CN102605070A - Lymphocyte gene rearrangement clonality assay kit and assay method thereof - Google Patents
Lymphocyte gene rearrangement clonality assay kit and assay method thereof Download PDFInfo
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Abstract
The invention belongs to the technical field of biological reagents, and relates to the assay of gene rearrangement clonality related to lymphoid neoplasm diagnosis. The development of a full-primer coverage, low-cost and easy-to-operate lymphoid tissue gene rearrangement assay kit capable of being popularized in ordinary hospitals is urgently needed clinically. The invention discloses a lymphocyte gene rearrangement clonality assay kit, which is all-liquid reagent based on combined primers, and the invention also provides a related assay method and a hierarchical optimization scheme. When the kit is used for assaying the clonality of lymphocyte gene rearrangement, the assay accuracy can reach more than 99 percent, moreover, the steps of the assay method are simple, the total cost is low, and the invention is highly worth popularizing clinically.
Description
Technical field
The invention belongs to the biological reagent technical field, be specifically related to a kind of detection kit and detection method thereof of lymphocyte rearrangement clone property.
Background technology
Lymphoma (lymphoma) is the common cancer that lymphadenosis causes; In recent years along with popularizing of industrialization degree and increasing the weight of of environmental pollution; What especially the indoor and outdoor decoration was polluted increases the weight of, and its sickness rate raises year by year, serious threat human body Health and Living quality.Though lymphadenomatous in recent years treatment has obtained some progress; But because of the difficulty in the early diagnosis, and the complicacy of lymphoma type, the problem of its survival rate does not still have the improvement of essence; A lot of patients have been in the late period of disease when confirming diagnosis, lost the optimal treatment chance.
Lymphoid tissue is made up of immunologically competent cells such as T, B, NK lymphocytes.Lymphocytic cell surface has and antibody or antigen-specific bonded acceptor molecule, comprise the Tegeline that bone-marrow-derived lymphocyte produces (Immunoglobin, IG), and the TXi Baoshouti that produces of T lymphocyte (T Cell Receptor, TCR).Lymphocyte is when undifferentiated state, and to be V, D, J, C gene hold 3 ' end to be that wire is discontinuous to be arranged on the dna single chain from 5 ' to the embryonal system form of IG and tcr gene; When lymphocyte is grown certain phase, under special recombinase effect, selectivity couples together V, D, J, C district gene by different order, and promptly producer is reset.Have only producer to reset and just possibly form a gene that expressive function is arranged, lymphocyte breaks up mature cell from parent cell need be through repeatedly rearrangement.Normal B and T lymphocyte are polyclonal IG and tcr gene rearrangement and the rearrangement dna fragmentation that occurs varying in size.The Lymphoid tissue malignant tumour be considered to B or the T lymphocyte is blocked a certain stage in its atomization and causes lymphocytic monoclonicity hyperplasia; Make its specific genes rearrangement form account for certain predominance, due to IG or the tcr gene that monoclonicity occurs reset.Deriving from the monoclonicity that IG and TCR appear respectively in B, the lymphocytic lymphoma of T resets; Deriving from NK cell and NK/T cell lymphoma does not then have IG and the rearrangement of TCR monoclonicity, and normal Lymphoid tissue and reactive lymphadenosis are the polyclone property rearrangement of IG gene and tcr gene.This is the theoretical basis of Lymphoid tissue oncogene diagnosis.
The Lymphoid tissue structure is special, can produce reactive hyperplasia in various degree when receiving antigenic stimulation, and normal configuration gets muddled, and only is difficult to confirm that with morphological observation it is very pernicious.Most of in fact lymphadenomatous early stage performances are exactly the non-specific hyperplasia of Lymphoid tissue, have brought difficulty to early diagnosis.Techtology is observed and the immunophenotype analysis can be clarified a diagnosis though most lymphadenosis venereal diseases accommodations are crossed; But still have more case to assist to confirm lesion nature through clone's property analysis of gene level; Particularly in the diagnosis of t cell lymphoma, only still difficult fixed it is very pernicious with the immunophenotype analysis.Add the complicacy of the pathomorphism variation that exists in the real work, the reasons such as unstable of immunohistochemical methods itself, utilization rearrangement is analyzed its clone's property and is then had important value.The rearrangement detection technique is early stage, difficult or micro-Lymphoid tissue sample confirms that diagnosis provides possibility; It is important supplement to morphological examination and immunohistochemical methods method; Its meaning is: (1) confirms that lymphoproliferative disease is very pernicious, distinguishes Lymphoid tissue reactive hyperplasia and lymphoma.(2) confirm the origin of lymphoma tumour cell.(3) treatment monitoring helps the small remaining kitchen range of inspection, confirms whether tumour recurs.
At present, the method that detects lymphocyte rearrangement clone property mainly adopts PCR method to IG and the detection of tcr gene clone property, and high relatively susceptibility is arranged.But the susceptibility of PCR method and specificity receive influence of various factors, and wherein the coverage rate of primer is the main bottleneck that restriction PCR detects positive rate always.Because complicacy, the variety of IG and tcr gene rearrangement process and adopt the limitation of design of primers in the past, false negative and false positive rate are all higher, make its application receive certain limitation.For solving the low problem of primer fraction of coverage; The panel of experts of compositions such as Europe seven state's blood pathology developed BIOMED-2 rearrangement primer detection system and test kit thereof in 2007, solved single general-purpose primer and the low problem of heminested PCR primer fraction of coverage effectively.But the problem of bringing is: (1) BIOMED-2 rearrangement primer detection system primer system is huge, and test kit is very expensive; (2) this detection architecture is to the having relatively high expectations of reaction system such as archaeal dna polymerase, and essentially uses high-quality enzyme system; (3) expenses standard of present domestic rearrangement is lower, can not mate this detection system; (4) this system recommendation interpretation of result system needs genescan, and pertinent instruments equipment is very expensive.So at home, have only 1-2 family's large hospital to be used for scientific research and clinical diagnosis at present, most of hospital can't be used for patient's diagnosis clinically, and clinical universal difficulty is very big.
Therefore; Present stage is domestic to press for that a kind of primer coverage rate of exploitation is complete, cost is low, easy and simple to handle and Lymphoid tissue rearrangement detection technique that equipment requirements is low; Can popularize in general hospital, change the existing situation of rearrangement detection range, thereby satisfy the needs of clinical diagnosis; For patient strives for early diagnosis, the chance of early treatment.
Summary of the invention
The object of the present invention is to provide a kind of test kit that can be used for detecting lymphocyte rearrangement clone property.
The present invention also aims to provide that a kind of primer coverage rate is complete, cost is low, easy and simple to handle and equipment requirements is low, the method for the lymphocyte rearrangement clone property detection that can popularize in general hospital.
The inventor is incomplete to the primer coverage that exists in the at present domestic existing detection technique to lymphocyte rearrangement clone property; False negative and false positive rate are high; Available reagent box expense is high, and the detection system cost is high, is difficult to problems such as general hospital's penetration and promotion at home; Through extensive and deep research; And, detect clone's property of lymphocyte rearrangement fast and easily through the common PCR technology through the combined primer system of one cover of experimental design repeatedly, it is very pernicious to confirm that the lymphadenosis venereal disease becomes.
The ultimate principle of test kit of the present invention and detection method is a round pcr; And be the basis with combined primer system; Broad covered area is added the combination of science, during pcr amplification; Just can detect as long as there is monoclonicity rearrangement to exist, thereby realize that the good virulent that the lymphadenosis venereal disease is become is definite.
The inventor is according to relevant lymphocyte rearrangement pertinent literature (van Dongen JJ; Langerak AW; Br ü ggemann M, et al.Design and standardization of PCR primers and protocols for detection of clonal immunoglobulin and T cell receptor gene recombinations in suspect lymphoproliferations:report of the Biomed-2 concerted action BMH4-C98-3936.Leukemia 2003; 17:2257-2317; Van Krieken JH; Langerak AW; Macintyre EA, Improved reliability of lymphoma diagnostics via PCR-based clonality testing:report of the BIOMED-2 Concerted Action BHM4-CT98-3936.Leukemia.2007; 21 (2): 201-206; Br ü ggemann M; White H, Gaulard P Powerful strategy for polymerase chain reaction-based clonality assessment in T-cell malignancies Report of the BIOMED-2 Concerted Action BHM4 CT98-3936.Leukemia.2007; 21 (2): 215-221; Evans PA; Port Ch; Groenen PJ, Significantly improved PCR-based clonality testing in B-cell malignancies by use of multiple immunoglobulin gene targets.Report of the BIOMED-2 Concerted Action BHM4-CT98-3936.Leukemia.2007; 21 (2): 207-214.) and the inventor and research team previous work basis thereof (Ni Canrong, Zhang Taiming, He Miaoxia etc. rearrangement detection technique operating body can reach precaution. clinical and experimental pathology magazine, 2007; 23 (4): 485-486.), expand the face of containing of primer to maximum range in conjunction with common Lymphoid tissue rearrangement pattern, the combination primer system that constitutes this detection kit detects clone's property of lymphocyte rearrangement.
Concrete rearrangement combination of primers relevant information is seen table 1.
The main primer sets zoarium of table 1 lymphocyte rearrangement and detected object and product clip size
| Molectron | Detected object | Combination of primers | The product fragment |
| Molectron 1 | IGH?VH-FR1-JH | IGH?VH1-6-FR1-JH | 310-360bp |
| Molectron 2 | IGH?VH-FR2-JH | IGH?VH1-7-FR2-JH | 250-290bp |
| Molectron 3 | IGH?VH-FR3-JH | IGH?VH1-7-FR3-JH | 100-170bp |
| Molectron 4 | IGH?DH-JH | IGH?DH1-6-JH | 110-290bp；390-420bp |
| Molectron 5 | IGH?DH-JH | IGH?DH7-JH | 100-130bp |
| Molectron 6 | IGK?Vκ-Jκ | IGK?Vκ1-7-Jκ | 120-160bp；190-210bp；260-300bp |
| Molectron 7 | IGK?Vκ-kde | IGH?Vκ1-7-kde | 210-250bp；270-300bp；350-390bp |
| Molectron 8 | IGL?Vλ-Jλ | IGH?Vλ1-3-Jλ1-3 | 140-165bp |
| Molectron 9 | TCRG?Vγ1-Jγ1 | TCRG?Vγ1-Jγ1 | 145-255bp |
| Molectron 10 | TCRG?Vγ2-Jγ2 | TCRG?Vγ2-Jγ2 | 80-220bp |
| Molectron 11 | TCRB?Vβ1-Jβ1 | TCRB?Vβ1-Jβ1 | 245-285bp |
| Molectron 12 | TCRB?Vβ2-Jβ2 | TCRB?Vβ2-Jβ2 | 245-285bp |
| Molectron 13 | TCRB?Dβ-Jβ | TCRB?Dβ-Jβ | 170-210bp；285-325bp |
| Molectron 14 | TCRD?Vδ-Dδ-Jδ | TCRD?Vδ-Dδ-Jδ | 120-280bp |
The combination primer design:
In the present invention; According to the characteristics and the multifarious fact that IG and tcr gene in IG and tcr gene structure and the lymphocyte atomization are reset, design contain might reset the combined primer system of mode, primer sequence is no more than 30bp; Amplified fragments is between 150-300bp; Susceptibility and specificity have all had obvious improvement and lifting, and available regular-PCR technology for detection, are convenient to apply in the general hospital.
In the present invention, described bone-marrow-derived lymphocyte dependency rearrangement design of primers site comprises:
(1) molectron 1:IGH VH-FR1-JH resets (IGH VH-FR1-JH).
(2) molectron 2:IGH VH-FR2-JH resets (IGH VH-FR2-JH).
(3) molectron 3:IGH VH-FR3-JH resets (IGH VH-FR3-JH).
(4) molectron 4:IGH DH1-6-JH resets (IGH DH1-6-JH).
(5) molectron 5:IGH DH7-JH resets (IGH DH7-JH).
(6) molectron 6:IGK V κ-J κ resets (IGK V κ-J κ).
(7) molectron 7:IGK V κ-Kde rearrangement (IGK V κ-Kde).
(8) molectron 8:IGL V λ-J λ resets (IGL V λ-J λ).
In the present invention, described T lymphocyte dependency rearrangement design of primers site comprises:
(9) molectron 9:TCRG V γ 1-J γ 1 resets (TCRG V γ 1-J γ 1).
(10) molectron 10:TCRG V γ 2-J γ 2 resets (TCRG V γ 2-J γ 2).
(11) molectron 11:TCRB V β 1-J β 1 resets (TCRB V β 1-J β 1).
(12) molectron 12:TCRB V β 2-J β 2 resets (TCRB V β 2-J β 2).
(13) molectron 13:TCRB D β-J β resets (TCRB D β-J β).
(14) molectron 14:TCRD V δ-J δ resets (TCRD V δ-J δ).
First aspect of the present invention is to provide a kind of test kit that can be used for detecting lymphocyte rearrangement clone property, and described test kit comprises contained following primer sets zoarium in container and the container:
Reverse primer shown in forward primer shown in the molectron 1:SEQ ID NO:1+SEQ ID NO:2.
Reverse primer shown in forward primer shown in the molectron 2:SEQ ID NO:3+SEQ ID NO:4.
Reverse primer shown in forward primer shown in the molectron 3:SEQ ID NO:5+SEQ ID NO:6.
Reverse primer shown in forward primer shown in the molectron 4:SEQ ID NO:7+SEQ ID NO:8.
Reverse primer shown in forward primer shown in the molectron 5:SEQ ID NO:9+SEQ ID NO:10.
Reverse primer shown in forward primer shown in the molectron 6:SEQ ID NO:11+SEQ ID NO:12.
Reverse primer shown in forward primer shown in the molectron 7:SEQ ID NO:13+SEQ ID NO:14.
Reverse primer shown in forward primer shown in the molectron 8:SEQ ID NO:15+SEQ ID NO:16.
Reverse primer shown in forward primer shown in the molectron 9:SEQ ID NO:17+SEQ ID NO:18.
Reverse primer shown in forward primer shown in the molectron 10:SEQ ID NO:19+SEQ ID NO:20.
Reverse primer shown in forward primer shown in the molectron 11:SEQ ID NO:21+SEQ ID NO:22.
Reverse primer shown in forward primer shown in the molectron 12:SEQ ID NO:23+SEQ ID NO:24.
Reverse primer shown in forward primer shown in the molectron 13:SEQ ID NO:25+SEQ ID NO:.26.
Reverse primer shown in forward primer shown in the molectron 14:SEQ ID NO:27+SEQ ID NO:28.
In the molectron of the present invention, the ratio of two kinds of primers is 1: 3-3: 1, and concentration is 10pmol/ul.
Second aspect of the present invention provides the above-mentioned test kit of a kind of usefulness to detect the method for Lymphoid tissue rearrangement clone property, said method comprising the steps of:
A, be template, use the primer arbitrary or combination that both are above among the following molectron 1-14 to carry out the PCR reaction respectively with Lymphoid tissue biopsy to be measured or specimens from pri genomic dna:
(1) reverse primer shown in the forward primer shown in the molectron 1:SEQ ID NO:1+SEQ ID NO:2.This molectron is reset (IGH VH-FR1-JH) corresponding to IGH VH-FR1-JH.
(2) reverse primer shown in the forward primer shown in the molectron 2:SEQ ID NO:3+SEQ ID NO:4.This molectron is reset (IGH VH-FR2-JH) corresponding to IGH VH-FR2-JH.
(3) reverse primer shown in the forward primer shown in the molectron 3:SEQ ID NO:5+SEQ ID NO:6.This molectron is reset (IGH VH-FR3-JH) corresponding to IGH VH-FR3-JH.
(4) reverse primer shown in the forward primer shown in the molectron 4:SEQ ID NO:7+SEQ ID NO:8.This molectron is reset (IGH DH1-6-JH) corresponding to IGH DH1-6-JH.
(5) reverse primer shown in the forward primer shown in the molectron 5:SEQ ID NO:9+SEQ ID NO:10.This molectron is reset (IGH DH7-JH) corresponding to IGH DH7-JH.
(6) reverse primer shown in the forward primer shown in the molectron 6:SEQ ID NO:11+SEQ ID NO:12.This molectron is reset (IGK V κ-J κ) corresponding to IGK V κ-J κ.
(7) reverse primer shown in the forward primer shown in the molectron 7:SEQ ID NO:13+SEQ ID NO:14.This molectron is reset (IGK V κ-Kde) corresponding to IGK V κ-Kde.
(8) reverse primer shown in the forward primer shown in the molectron 8:SEQ ID NO:15+SEQ ID NO:16.This molectron is reset (IGL V λ-J λ) corresponding to IGL V λ-J λ.
(9) reverse primer shown in the forward primer shown in the molectron 9:SEQ ID NO:17+SEQ ID NO:18.This molectron is reset (TCRG V γ 1-J γ 1) corresponding to TCRG V γ 1-J γ 1.
(10) reverse primer shown in the forward primer shown in the molectron 10:SEQ ID NO:19+SEQ ID NO:20.This molectron is reset (TCRG V γ 2-J γ 2) corresponding to TCRG V γ 2-J γ 2.
(11) reverse primer shown in the forward primer shown in the molectron 11:SEQ ID NO:21+SEQ ID NO:22.This molectron is reset (TCRB V β 1-J β 1) corresponding to TCRB V β 1-J β 1.
(12) reverse primer shown in the forward primer shown in the molectron 12:SEQ ID NO:23+SEQ ID NO:24.This molectron is reset (TCRB V β 2-J β 2) corresponding to TCRB V β 2-J β 2.
(13) reverse primer shown in the forward primer shown in the molectron 13:SEQ ID NO:25+SEQ ID NO:26.This molectron is reset (TCRB D β-J β) corresponding to TCRB D β-J β.
(14) reverse primer shown in the forward primer shown in the molectron 14:SEQ ID NO:27+SEQ ID NO:28.This molectron is reset (TCRD V δ-J δ) corresponding to TCRD V δ-J δ.
Clone's property of rearrangement is judged in B, detection corresponding to the gel electrophoresis strip of the PCR reaction product of each molectron.
When the gel electrophoresis strip of PCR reaction product is that bandwidth is about 1mm corresponding to the segmental single band of purpose, brightness is strong, and edge clear is neat, judges then that this tissue lymph's cytogene is reset to be monoclonicity.
When the gel electrophoresis strip of PCR reaction product for band occurring in non-purpose fragment position, or bandwidth is greater than 1mm, a little less than the brightness, also can be strong, edge fog is irregular, all is judged as non-specific assorted band, then this tissue lymph's cytogene is reset and is polyclone property.
When the gel electrophoresis strip of PCR reaction product for smearing shape, or do not have obvious band and this tissue lymph's cytogene occurs then and reset and be polyclone property.
Do not have primer dimer when the gel electrophoresis of PCR reaction product and occur, do not have obvious internal reference gene purpose fragment band yet and occur, then be PCR reaction failure.
Sample of the present invention is fit to the tissues such as biopsy, excision tissue, peripheral blood, marrow and dropsy of serous cavity of tissue.In the above-mentioned tissue that is detected, extract genomic dna by ordinary method; As pcr template; Mix mutually with the aforementioned combination of primers that designs; Whether whether carry out PCR reaction, detect in the PCR reaction product through gel electrophoresis (SEPIGEL 305 or agarose gel electrophoresis) and occur and the corresponding monoclonicity band of purpose fragment, judge that institute is detected organizing with this is the malignant tumour that Lymphoid tissue is originated.
In order to realize the judgement of accuracy clone property, in preferred version of the present invention, also be provided with clone's property positive control or standard substance.Each rearrangement mode has all disposed the positive control or the standard substance of monoclonicity.Confirming the rearrangement preferred version of required detection, various positive controls or standard substance all adopt conventional PCR method.
In order to realize the judgement of accuracy clone property, in test kit of the present invention, also be provided with PCR reaction internal reference (β-actin).
The third aspect of the invention; A kind of preferred version that detects the various combination primer of lymphocyte rearrangement clone property is provided; Help lymphadenomatous quick, the simple and direct diagnosis of originating of the different lymphocytes of clinical signs of suspected: when clinical signs of suspected is B cell lymphoma; 1. at first select molectron 1+ molectron 2+ molectron 3+ molectron 6+ molectron 7 to carry out the PCR reaction; When the monoclonicity band occurring, then be diagnosed as bone-marrow-derived lymphocyte source property lymphoma corresponding to any one selected molectron PCR reaction product; 2. when no monoclonicity band occurs, select molectron 4+ molectron 5+ molectron 8 to carry out the PCR reaction again, when the monoclonicity band occurring, then be diagnosed as bone-marrow-derived lymphocyte source property lymphoma corresponding to any one selected molectron PCR reaction product; 3. when no monoclonicity band occurs, then can get rid of the possibility of B cell lymphoma.
When clinical signs of suspected is t cell lymphoma, 1. at first select molectron 9+ molectron 11+ molectron 12 to carry out the PCR reaction, when the monoclonicity band occurring, then be diagnosed as T lymphocyte source property lymphoma corresponding to any one selected molectron PCR reaction product; 2. select molectron 10+ molectron 13+ molectron 14 to carry out the PCR reaction again, when the monoclonicity band occurring, then be diagnosed as T lymphocyte source property lymphoma corresponding to any one selected molectron PCR reaction product; 3. when no monoclonicity band occurs, then can get rid of the possibility of t cell lymphoma.4. when highly suspecting
γ δT lymphocyte lymphoma person then can directly select molectron 14 to carry out PCR reaction, if then be diagnosed as when the monoclonicity band occurring
γ δT lymphocyte lymphoma; If when no monoclonicity band occurs, then can get rid of basically
γ δT lymphocyte lymphoma is selected by the scheme of above-mentioned suspection t cell lymphoma again.
When clinical suspection lymphoma; In the time of can't confirming that again B still is a T lymphocyte source possibility: 1. at first select molectron 2+ molectron 6+ molectron 7+ molectron 9+ molectron 10 to carry out the PCR reaction: when the monoclonicity band occurring corresponding to any one selected molectron PCR reaction product of first three molectron; Then be diagnosed as bone-marrow-derived lymphocyte source property lymphoma; When the monoclonicity band occurring, then be diagnosed as T lymphocyte source property lymphoma corresponding to any one selected molectron PCR reaction product of latter two molectron; 2. when no monoclonicity band occurs; Select molectron 1+ molectron 3+ molectron 4+ molectron 11+ molectron 12 again: when the monoclonicity band occurring corresponding to any one selected molectron PCR reaction product of first three molectron; Then be diagnosed as bone-marrow-derived lymphocyte source property lymphoma; When the monoclonicity band occurring, then be diagnosed as T lymphocyte source property lymphoma corresponding to any one selected molectron PCR reaction product of latter two molectron; 3. when no monoclonicity band occurs; Select molectron 5+ molectron 8+ molectron 13+ molectron 14 again: when the monoclonicity band occurring corresponding to any one selected molectron PCR reaction product of preceding two molectrons; Then be diagnosed as bone-marrow-derived lymphocyte source property lymphoma; When the monoclonicity band occurring, then be diagnosed as T lymphocyte source property lymphoma corresponding to any one selected molectron PCR reaction product of latter two molectron; 4. when no monoclonicity band occurs, then can get rid of lymphadenomatous possibility.
Technical scheme major advantage of the present invention is:
(1) adopt test kit of the present invention to carry out clone's property detection of lymphocyte rearrangement, can be simultaneously the rearrangement of different modes be detected, the reaction conditions homogeneous during detection, accuracy in detection can reach more than 99%.
(2) detection method step of the present invention is simple, thereby has avoided many uncertain factors of existing in the complex operations process, thereby can improve the detection accuracy rate greatly.
(3) primer system of the present invention has covered all common rearrangement modes of IG and TCR, and the false negative of having avoided single in the past primer or familial primer system to cause has improved the detection accuracy rate greatly.
(4) detection architecture cost of the present invention is low, and low for equipment requirements, and the layering preferred version to different cases is provided, and meets the clinical practice need of work especially, is adapted at general coventional type hospital and promotes the use of.
Others of the present invention are because the disclosure of this paper is conspicuous as far as those skilled in the art.
Description of drawings
Fig. 1 is molectron 1,2,3 design of primers sites (IGH FR1, IGH FR2, IGH FR3)
Fig. 2 is molectron 4,5 design of primers sites (IGH DH1-6, IGH DH7)
Fig. 3 is molectron 6 design of primers sites (IGK V κ-J κ)
Fig. 4 is molectron 7 design of primers sites (IGK V κ-kde)
Fig. 5 is molectron 8 design of primers sites (IGL V λ-J λ)
Fig. 6 is molectron 9,10 design of primers sites (TCRG V γ-J γ)
Fig. 7 is molectron 11,12 design of primers sites (TCRG V β-J β)
Fig. 8 is molectron 13 design of primers sites (TCRG D β-J β)
Fig. 9 is molectron 14 design of primers sites (TCRD V γ-D γ-J γ)
Figure 10 is polyacrylate hydrogel electrophoresis (left side) and agarose gel electrophoresis (right side) PCR product analysis figure
Single band (arrow is represented) is the monoclonicity band among the figure, and the band clear and definite is easy to judge.
Specific embodiments
Below in conjunction with specific embodiment and accompanying drawing, further set forth the present invention.Should understand these embodiment and only be used to explain the present invention, and be not used in restriction range of application of the present invention.The experimental technique of the concrete working conditions of unreceipted embodiment among the following embodiment; Usually according to people such as normal condition such as Sambrook; Molecular cloning: lab guide (New York:Cold Spring Harbor Labarotary Press; 1989) condition described in, or by condition that manufacturer provided.Unless otherwise indicated, otherwise per-cent and umber all calculate by weight.
Only if definition separately, the same meaning that employed all specialties and scientific words and this area professional are familiar with in the literary composition.In addition, any with the institute similar content of putting down in writing or the equalization method and material all can be applicable among the present invention.The usefulness that preferable implementation method described in the literary composition and material only present a demonstration.
Embodiment 1: a kind of lymph group cytogene is reset the detection kit of clone's property
A kind of practicality and convenience detect the detection kit that lymph group cytogene is reset clone's property; Described test kit contain a plurality of independently containers or cut apart each other the hole, it is fit that the following primer sets of being made up of two primers respectively is housed in each container or the hole:
Reverse primer shown in forward primer shown in the molectron 1:SQE ID NO.1+SQE ID NO.2.
Reverse primer shown in forward primer shown in the molectron 2:SQE ID NO.3+SQE ID NO.4.
Reverse primer shown in forward primer shown in the molectron 3:SQE ID NO.5+SQE ID NO.6.
Reverse primer shown in forward primer shown in the molectron 4:SQE ID NO.7+SQE ID NO.8.
Reverse primer shown in forward primer shown in the molectron 5:SQE ID NO.9+SQE ID NO.10.
Reverse primer shown in forward primer shown in the molectron 6:SQE ID NO.11+SQE ID NO.12.
Reverse primer shown in forward primer shown in the molectron 7:SQE ID NO.13+SQE ID NO.14.
Reverse primer shown in forward primer shown in the molectron 8:SQE ID NO.15+SQE ID NO.16.
Reverse primer shown in forward primer shown in the molectron 9:SQE ID NO.17+SQE ID NO.18.
Reverse primer shown in forward primer shown in the molectron 10:SQE ID NO.19+SQE ID NO.20.
Reverse primer shown in forward primer shown in the molectron 11:SQE ID NO.21+SQE ID NO.22.
Reverse primer shown in forward primer shown in the molectron 12:SQE ID NO.23+SQE ID NO.24.
Reverse primer shown in forward primer shown in the molectron 13:SQE ID NO.25+SQE ID NO.26.
Reverse primer shown in forward primer shown in the molectron 14:SQE ID NO.27+SQE ID NO.28.
In the molectron of the present invention, the ratio of two kinds of primers is 1: 3-3: 1, and concentration is 10pmol/ul.
When described test kit detects; Can be with the genomic dna (as pcr template) of testing sample; Or positive control (as pcr template) is joined in the test kit each contain respectively in the fit container of different primer sets; Add other again and carry out the required conventional ingredient of PCR reaction (like the Taq enzyme etc.) and carry out the PCR reaction, provided the internal reference primer to react simultaneously by the quality that guarantees template DNA can add test kit simultaneously.Reaction finishes the back through whether existing corresponding to the segmental monoclonicity band of purpose in gel electrophoresis (SEPIGEL 305 or agarose gel electrophoresis) the detection PCR reaction product; With this judge the sample that detected whether be the malignant tumour in Lymphoid tissue source and their origin of cell, what help that clinical definite lymphadenosis venereal disease becomes is very pernicious.
Embodiment 2: clone's property of Lymphoid tissue rearrangement confirms in the tissue to be detected
1, the synthetic design (shown in Fig. 1-9) that reaches detection method of concrete primer
According to 14 selected rearrangement modes, in conjunction with reference, to use Oligo 6.2 primer-design softwares and design 14 molectrons of 14 groups of primers formation, the primer system entrusts specialized company's (Shanghai Sani's bio tech ltd) synthetic.
In the primer system, the ratio of two kinds of primers is 1: 3-3: 1, and concentration is 10pmol/ul.
(1) molectron 1:
SEQ?ID?NO：1?5’-GGCCTCAGTGAAGGTCTCCTGCAAG-3’
SEQ?ID?NO：2?3’-CCAGTGGCAGAGGAGTCCATTC-5’
Product magnitude range 310-360bp
(2) molectron 2:
SEQ?ID?NO：3?5’-CTGGGTGCGACAGGCCCCTGGACAA-3’
SEQ?ID?NO：4?3’-CCAGTGGCAGAGGAGTCCATTC-5’
Product magnitude range 250-290bp
(3) molectron 3:
SEQ?ID?NO：5 5’-TGGAGCTGAGCAGCCTGAGATCTGA-3’
SEQ?ID?NO：6 3’-CCAGTGGCAGAGGAGTCCATTC-5’
Product magnitude range 100-170bp
(4) molectron 4:
SEQ?ID?NO：7 5’-GGCGGAATGTGTGCAGGC-3’
SEQ?ID?NO：8 3’-CCAGTGGCAGAGGAGTCCATTC-5’
Product magnitude range 110-290bp; 390-420bp
(5) molectron 5:
SEQ?ID?NO：9 5’-CACAGGCCCCCTACCAGC-3’
SEQ?ID?NO：10?3’-CCAGTGGCAGAGGAGTCCATTC-5’
Product magnitude range 100-130bp
(6) molectron 6:
SEQ?ID?NO：11 5’-TCAAGGTTCAGCGGCAGTGGATCTG-3’
SEQ?ID?NO：12 3’-CCCTGGTTCCACCTCTAGTTTGCATTC-5’
Product magnitude range 120-160bp; 190-210bp; 260-300bp
(7) molectron 7:
SEQ?ID?NO：13 5’-CGTGGCACCGCGAGCTGTAGAC-3’
SEQ?ID?NO：14 3’-ATCCTGTTGGACGAGACTGGAGACTCC-5’
Product magnitude range 210-250bp; 270-300bp; 350-390bp
(8) molectron 8:
SEQ?ID?NO：15 5’-ATTCTCTGGCTCCAAGTCTGGC-3’
SEQ?ID?NO：16 3’-CCCTGGTTCGAGTGGCAGGATC-5’
Product magnitude range 140-165bp
(9) molectron 9:
SEQ?ID?NO：17 5’-GGAAGGCCCCACAGCRTCTT-3’
SEQ?ID?NO：18 3’-CGAGTATCATTGAAGCGGACCATT-5’
Product magnitude range 145-255bp
(10) molectron 10:
SEQ?ID?NO：19?5’-CGGCACTGTCAGAAAGGAATC-3’
SEQ?ID?NO：20?3’-GAGAAACCGTCACCTTGTTGTG-5’
Product magnitude range 80-220bp
(11) molectron 11:
SEQ?ID?NO：21?5’-AACTATGTTTTGGTATCGTCA-3’
SEQ?ID?NO：22?3’-GTGGTCTAAGTGTCAACATCCATTC-5’
Product magnitude range 245-285bp
(12) molectron 12:
SEQ?ID?NO：23?5’-CACGATGTTCTGGTACCGTCAGCA-3’
SEQ?ID?NO：24?3’-AGTGGCACGATCCATTCTTCC-5’
Product magnitude range 245-285bp
(13) molectron 13:
SEQ?ID?NO：25?5’-GCCAAACAGCCTTACAAAGAC-3’
SEQ?ID?NO：26?3’-CTGGTCCAATTGGCAACATCCATTC-5’
Product magnitude range 170-210bp; 285-325bp
(14) molectron 14:
SEQ?ID?NO：27?5’-ATGCAAAAAGTGGTCGCTATT-3’
SEQ?ID?NO：28?3’-CTTGGGCACACTGACACCTTG-5’
Product magnitude range 120-280bp
Prepare the detection reaction system by following scheme:
Carry out the PCR reaction by following reaction conditions:
In each reaction positive control is set, internal reference and no template contrast.
2, the preparation of sample to be tested, test:
Get the paraffin-embedded tissue section of sample to be tested; With phenol chloroform extraction method (the Zhu ZZ et al.A p53 polymorphism modifies the risk of hepatocellular carcinoma among non-carriers but not carriers of chronic hepatisis B virus infection after improving; Cancer letter; 2005; 229:77-83), extracting genomic dna from biopsy or excision tissue, stdn aqueous dna concentration is to 25ng/ul.The capable gel electrophoresis of the aqueous dna that takes a morsel (SEPIGEL 305 or agarose gel electrophoresis).
3, the test of PCR reaction product:
Get PCR reaction product row gel electrophoresis (SEPIGEL 305 or agarose gel electrophoresis) (shown in figure 10):
When the gel electrophoresis strip of PCR reaction product is that bandwidth is about 1mm corresponding to the segmental single band of purpose, brightness is strong, and edge clear is neat, then judges the monoclonicity that is of this Lymphoid tissue rearrangement.
When the gel electrophoresis strip of PCR reaction product for band occurring in non-purpose fragment position, or bandwidth is greater than 1mm, a little less than the brightness, also can be strong, edge fog is irregular, all is judged as non-specific assorted band, then this Lymphoid tissue rearrangement is polyclone property.
When the gel electrophoresis strip of PCR reaction product for smearing shape, or do not have that then this Lymphoid tissue rearrangement appears in obvious band be polyclone property.
Do not have primer dimer when the gel electrophoresis of PCR reaction product and occur, do not have obvious internal reference gene purpose fragment band yet and occur, then be PCR reaction failure.
Embodiment 3: the detection instance one of test kit of the present invention
When clinical signs of suspected is B cell lymphoma: 1. at first select molectron 1+ molectron 2+ molectron 3+ molectron 6+ molectron 7 to carry out the PCR reaction; When the monoclonicity band occurring, then be diagnosed as bone-marrow-derived lymphocyte source property lymphoma corresponding to any one selected molectron PCR reaction product; 2. when no monoclonicity band occurs, select molectron 4+ molectron 5+ molectron 8 to carry out the PCR reaction again, when the monoclonicity band occurring, then be diagnosed as bone-marrow-derived lymphocyte source property lymphoma corresponding to any one selected molectron PCR reaction product; 3. when no monoclonicity band occurs, then can get rid of the possibility of B cell lymphoma.
Concrete case is following:
1. medical history: 53/F finds cheek district, right side lump half a year, enlargement gradually
2. pathologic finding: one of right side parotid gland tissues, size 4.5 * 3 * 2cm, tangent plane see the irregular shape thing that swell, big or small 3.5 * 3 * 1.5cm, pearl, quality is medium, owes clearly with the surrounding tissue boundary.A large amount of lymphocytic infiltrations in the parotid gland tissues are found in microscopic examination, the local nodositas structure that forms, and the visible lymph follicle in subregion forms, the visible more plasmocyte infiltrating in subregion.Binding immunoassay group result suspects B lymphocytic lymphoma.
3. with the phenol chloroform extraction method after improving, section extracting genomic dna from the paraffin-embedded tissue of the swollen thing of the parotid gland of excision, stdn aqueous dna concentration is to 25ng/ul.The capable gel electrophoresis of the aqueous dna that takes a morsel (SEPIGEL 305 or agarose gel electrophoresis) detects the DNA quality.
4. prepare the detection reaction system by following scheme:
5. carry out the PCR reaction by following reaction conditions:
In each reaction positive control is set, internal reference and no template contrast.
Carry out the PCR reaction by the layering of aforesaid combination scheme.
6. get PCR reaction product row agarose gel electrophoresis; Find that according to embodiment 2-3 the gel electrophoresis strip of PCR reaction product corresponds respectively to molectron 2 and the segmental single band of molectron 6 purposes; Bandwidth is about 1mm; Brightness is strong, and edge clear is neat, judges that then this Lymphoid tissue rearrangement result is the IG monoclonicity.
7. pathological diagnosis: (right side cheek district) bone-marrow-derived lymphocyte property lymphoma, combining form and immunohistochemical methods result are mucous membrane relevant edge district B cell lymphoma
Embodiment 3: the detection instance two of test kit of the present invention
When clinical signs of suspected is t cell lymphoma: 1. at first select molectron 9+ molectron 11+ molectron 12 to carry out the PCR reaction, when the monoclonicity band occurring, then be diagnosed as T lymphocyte source property lymphoma corresponding to any one selected molectron PCR reaction product; 2. select molectron 10+ molectron 13+ molectron 14 to carry out the PCR reaction again, when the monoclonicity band occurring, then be diagnosed as T lymphocyte source property lymphoma corresponding to any one selected molectron PCR reaction product; 3. when no monoclonicity band occurs, then can get rid of the possibility of t cell lymphoma.
Concrete case is following:
1. medical history: 71/M finds that the bilateral neck lymphadenectasis is surplus March
2. pathologic finding: one piece of right side cervical lymph node, size 3.5 * 2 * 1.5cm, the visible coating of tangent plane, pearl, quality is soft, evenly.Microscopic examination lymph node structure partial destruction, paracortex obviously enlarges a large amount of lymphocytosis, cell multiform, little blood vessel hyperplasia, the visible plasmocyte in subregion, eosinophilic granulocyte and histiocytic infiltrate.Binding immunoassay group result suspects T lymphocyte lymphoma.
3. with the phenol chloroform extraction method after improving, section extracting genomic dna from the paraffin-embedded tissue of the right cervical lymph node of excision, stdn aqueous dna concentration is to 25ng/ul.The capable gel electrophoresis of the aqueous dna that takes a morsel (SEPIGEL 305 or agarose gel electrophoresis) detects the DNA quality.
4. prepare the detection reaction system by following scheme:
5. carry out the PCR reaction by following reaction conditions:
In each reaction positive control is set, internal reference and no template contrast.
Carry out the PCR reaction by the layering of aforesaid combination scheme.
6. get PCR reaction product row agarose gel electrophoresis; Find that according to embodiment 2-3 the gel electrophoresis strip of PCR reaction product corresponds respectively to molectron 10 and the segmental single band of molectron 13 purposes; Bandwidth is about 1mm; Brightness is strong, and edge clear is neat, judges that then this Lymphoid tissue rearrangement result is the TCR monoclonicity.
7. pathological diagnosis: (right cervical lymph node) T lymphocytic lymphoma, combining form and immunohistochemical methods result are lymphoma peripheral T cell (the non-type that refers in particular to)
Embodiment 5: the detection instance three of test kit of the present invention
When clinical suspection lymphoma; In the time of can't confirming that again B still is a T lymphocyte source possibility: 1. at first select molectron 2+ molectron 6+ molectron 7+ molectron 9+ molectron 10 to carry out the PCR reaction: when the monoclonicity band occurring corresponding to any one selected molectron PCR reaction product of first three molectron; Then be diagnosed as bone-marrow-derived lymphocyte source property lymphoma; When the monoclonicity band occurring, then be diagnosed as T lymphocyte source property lymphoma corresponding to any one selected molectron PCR reaction product of latter two molectron; 2. when no monoclonicity band occurs; Select molectron 1+ molectron 3+ molectron 4+ molectron 11+ molectron 12 again: when the monoclonicity band occurring corresponding to any one selected molectron PCR reaction product of first three molectron; Then be diagnosed as bone-marrow-derived lymphocyte source property lymphoma; When the monoclonicity band occurring, then be diagnosed as T lymphocyte source property lymphoma corresponding to any one selected molectron PCR reaction product of latter two molectron; 3. when no monoclonicity band occurs; Select molectron 5+ molectron 8+ molectron 13+ molectron 14 again: when the monoclonicity band occurring corresponding to any one selected molectron PCR reaction product of preceding two molectrons; Then be diagnosed as bone-marrow-derived lymphocyte source property lymphoma; When the monoclonicity band occurring, then be diagnosed as T lymphocyte source property lymphoma corresponding to any one selected molectron PCR reaction product of latter two molectron; 4. when no monoclonicity band occurs, then can get rid of lymphadenomatous possibility.
Concrete case is following:
1. medical history: 42/M generates heat and accompanies the lymph nodes of body as a whole enlargement surplus January
2. pathologic finding: one piece of left side cervical lymph node, size 3 * 2 * 1cm, the visible coating of tangent plane, greyish white bois de rose, quality is soft, evenly.The microscopic examination lymph node structure is visible, and lymph foilicie hyperplasia is obvious, and paracortex obviously enlarges, a large amount of lymphocytosis, and the cell polymorphism is obvious, little blood vessel hyperplasia, subregion visible tissue cell and eosinophilic granulocyte soak into.Binding immunoassay group result suspects lymphoma.
3. with the phenol chloroform extraction method after improving, section extracting genomic dna from the paraffin-embedded tissue of the right cervical lymph node of excision, stdn aqueous dna concentration is to 25ng/ul.The capable gel electrophoresis of the aqueous dna that takes a morsel (SEPIGEL 305 or agarose gel electrophoresis) detects the DNA quality.
4. prepare the detection reaction system by following scheme:
5. carry out the PCR reaction by following reaction conditions:
In each reaction positive control is set, internal reference and no template contrast.
Carry out the PCR reaction by the layering of aforesaid combination scheme.
6. get PCR reaction product row agarose gel electrophoresis, do not find that according to embodiment 2-3 judgement criteria the gel electrophoresis strip of PCR reaction product is a monoclonicity.
7. pathological diagnosis: (left neck lymphoglandula) combines clinical, pathomorphism, immunohistochemical methods and rearrangement result is reactive hyperplasia of lymph node
The present invention adopts routine to be used for the PCR method of rearrangement; Use a cover associativity primer system; Be provided with positive control and internal reference, and designed accordingly, accept clinical common biopsy, the paraffin-embedded tissue of excision sample according to the alternative layering preferred version of the particular case of different cases; And the detection of the lymphocyte rearrangement clone property of peripheral blood, marrow, dropsy of serous cavity, carry out interpretation of result with regular-PCR amplification appearance with gel electrophoresis commonly used.The present invention detects true reflection clinical samples, and positive rate and rate of accuracy reached are more than 95%.
Some results of study according to before bibliographical information and the present invention show, the detection of the clone of lymphocyte rearrangement in the past property, and domestic existing problems are exactly that the primer coverage rate is low, and the available reagent box is expensive, and cost is high, can't popularize in general coventional type hospital.The present invention has overcome above shortcoming; The primer maximization covers all possible rearrangement mode; Detection method adopts regular-PCR amplification appearance to carry out with gel electrophoresis commonly used, greatly reduces total cost, is adapted at general coventional type hospital and carries out clinical application.
All documents that the present invention mentions are incorporated by reference in this application all.Should be understood that in addition after having read above-mentioned teachings of the present invention, this area professional can make various changes or modification to the present invention, these equivalent form of values fall within the application's book appended claims institute restricted portion equally.
Claims (13)
1. one kind is used to detect the test kit that property is cloned in lymphocyte rearrangement, and described test kit comprises contained following primer sets zoarium in container and the container:
Reverse primer shown in forward primer shown in the molectron 1:SEQ ID NO:1+SEQ ID NO:2;
Reverse primer shown in forward primer shown in the molectron 2:SEQ ID NO:3+SEQ ID NO:4;
Reverse primer shown in forward primer shown in the molectron 3:SEQ ID NO:5+SEQ ID NO:6;
Reverse primer shown in forward primer shown in the molectron 4:SEQ ID NO:7+SEQ ID NO:8;
Reverse primer shown in forward primer shown in the molectron 5:SEQ ID NO:9+SEQ ID NO:10;
Reverse primer shown in forward primer shown in the molectron 6:SEQ ID NO:11+SEQ ID NO:12;
Reverse primer shown in forward primer shown in the molectron 7:SEQ ID NO:13+SEQ ID NO:14;
Reverse primer shown in forward primer shown in the molectron 8:SEQ ID NO:15+SEQ ID NO:16;
Reverse primer shown in forward primer shown in the molectron 9:SEQ ID NO:17+SEQ ID NO:18;
Reverse primer shown in forward primer shown in the molectron 10:SEQ ID NO:19+SEQ ID NO:20;
Reverse primer shown in forward primer shown in the molectron 11:SEQ ID NO:21+SEQ ID NO:22;
Reverse primer shown in forward primer shown in the molectron 12:SEQ ID NO:23+SEQ ID NO:24;
Reverse primer shown in forward primer shown in the molectron 13:SEQ ID NO:25+SEQ ID NO:.26;
Reverse primer shown in forward primer shown in the molectron 14:SEQ ID NO:27+SEQ ID NO:28.
2. a kind of test kit that is used to detect lymphocyte rearrangement clone property according to claim 1 is characterized in that the ratio of two kinds of primers is 1 in the molectron: 3-3: 1, and concentration is 10pmol/ul.
3. as claimed in claim 1 being used to detected the detection method that the test kit of property is cloned in lymphocyte rearrangement, may further comprise the steps:
A, be template, use that the primer arbitrary or combination more than both carries out the PCR reaction respectively in the molectron 1 to 14 with Lymphoid tissue biopsy to be measured or specimens from pri genomic dna;
Clone's property of rearrangement is judged in B, detection corresponding to the gel electrophoresis strip of the PCR reaction product of each molectron 1 to 14:
The PCR reaction product magnitude range 310-360bp of molectron 1;
The PCR reaction product magnitude range 250-290bp of molectron 2;
The PCR reaction product magnitude range 100-170bp of molectron 3;
The PCR reaction product magnitude range 110-290bp of molectron 4; 390-420bp;
The PCR reaction product magnitude range 100-130bp of molectron 5;
The PCR reaction product magnitude range 120-160bp of molectron 6; 190-210bp; 260-300bp;
The PCR reaction product magnitude range 210-250bp of molectron 7; 270-300bp; 350-390bp;
The PCR reaction product magnitude range 140-165bp of molectron 8;
The PCR reaction product magnitude range 145-255bp of molectron 9;
The PCR reaction product magnitude range 80-220bp of molectron 10;
The PCR reaction product magnitude range 245-285bp of molectron 11;
The PCR reaction product magnitude range 245-285bp of molectron 12;
The PCR reaction product magnitude range 170-210bp of molectron 13; 285-325bp;
The PCR reaction product magnitude range 120-280bp of molectron 14;
When the gel electrophoresis strip of PCR reaction product is that bandwidth is about 1mm corresponding to the segmental single band of purpose, brightness is strong, and edge clear is neat, judges then that this tissue lymph's cytogene is reset to be monoclonicity;
When the gel electrophoresis strip of PCR reaction product for band occurring in non-purpose fragment position, or bandwidth is greater than 1mm, a little less than the brightness, also can be strong, edge fog is irregular, all is judged as non-specific assorted band, then this tissue lymph's cytogene is reset and is polyclone property;
When the gel electrophoresis strip of PCR reaction product for smearing shape, or do not have obvious band and this tissue lymph's cytogene occurs then and reset and be polyclone property;
Do not have primer dimer when the gel electrophoresis of PCR reaction product and occur, do not have obvious internal reference gene purpose fragment band yet and occur, then be PCR reaction failure.
4. according to claim 3 being used to detected the detection method that the test kit of property is cloned in lymphocyte rearrangement, it is characterized in that, the reaction system of PCR reaction is following in the steps A:
The reaction conditions of PCR reaction is following:
5. detect the detection method that the test kit of property is cloned in lymphocyte rearrangement according to claim 3 or 4 described being used to, it is characterized in that gel electrophoresis is SEPIGEL 305 or agarose gel electrophoresis among the step B.
6. detect the detection method that the test kit of property is cloned in lymphocyte rearrangement according to claim 3 or 4 described being used to, it is characterized in that, select molectron 1+ molectron 2+ molectron 3+ molectron 6+ molectron 7 to carry out the PCR reaction in the steps A.
7. detect the detection method that the test kit of property is cloned in lymphocyte rearrangement according to claim 3 or 4 described being used to, it is characterized in that, select molectron 4+ molectron 5+ molectron 8 to carry out the PCR reaction in the steps A.
8. detect the detection method that the test kit of property is cloned in lymphocyte rearrangement according to claim 3 or 4 described being used to, it is characterized in that, select molectron 9+ molectron 11+ molectron 12 to carry out the PCR reaction in the steps A.
9. detect the detection method that the test kit of property is cloned in lymphocyte rearrangement according to claim 3 or 4 described being used to, it is characterized in that, select molectron 10+ molectron 13+ molectron 14 to carry out the PCR reaction in the steps A.
10. detect the detection method that the test kit of property is cloned in lymphocyte rearrangement according to claim 3 or 4 described being used to, it is characterized in that, select molectron 14 to carry out the PCR reaction in the steps A.
11. detect the detection method that the test kit of property is cloned in lymphocyte rearrangement according to claim 3 or 4 described being used to, it is characterized in that, select molectron 2+ molectron 6+ molectron 7+ molectron 9+ molectron 10 to carry out the PCR reaction in the steps A.
12. detect the detection method that the test kit of property is cloned in lymphocyte rearrangement according to claim 3 or 4 described being used to, it is characterized in that, select molectron 1+ molectron 3+ molectron 4+ molectron 11+ molectron 12 to carry out the PCR reaction in the steps A.
13. detect the detection method that the test kit of property is cloned in lymphocyte rearrangement according to claim 3 or 4 described being used to, it is characterized in that, select molectron 5+ molectron 8+ molectron 13+ molectron 14 to carry out the PCR reaction in the steps A.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2015063121A1 (en) * | 2013-10-29 | 2015-05-07 | Region Syddanmark | Method for analyzing body fluid samples |
| CN109897898A (en) * | 2019-02-14 | 2019-06-18 | 厦门飞朔生物技术有限公司 | A kind of construction method and its application for resetting library for high-flux sequence detection lymthoma IG and TCR |
| CN110656161A (en) * | 2018-06-28 | 2020-01-07 | 苏州云泰生物医药科技有限公司 | Capillary electrophoresis kit for detecting human immunoglobulin gene rearrangement and use method thereof |
| CN112210595A (en) * | 2020-08-11 | 2021-01-12 | 广州君瑞康生物科技有限公司 | Method for detecting minimal residual disease |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1965089A (en) * | 2002-10-11 | 2007-05-16 | 鹿特丹伊拉兹马斯大学 | Nucleic acid amplification primers for PCR-based clonality studies |
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Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1965089A (en) * | 2002-10-11 | 2007-05-16 | 鹿特丹伊拉兹马斯大学 | Nucleic acid amplification primers for PCR-based clonality studies |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2015063121A1 (en) * | 2013-10-29 | 2015-05-07 | Region Syddanmark | Method for analyzing body fluid samples |
| US10227655B2 (en) | 2013-10-29 | 2019-03-12 | Region Syddanmark | Method for analyzing body fluid samples |
| CN110656161A (en) * | 2018-06-28 | 2020-01-07 | 苏州云泰生物医药科技有限公司 | Capillary electrophoresis kit for detecting human immunoglobulin gene rearrangement and use method thereof |
| CN110656161B (en) * | 2018-06-28 | 2022-12-13 | 苏州云泰生物医药科技有限公司 | Capillary electrophoresis kit for detecting human immunoglobulin gene rearrangement and use method thereof |
| CN109897898A (en) * | 2019-02-14 | 2019-06-18 | 厦门飞朔生物技术有限公司 | A kind of construction method and its application for resetting library for high-flux sequence detection lymthoma IG and TCR |
| CN112210595A (en) * | 2020-08-11 | 2021-01-12 | 广州君瑞康生物科技有限公司 | Method for detecting minimal residual disease |
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Application publication date: 20120725 |