to each polymorphic allele mark, need two pairs of primers of design, one is directed against two allelic common sites, and one to unique polymorphic site.TaqMan probe probe is selected between two allelotrope; Then each PCR product is carried out the melting curve analysis.
Table 1. is used for the characteristic of the specific mark of real-time quantitative PCR
In the table 1, " ND " expression does not design the Probe probe;
The Informativity informative site quantity that (informedness)=this mark provided/all D-As that screened are to quantity
S3. real-time quantitative PCR detects hemopoietic chimera:
Utilize the real-time quantitative PCR of SYBR green and TaqMan to detect, each reacts 20 microlitres, in Rotor gene3000 quantitative real time PCR Instrument, carries out.The PCR reaction system is following:
Before quantitatively, screening earlier is applicable to the polymorphism mark of this donor and acceptor.Transplanting preceding DNA with donor and acceptor is substrate; From 12 polymorphum genetic markers, select an optimal allelotrope site; Positive allelotrope is defined as Ct value (cycle number that Ct value is experienced when referring to the thresholding of the fluorescent signal arrival setting in each reaction tubes) between 20～25, and negative allelotrope is defined as Ct value＞36.When donor and acceptor demonstrate different PCR products, explain that donor and acceptor have different allelotrope, this mark can be used as the polymorphism mark of detection by quantitative hemopoietic chimera.
Wherein, the PCR condition is following:
To include double-stranded DNA in be nonspecific in order to remedy optical dye SYBR green; Move the specificity that melting curve (melting curve) can be used for verifying the PCR product simultaneously; As shown in Figure 2; Two allelotrope that the PCR product produces demonstrate two visibly different melting curves, and one at 78 ℃, and one at 80 ℃.Polymorphism mark 4a and 4b are positive in acceptor, and donor has only 4a positive, so polymorphism mark 4b is regarded as an information flag (informative mark).
S4. the hemopoietic chimera of 12 different weaker concns of synthetic:
Further; With the hemopoietic chimera (from 0.01% to 100%) of 12 different weaker concns of synthetic-dilute receptor dna or receptor dna dilutes donor dna with donor dna; The amplification curve of standard is made by the increase hemopoietic chimera of above synthetic of the PCR in the allelotrope site of donor or receptor-specific; As shown in Figure 3, wherein:
A representes the quantitative curve of SYBR green Q-PCR, and the X axle representes that PCR begins to produce the cycle index of fluorescent signal, and the high more gene copy number of representative more early appears in signal;
B representes the standard amplified curve that the linear corresponding relation of quantity drawn according to receptor marker Ct value and acceptor/donor dna fragment, nucleic acid precision correlation coefficient r=0.997, and PCR efficient is 0.97.
The negative allelotrope of donor or acceptor can be used as negative control, and the per-cent of the donorcells after the transplanting can calculate from the amplification curve of standard.
S5. statistical analysis
We compare the real-time quantitative PCR that utilizes this method and TaqMan probe, its comparative result such as following table 2; We have detected 18 cases with this novel method, and detected result and result with traditional M icrosatellite/FISH are that consistent (as shown in table 3, wherein, " NA " expression is inapplicable; " ND " expression is not done; Case 1-9 is the unmatched transplanting of sex, compares with FISH result; Case 10-18 is other transplanting of the same sex, compares with Microsatellite analysis result.)
The real-time quantitative PCR result's of table 2.SYBR green and TaqMan probe comparison
| Donorcells (%) | SYBR?green?based?Q-PCR | TaqMan?probe?based?Q-PCR |
| 0.03 | 0.03±0.003 | 0.03±0.005 |
| 0.3 | 0.22±0.06 | 0.21±0.04 |
| 1.5 | 1.48±0.11(0.9-2.2) | 1.46±0.13(0.9-2.2) |
| 15 | 16.70±1.19(9.1-25.2) | 17.80±1.59(10.7-24.4) |
| 30 | 33.36±1.98(21.6-45.5) | 35.30±1.92(19.9-45.4) |
| 50 | 53.61±3.79(37.8-74.3) | 59.24±2.15(49.0-70.7) |
| 70 | 83.95±5.40(55.6-100) | 64.61±6.23(51.9-87.2) * |
| 85 | 100.41±7.57(55-100) | 82.63±4.41(58.7-100) * |
Values?are?the?mean±SE，n＝12.
*，P＜0.05，
SYBR green based Q-PCR: based on the real time fluorescent quantitative of optical dye SYBR green;
TaqMan probe based Q-PCR: based on the real time fluorescent quantitative of TaqMan probe.
The result's of table 3.SYBR green real time quantitative PCR method and Microsatellite/FISH comparison
Comparing result by above-mentioned shows that the sensitivity of method of the present invention and accuracy are the same high with the real time quantitative PCR method of TaqMan probe.Because Sybr green low price, long quality-guarantee period makes it in practical application, more be superior to Taqman PCR.But present method is widely used, and is applicable to all kinds of allosome stem cell transplantations (comprising with sex and different sexes), and sensitivity is higher than two kinds of methods in addition of prior art far away.The method of the detection hemopoietic chimera that the present invention in sum, set up is a kind of shortcut and simple and reliable method.
The present invention is not limited to described embodiment, and those skilled in the art still can do some corrections or change, so rights protection scope of the present invention is as the criterion with claims restricted portion not breaking away from spirit of the present invention promptly openly in the scope.
Claims (6)
1. specimen collection method of utilizing real-time quantitative PCR that optical dye SYBR green carries out hemopoietic chimera to detect may further comprise the steps:
Transplant before and transplant back one month blood sample through clinical collection donor and acceptor;
Therefrom directly extract genomic dna with ordinary method; And
Measure DNA concentration with spectrophotometer, then be stored in 4 ℃ subsequent use.
2. real-time quantitative PCR detection method that utilizes optical dye SYBR green to carry out hemopoietic chimera, it comprises:
S1. method according to claim 1 makes a collection of specimens;
S2. select polymorphum genetic marker design primer and probe;
S3. real-time quantitative PCR detects hemopoietic chimera;
S4. the hemopoietic chimera of the different weaker concns of synthetic; And
S5. statistical analysis.
3. the real-time quantitative PCR detection method of hemopoietic chimera as claimed in claim 2; Wherein, Among the said step S2, select the choice criteria of polymorphum genetic marker design primer and probe to be: to comprise and contain at least two different polymorphic alleles of above successive base.
4. the real-time quantitative PCR detection method of hemopoietic chimera as claimed in claim 2 wherein, among the said step S4, is to utilize donor dna to dilute receptor dna or receptor dna dilutes the hemopoietic chimera that donor dna carries out 12 different weaker concns of synthetic.
5. the real-time quantitative PCR detection method of hemopoietic chimera as claimed in claim 4, wherein, the concentration from 0.01% to 100% of the hemopoietic chimera of 12 different weaker concns of said synthetic.
6. the real-time quantitative PCR detection method of hemopoietic chimera as claimed in claim 4 wherein, also comprises the specificity of moving melting curve checking PCR product simultaneously.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2011103629365A CN102399879A (en) | 2011-11-14 | 2011-11-14 | Hematopoietic chimera real-time quantitative PCR (Polymerase Chain Reaction) detection and specimen collection method |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2011103629365A CN102399879A (en) | 2011-11-14 | 2011-11-14 | Hematopoietic chimera real-time quantitative PCR (Polymerase Chain Reaction) detection and specimen collection method |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN102399879A true CN102399879A (en) | 2012-04-04 |
Family
ID=45882522
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN2011103629365A Pending CN102399879A (en) | 2011-11-14 | 2011-11-14 | Hematopoietic chimera real-time quantitative PCR (Polymerase Chain Reaction) detection and specimen collection method |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN102399879A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102399880A (en) * | 2011-11-14 | 2012-04-04 | 江苏迈健生物科技发展有限公司 | Real-time quantitative polymerase chain reaction (PCR) detection method for hematopoietic chimeras |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1997046706A1 (en) * | 1996-06-03 | 1997-12-11 | University Of Alberta | Methods for detection of rearranged dna |
| CN102443633A (en) * | 2011-11-14 | 2012-05-09 | 江苏迈健生物科技发展有限公司 | Method for real time quantitative PCR detection of hematopoietic chimeras and designing of genetic marker primers of hematopoietic chimeras |
-
2011
- 2011-11-14 CN CN2011103629365A patent/CN102399879A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1997046706A1 (en) * | 1996-06-03 | 1997-12-11 | University Of Alberta | Methods for detection of rearranged dna |
| CN102443633A (en) * | 2011-11-14 | 2012-05-09 | 江苏迈健生物科技发展有限公司 | Method for real time quantitative PCR detection of hematopoietic chimeras and designing of genetic marker primers of hematopoietic chimeras |
Non-Patent Citations (1)
| Title |
|---|
| 原丽: "实时荧光定量PCR-SNP检测移植后嵌合体方法的建立", 《中国优秀博硕士学位论文全文数据库（硕士）医药卫生科技辑》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102399880A (en) * | 2011-11-14 | 2012-04-04 | 江苏迈健生物科技发展有限公司 | Real-time quantitative polymerase chain reaction (PCR) detection method for hematopoietic chimeras |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US11519032B1 (en) | Transposition of native chromatin for personal epigenomics | |
| CN105506111B (en) | Method for detecting CNV (CNV) marker of MAPK10 gene of Nanyang cattle and application of CNV marker | |
| CN104232781A (en) | TaqMan probe real-time fluorescence PCR (Polymerase Chain Reaction) method for detecting HLA (Human Leukocyte Antigen)-B*5801 alleles | |
| DK3105324T3 (en) | NGS SYSTEM CONTROL AND PROCEDURES COMPREHENSIVE THESE | |
| KR101979218B1 (en) | Composition for determining bud mutation cultivar of Fuji apple | |
| CN107058538B (en) | Primer composition, kit composed of primer composition and application of kit | |
| Luthra et al. | TaqMan RT-PCR assay coupled with capillary electrophoresis for quantification and identification of bcr-abl transcript type | |
| CN103740702B (en) | A kind of SNP marker relevant to bay scallop heat tolerance and authentication method thereof and potential application | |
| US20190185943A1 (en) | Methods of mast cell tumor prognosis and uses thereof | |
| Bidartondo et al. | Fungal diversity in molecular terms: profiling, identification, and quantification in the environment | |
| CN110564861A (en) | Fluorescence labeling composite amplification kit for human Y chromosome STR locus and InDel locus and application thereof | |
| JP6819848B2 (en) | Comparative analysis method and device for miRNA expression level | |
| CN102286616A (en) | Method and kit for detecting mycobacterium tuberculosis isoniazide drug resistant gene mutation | |
| CN102443633A (en) | Method for real time quantitative PCR detection of hematopoietic chimeras and designing of genetic marker primers of hematopoietic chimeras | |
| CN103468790A (en) | Primers used for detecting polymorphism of saccharum arundinaceum, and detection method and applications | |
| CN105368950B (en) | It is a kind of detection cyanobacteria plasmid quantitative PCR kit and application | |
| US20040048297A1 (en) | Nucleic acid detection assay control genes | |
| CN102443632A (en) | PCR (polymerase chain reaction) reaction system for real-time quantitative PCR detection method for hematopoietic chimerism | |
| CN101525665A (en) | SNP G10320A molecular marker of mitochondrion ND3 gene of senescence-associated degenerative disease, detecting method and kit thereof | |
| KR20170039918A (en) | Composition and method for prediction of swine fecundity using genomic differentially methylated region | |
| CN102399879A (en) | Hematopoietic chimera real-time quantitative PCR (Polymerase Chain Reaction) detection and specimen collection method | |
| CN101775435B (en) | Method and kit for detecting polymorphism of mitochondrial ND1 gene mononucleotide, and application of kit | |
| CN102399881A (en) | Method for synthesizing chimera in hemopoiesis chimera real-time quantitative polymerase chain reaction (PCR) detection | |
| CN109576350B (en) | Kit and method for simultaneously quantifying DNA and RNA and quality control method | |
| CN101532058A (en) | Ageing related degenerative diseases chondriosome ND3 gene SNP G10310A molecular marker, detecting method and kit |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C53 | Correction of patent of invention or patent application | ||
| CB03 | Change of inventor or designer information |
Inventor after: Jin Xingliang Inventor after: Bai Lijun Inventor after: Chang Jing Inventor before: Yu Qiang |
|
| COR | Change of bibliographic data |
Free format text: CORRECT: INVENTOR; FROM: YU QIANG TO: JIN XINGLIANG BAI LIJUN CHANG JING |
|
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C02 | Deemed withdrawal of patent application after publication (patent law 2001) | ||
| WD01 | Invention patent application deemed withdrawn after publication |
Application publication date: 20120404 |