CN106148488B - ERCC1 gene mutation detection specific primer and liquid chip kit - Google Patents
ERCC1 gene mutation detection specific primer and liquid chip kit Download PDFInfo
- Publication number
- CN106148488B CN106148488B CN201510151622.9A CN201510151622A CN106148488B CN 106148488 B CN106148488 B CN 106148488B CN 201510151622 A CN201510151622 A CN 201510151622A CN 106148488 B CN106148488 B CN 106148488B
- Authority
- CN
- China
- Prior art keywords
- seq
- site
- sequence
- primer
- sequence consisting
- 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.)
- Active
Links
- 238000001514 detection method Methods 0.000 title claims abstract description 68
- 101150050700 ERCC1 gene Proteins 0.000 title claims abstract description 43
- 206010064571 Gene mutation Diseases 0.000 title claims abstract description 26
- 239000007788 liquid Substances 0.000 title claims abstract description 23
- 241000183024 Populus tremula Species 0.000 claims abstract description 39
- 239000004005 microsphere Substances 0.000 claims abstract description 34
- 230000035772 mutation Effects 0.000 claims abstract description 26
- 230000003321 amplification Effects 0.000 claims abstract description 8
- 238000003199 nucleic acid amplification method Methods 0.000 claims abstract description 8
- 125000006850 spacer group Chemical group 0.000 claims description 12
- 108090000623 proteins and genes Proteins 0.000 claims description 11
- 239000007791 liquid phase Substances 0.000 abstract description 14
- 238000012163 sequencing technique Methods 0.000 abstract description 6
- 238000006243 chemical reaction Methods 0.000 description 16
- 239000000523 sample Substances 0.000 description 14
- 238000000034 method Methods 0.000 description 13
- 238000012360 testing method Methods 0.000 description 13
- 238000005516 engineering process Methods 0.000 description 12
- 238000013461 design Methods 0.000 description 10
- 230000000694 effects Effects 0.000 description 9
- 239000000047 product Substances 0.000 description 8
- 238000004458 analytical method Methods 0.000 description 7
- 238000009396 hybridization Methods 0.000 description 7
- 239000012224 working solution Substances 0.000 description 7
- YBJHBAHKTGYVGT-ZKWXMUAHSA-N (+)-Biotin Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 description 6
- 101000876529 Homo sapiens DNA excision repair protein ERCC-1 Proteins 0.000 description 5
- 230000008569 process Effects 0.000 description 5
- 239000011550 stock solution Substances 0.000 description 5
- 102100035186 DNA excision repair protein ERCC-1 Human genes 0.000 description 4
- 239000007983 Tris buffer Substances 0.000 description 4
- 230000008901 benefit Effects 0.000 description 4
- 230000015572 biosynthetic process Effects 0.000 description 4
- 239000000872 buffer Substances 0.000 description 4
- 238000002360 preparation method Methods 0.000 description 4
- 230000035945 sensitivity Effects 0.000 description 4
- 239000000243 solution Substances 0.000 description 4
- 238000003786 synthesis reaction Methods 0.000 description 4
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 4
- 108090000790 Enzymes Proteins 0.000 description 3
- 102000004190 Enzymes Human genes 0.000 description 3
- 229960002685 biotin Drugs 0.000 description 3
- 235000020958 biotin Nutrition 0.000 description 3
- 239000011616 biotin Substances 0.000 description 3
- 239000011248 coating agent Substances 0.000 description 3
- 238000000576 coating method Methods 0.000 description 3
- 230000000295 complement effect Effects 0.000 description 3
- 238000003364 immunohistochemistry Methods 0.000 description 3
- 238000007403 mPCR Methods 0.000 description 3
- 238000001840 matrix-assisted laser desorption--ionisation time-of-flight mass spectrometry Methods 0.000 description 3
- 239000000203 mixture Substances 0.000 description 3
- 102000054765 polymorphisms of proteins Human genes 0.000 description 3
- 238000007400 DNA extraction Methods 0.000 description 2
- 230000033616 DNA repair Effects 0.000 description 2
- LYCAIKOWRPUZTN-UHFFFAOYSA-N Ethylene glycol Chemical compound OCCO LYCAIKOWRPUZTN-UHFFFAOYSA-N 0.000 description 2
- 239000007987 MES buffer Substances 0.000 description 2
- 206010028980 Neoplasm Diseases 0.000 description 2
- 238000012408 PCR amplification Methods 0.000 description 2
- 238000002512 chemotherapy Methods 0.000 description 2
- RGWHQCVHVJXOKC-SHYZEUOFSA-J dCTP(4-) Chemical compound O=C1N=C(N)C=CN1[C@@H]1O[C@H](COP([O-])(=O)OP([O-])(=O)OP([O-])([O-])=O)[C@@H](O)C1 RGWHQCVHVJXOKC-SHYZEUOFSA-J 0.000 description 2
- 239000012154 double-distilled water Substances 0.000 description 2
- 239000003814 drug Substances 0.000 description 2
- 238000001976 enzyme digestion Methods 0.000 description 2
- 238000009472 formulation Methods 0.000 description 2
- 239000012634 fragment Substances 0.000 description 2
- 238000002156 mixing Methods 0.000 description 2
- 238000010369 molecular cloning Methods 0.000 description 2
- 239000013642 negative control Substances 0.000 description 2
- 102000039446 nucleic acids Human genes 0.000 description 2
- 108020004707 nucleic acids Proteins 0.000 description 2
- 150000007523 nucleic acids Chemical class 0.000 description 2
- BASFCYQUMIYNBI-UHFFFAOYSA-N platinum Chemical compound [Pt] BASFCYQUMIYNBI-UHFFFAOYSA-N 0.000 description 2
- 238000004393 prognosis Methods 0.000 description 2
- 102000004169 proteins and genes Human genes 0.000 description 2
- 230000008439 repair process Effects 0.000 description 2
- 108091008146 restriction endonucleases Proteins 0.000 description 2
- 238000007894 restriction fragment length polymorphism technique Methods 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- 239000000725 suspension Substances 0.000 description 2
- VLEIUWBSEKKKFX-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;2-[2-[bis(carboxymethyl)amino]ethyl-(carboxymethyl)amino]acetic acid Chemical compound OCC(N)(CO)CO.OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O VLEIUWBSEKKKFX-UHFFFAOYSA-N 0.000 description 1
- 108700028369 Alleles Proteins 0.000 description 1
- 206010059866 Drug resistance Diseases 0.000 description 1
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 1
- 229920001213 Polysorbate 20 Polymers 0.000 description 1
- ATJFFYVFTNAWJD-UHFFFAOYSA-N Tin Chemical compound [Sn] ATJFFYVFTNAWJD-UHFFFAOYSA-N 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- 230000000259 anti-tumor effect Effects 0.000 description 1
- 239000002246 antineoplastic agent Substances 0.000 description 1
- 229940041181 antineoplastic drug Drugs 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 238000007405 data analysis Methods 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 238000001962 electrophoresis Methods 0.000 description 1
- 230000001036 exonucleolytic effect Effects 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 210000003917 human chromosome Anatomy 0.000 description 1
- WGCNASOHLSPBMP-UHFFFAOYSA-N hydroxyacetaldehyde Natural products OCC=O WGCNASOHLSPBMP-UHFFFAOYSA-N 0.000 description 1
- 230000002055 immunohistochemical effect Effects 0.000 description 1
- 229920002521 macromolecule Polymers 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 230000020520 nucleotide-excision repair Effects 0.000 description 1
- 230000002688 persistence Effects 0.000 description 1
- 229910052697 platinum Inorganic materials 0.000 description 1
- 238000003752 polymerase chain reaction Methods 0.000 description 1
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 description 1
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 238000004451 qualitative analysis Methods 0.000 description 1
- 238000004445 quantitative analysis Methods 0.000 description 1
- 238000003753 real-time PCR Methods 0.000 description 1
- 239000007790 solid phase Substances 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 239000013589 supplement Substances 0.000 description 1
- 230000001360 synchronised effect Effects 0.000 description 1
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses an ERCC1 gene mutation detection liquid chip and a specific primer, wherein the liquid chip mainly comprises: each ASPE primer consists of a tag sequence at the 5 'end and a specific primer sequence at the 3' end aiming at a target gene mutation site, wherein the specific primer sequences are as follows: SEQ ID NO.11 and SEQ ID NO.12 for the T354C site, SEQ ID NO.13 and SEQ ID NO.14 for the G262T site, SEQ ID NO.15 and SEQ ID NO.16 for the G197T site, SEQ ID NO.17 and SEQ ID NO.18 for the T931G site, and SEQ ID NO.19 and SEQ ID NO.20 for the A1187G site; microspheres coated with anti-tag sequences; and (3) an amplification primer. The coincidence rate of the detection result of the detection liquid phase chip provided by the invention and a sequencing method is up to 100 percent, and the independent and parallel detection of wild types and mutant types of a plurality of mutation sites can be realized.
Description
Technical Field
The invention belongs to the field of molecular biology, relates to medicine and biotechnology, and particularly relates to an ERCC1 gene mutation detection specific primer and a liquid chip kit.
Background
The nucleotide Excision Repair Cross-Complementing group1 (precision Repair Cross completion group1, ERCC1) is located on human chromosome 19q13.2, has the length of 15kb, encodes a protein consisting of 297 amino acids, is an important member in an exonucleolytic Repair family, is a key member of an NER system, and is involved in DNA chain cutting and damage recognition. Meanwhile, the ERCC1 gene polymorphism is not only related to the chemotherapy curative effect of the tumor, but also has an influence on prognosis. In the process of treating tumors, the enhancement of DNA repair capacity influences the exertion of the curative effect of anti-tumor drugs and is easy to generate drug resistance; the reduced DNA repair capacity contributes to the persistence of the function of the platinum-DNA adduct with antitumor activity, thus having a good impact on prognosis. A plurality of documents report that the SNP of ERCC1 has a significant relation with the chemotherapy sensitivity of platinum drugs.
At present, the detection technology of ERCC1 gene polymorphism mainly comprises the following steps: real-time PCR based on PCR technology, PCR-RFLP technology, matrix assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) technology and immunohistochemical technology (IHC). The PCR-RFLP method is based on the change of restriction enzyme recognition site caused by gene mutation, such as site loss or new site generation, a certain specific segment is amplified through PCR, the amplified product is cut by restriction enzyme, and the size of the segment is observed through electrophoresis. The matrix-assisted laser desorption ionization time-of-flight mass spectrometry technology is a soft ionization technology, has strong and mature functions in the detection of protein and other biological macromolecules, but in the field of nucleic acid detection, the detection is limited to a certain extent due to the particularity of nucleic acid molecules. Although immunohistochemistry has the advantages of specificity, strong sensitivity, simple operation and the like, the antibody use, the operation steps, the result judgment and the like of immunohistochemistry have no unified standard, thereby influencing the clinical popularization and application of immunohistochemistry.
The liquid phase chip, also known as flow fluorescence technology and suspension array, has the advantages of fast detection speed, high result accuracy, simple operation, powerful functions, low cost, etc. However, in order to realize gene mutation detection by using a liquid chip, the detection result has many parameters, including the design of primers, the combination with tag sequences, the cross reaction with PCR primers, the cross reaction with multiple sequences at other sites, the coordination of Tm values of the whole detection platform, and the like, and when designing a liquid chip for detecting the target gene mutation, the key consideration is that the liquid chip can be used for carrying out detection independently and also can be used for carrying out synchronous detection with other sites. At present, the development of an ERCC1 gene mutation detection liquid chip kit which can realize independent detection and parallel detection is urgently needed.
Disclosure of Invention
One of the purposes of the invention is to provide an ERCC1 gene mutation detection liquid chip kit which can be used for detecting wild types and mutant types of five common genotypes of an ERCC1 gene, namely T354C, G262T, G197T, T931G and A1187G independently or in parallel.
The technical scheme for achieving the purpose is as follows.
A liquid chip kit for detecting ERCC1 gene mutation comprises:
(A) wild type and mutant ASPE primer pairs designed for different mutation sites of ERCC1 gene, respectively: each ASPE primer consists of a tag sequence at the 5 'end and a specific primer sequence at the 3' end aiming at the mutation site of the target gene, wherein the specific primer sequence is selected from at least one pair of the following: SEQ ID NO.11 and SEQ ID NO.12 for the T354C site, SEQ ID NO.13 and SEQ ID NO.14 for the G262T site, SEQ ID NO.15 and SEQ ID NO.16 for the G197T site, SEQ ID NO.17 and SEQ ID NO.18 for the T931G site, and SEQ ID NO.19 and SEQ ID NO.20 for the A1187G site; the tag sequence is selected from SEQ ID NO.1-SEQ ID NO. 10;
(B) microspheres coated by the anti-tag sequence and having different color codes, wherein a spacer arm sequence is further arranged between the anti-tag sequence and the microspheres; the anti-tag sequence is selected from SEQ ID NO.21-SEQ ID NO.30, and the anti-tag sequence can be complementarily paired with the tag sequence selected in the step (A) correspondingly;
(C) a primer for amplifying a target sequence to be detected having a mutation site corresponding to the ASPE primer.
In some of these embodiments, the amplification primers are selected from at least one of the following pairs: SEQ ID NO.31 and SEQ ID NO.32 for the T354C site, SEQ ID NO.33 and SEQ ID NO.34 for the G262T site, SEQ ID NO.35 and SEQ ID NO.36 for the G197T site, SEQ ID NO.37 and SEQ ID NO.38 for the T931G site, and SEQ ID NO.39 and SEQ ID NO.40 for the A1187G site.
In some of these embodiments, the ASPE primer pair is selected from at least one of the following pairs: a sequence consisting of SEQ ID NO.1 and SEQ ID NO.11 and a sequence consisting of SEQ ID NO.2 and SEQ ID NO.12 directed to position T354C; the sequence consisting of SEQ ID NO.3 and SEQ ID NO.13 and the sequence consisting of SEQ ID NO.4 and SEQ ID NO.14 for position G262T; a sequence consisting of SEQ ID NO.5 and SEQ ID NO.15 and a sequence consisting of SEQ ID NO.6 and SEQ ID NO.16 for the G197T site; a sequence consisting of SEQ ID NO.7 and SEQ ID NO.17 and a sequence consisting of SEQ ID NO.8 and SEQ ID NO.18 for the G197T site; and a sequence consisting of SEQ ID NO.9 and SEQ ID NO.19 and a sequence consisting of SEQ ID NO.10 and SEQ ID NO.20 to the G197T site.
Another purpose of the invention is to provide a specific primer for ERCC1 gene mutation detection.
The technical scheme for realizing the purpose is as follows:
specific primers for detecting ERCC1 gene mutation are selected from at least one pair of the following: SEQ ID NO.11 and SEQ ID NO.12 for the T354C site, SEQ ID NO.13 and SEQ ID NO.14 for the G262T site, SEQ ID NO.15 and SEQ ID NO.16 for the G197T site, SEQ ID NO.17 and SEQ ID NO.18 for the T931G site, and SEQ ID NO.19 and SEQ ID NO.20 for the A1187G site.
The main advantages of the invention are:
1. the coincidence rate of the detection result of the ERCC1 gene mutation detection liquid chip kit provided by the invention and a sequencing method is up to 100%, and the time required by detection is far shorter than that of a common sequencing technology, thereby being particularly in line with the requirements of practical application. The prepared ERCC1 gene mutation detection liquid phase chip has very good signal-to-noise ratio, no cross reaction exists between the designed probe and the anti-tag sequence basically, a detection platform with uniform detection parameters is constructed by selecting the specific primer, the tag sequence and the anti-tag sequence, the cross reaction can be avoided, and the single and parallel detection of a plurality of mutation sites can be realized.
2. The invention selects the optimal combination from a plurality of specific primers through the design experience accumulated by the inventor for a long time and a large amount of experimental operations, and the ASPE primer specific primer designed by the invention can sensitively and specifically identify the mutation site detected by a target and accurately distinguish the genotypes of various types; in the same reaction system, cross reaction basically does not exist among different specific primers and between the specific primers and a PCR amplification product for non-target detection, the detection specificity is good, and the cross reaction rate is lower than 3%; besides the mutation condition of a single site, the mutation conditions of a plurality of mutation sites can be simultaneously detected in parallel, and the detection effects are consistent.
3. The detection method has simple steps, 5 mutant sites can be detected by one-step PCR to complete the amplification of 5 target sequences containing the mutant sites, and a plurality of uncertain factors existing in the complex operation processes of repeated PCR and the like are avoided, so that the detection accuracy can be greatly improved, and the accurate and simultaneously qualitative and quantitative analysis characteristics are embodied.
4. The invention not only overcomes the defects of low sensitivity and poor repeatability of detection results of the traditional solid phase chip kit, but also improves the existing liquid phase chip technology, so that the prepared microspheres can be suitable for different detection items and have strong expansibility. The detected fluorescence signal value is greatly improved, so that the detection sensitivity is further improved, the signal-to-noise ratio is enhanced, and the detection result is more accurate and reliable.
Detailed Description
In order to clearly understand the technical contents of the present invention, the following examples are given in detail. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Experimental procedures without specific conditions noted in the following examples, generally followed by conventional conditions, such as Sambrook et al, molecular cloning: the conditions described in the Laboratory Manual (New York: Cold spring Harbor Laboratory Press,1989), or according to the manufacturer's recommendations. The various chemicals used in the examples are commercially available.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used herein, the term "and/or" includes any and all combinations of one or more of the associated listed items.
Example 1
The ERCC1 gene mutation detection liquid chip kit of the embodiment mainly comprises:
third, ASPE primer
Specific primer sequences are designed aiming at wild types and mutant types of five common genotypes of an ERCC1 gene, namely T354C, G262T, G197T, T931G and A1187G respectively. The ASPE primer consists of a tag sequence and a specific primer sequence. The ASPE primer sequences are shown in the following table:
TABLE 1 ASPE primer sequence (tag sequence + specific primer sequence) of ERCC1 Gene
Each ASPE primer comprises two parts, the 5 'end is a specific tag sequence aiming at an anti-tag sequence on the corresponding microsphere, and the 3' end is a mutant type or wild type specific primer segment (as shown in the table 1). Wherein, the base marked by the frame is used as the wild type and mutant base of the target detection mutation site, and all ASPE primers are synthesized by Shanghai biological engineering technology service Limited. Each primer after synthesis was prepared into 100pmol/mL stock solution with 10mmol/L Tris Buffer. Two, anti-tag sequence coated microsphere
Selecting tag sequences according to the designed ASPE specific primer fragments, and reducing secondary structures possibly formed between anti-tag sequences of the microspheres and tag and the ASPE specific primer fragments to the maximum extent, wherein the numbers of the selected 10 microspheres and the corresponding anti-tag sequences on the microspheres are shown in Table 2:
TABLE 2 numbering of microspheres and corresponding anti-tag sequences on microspheres
Selected 10 microspheres were purchased from Luminex, usa and were coated with the anti-tag sequence. 5-10T spacer arm sequences are connected between the anti-tag sequences and the microspheres, namely a 5-10T spacer arm sequence is added in front of each anti-tag sequence, and the anti-tag sequences are synthesized by Shanghai Bioengineering technology service GmbH. The synthetic anti-tag sequence was treated with sterile ddH2O is prepared into a 100nmol/ml stock solution. The spacer arm is used for spacing the anti-tag from the surface of the microsphere or placing the anti-tag in a hydrophilic environmentAnd (4) columns. By arranging a spacer arm sequence with proper length between the anti-tag sequence and the microsphere, the steric hindrance can be reduced, and the efficiency of hybridization reaction and the specificity of the hybridization reaction are improved. Common spacer sequences include poly-dT, i.e., poly (dT), oligo-tetrapolyethylene glycol, and (CH2) n spacers (n.gtoreq.3), such as (CH2)12, (CH2)18, and the like. In addition, if a poly (dA) interference is present, poly (TTG) may also be used as a spacer. The spacer arm of the invention is preferably 5-10T, and the process of coating the microspheres is as follows:
respectively taking 5 × 106Each of the above numbered carboxylated microspheres (from Luminex) was suspended in 50ul of 0.1mol/L MES solution (pH4.5) and 10ul of synthetic anti-tag molecule (100nmol/ml) was added. 10ng/ml of working solution of EDC (N- (3-methylenepropyl-N-ethylenecarboxyl) (available from Pierce Chemical Co.) 2.5ul of working solution of EDC was added to the microsphere suspension, incubated at constant temperature for 30 minutes, 2.5ul of working solution of EDC was added, incubated at constant temperature for 30 minutes, washed once with 0.02% Tween-20 and then 0.1% SDS after the completion of the reaction, and the washed microspheres coated with the anti-tag sequence were resuspended in 100ul of Tris-EDTA solution [10mmol/LTris (pH8.0) ]]In 1mmol/L EDTA, storing at 2-8 deg.c in dark.
First, primer for amplifying target sequence containing mutation site
Aiming at five common genotypes of the ERCC1 gene, namely T354C, G262T, G197T, T931G and A1187G, amplification primer pairs (shown in Table 3) are designed, and 5 target sequences containing 5 mutation sites are amplified in parallel at one time.
TABLE 3 primers for amplifying target sequences with mutation sites
All primers were synthesized by Shanghai Biotechnology engineering services, Inc. Each primer after synthesis was prepared into 100pmol/mL stock solution with 10mmol/L Tris Buffer.
Example 2 detection of samples Using the ERCC1 Gene mutation detection liquid chip kit described in example 1
The formulations of the various solutions are as follows:
50mM MES buffer (pH5.0) formulation (250 ml):
2 XTM hybridization buffer
After filtration, the mixture was stored at 4 ℃.
The ExoSAP-IT kit was purchased from U.S. USB.
Biotin-labeled dCTP was purchased from Shanghai Biotechnology engineering services, Inc.
Firstly, DNA extraction of a sample:
the DNA to be detected is obtained by referring to the related method of DNA extraction in molecular cloning.
Second, PCR amplification of the sample to be tested
5 target sequences respectively containing five target detection mutation sites of T354C, G262T, G197T, T931G and A1187G of ERCC1 gene are amplified in one step by using 5 pairs of primers, the sizes of products are 347bp, 326bp, 301bp, 332bp and 322bp respectively, and the primer sequences (SEQ ID NO.31-40) are shown in the table 3.
Firstly, preparing a multiplex PCR primer working solution: 50ul of the primer stock solutions of SEQ ID NO.31-40 were taken out and placed in 1.5mL microcentrifuge tubes, and 10mmol/L Tris buffer r was used to prepare multiplex PCR primer working solutions each having a final concentration of 10 pmol/mL. The multiplex PCR reaction system is as follows:
the PCR amplification procedure was: 3min at 95 ℃; 30 cycles of 94 ℃ for 30s, 56 ℃ for 30s, 72 ℃ for 40 s; 10min at 72 ℃; storing at 4 deg.C for use.
Thirdly, enzyme digestion treatment of PCR product
1. Taking 7.5ul of the product after PCR reaction, adding 1ul of 10 XSAP buffer solution, 1ul of SAP enzyme and 0.5ul of Exo-I enzyme;
incubate at 2.37 ℃ for 15min, incubate at 80 ℃ for 15min, inactivate excess enzyme. The product after enzyme digestion is directly used for the subsequent ASPE primer extension reaction.
Site-specific primer extension reaction (ASPE)
The primer extension reaction was performed using the ASPE primer designed in example 1, and biotin-labeled dCTP was incorporated during the reaction, thereby allowing the product after the reaction to be labeled with a plurality of biotin.
Firstly, preparing mixed ASPE primer working solution: respectively taking 10ul of wild type and mutant ASPE primer stock solution corresponding to the gene to be detected, adding 10mmol/L Tris Buffer to supplement to 200ul, and uniformly mixing to obtain the ASPE mixed primer working solution. The system for the ASPE reaction is as follows:
the reaction procedure is as follows: 2min at 96 ℃; 30 cycles of 94 ℃ for 30s, 54 ℃ for 1min, 72 ℃ for 2 min; storing at 4 deg.C for use.
Fifthly, hybridization reaction
1. Based on the designed ASPE primers, 10 corresponding coated microspheres (as described in example 1) were selected per group, each at a concentration of 2.5X 105Per ml;
2. 1ul of microspheres with each number are respectively taken and put in a 1.5ml microcentrifuge tube;
3. centrifuging the microspheres at a speed of more than or equal to 10000g for 1-2 min;
4. discarding the supernatant, resuspending the microspheres in 100ul of 2 XTM hybridization buffer, and mixing by vortex;
5. suspending 25ul of the above microspheresAdd 25ul ddH to control wells in corresponding wells of a 96-well filter plate2O；
6. Taking 5-25ul ASPE reaction solution into corresponding holes, and using ddH2O is complemented to 50 ul;
7. wrapping a 96-well plate with tin foil paper to avoid light, and incubating and hybridizing at 95 ℃ for 60s and 37 ℃ for 15 min;
8. centrifuging the hybridized microspheres for 2-5 min at a speed of more than or equal to 3000 g;
9. removing supernatant, and suspending the microspheres in 75ul of 1 XTM hybridization buffer;
10. centrifuging the microspheres at a speed of more than or equal to 3000g for 2-5 min;
11. resuspend the microspheres in 75ul of 1 XTM hybridization buffer, add 15ul of streptavidin-phycoerythrin (SA-PE) at 10 ug/ml;
incubate at 12.37 ℃ for 15min and detect on Luminex instruments.
Sixthly, result detection and data analysis
And detecting the product after reaction by a Luminex series analytical instrument. The results of the measurements are shown in tables 4, 5 and 6. The following requirements are placed on the fluorescence values (MFI) and data processing:
1. each locus needs to have at least one allele MFI greater than 300 and greater than 10 XPCR negative control MFI;
2. NET MFI-sample MFI-PCR negative control MFI (NET MFI less than 0 indicated as 0);
3. the mutation ratio is calculated according to the following formula from the data satisfying the above two conditions:
mutation ratio (mutant NET MFI + wild-type NET MFI)
4. A threshold (cut-off value) is empirically determined for the mutation ratio at each detection site to classify wild-type homozygotes, heterozygotes, and mutant homozygotes.
By using the method to detect the SNP locus of the ERCC1 gene in 20 samples, the experimental data meet the requirements, and therefore, the mutation ratio of the ERCC1 gene can be calculated. The threshold value (cut-off value) is set as follows: the mutation ratio ranging from 0% to 20% is regarded as wild type homozygote; 30% -70% are considered heterozygotes; 80% -100% are considered variant homozygotes. The results of the sequencing method detection and the liquid phase chip are compared, and the coincidence rate of the detection results of the typing method provided by the invention is calculated. The method has the advantage that the coincidence rate of the ERCC1 genotype detection result and the sequencing result of 20 samples reaches 100%. Therefore, the ERCC1 gene SNP detection liquid-phase chip provided by the invention can accurately detect the SNP type of the ERCC1, and the result is stable and reliable.
TABLE 4 sample test results (MFI)
TABLE 5 sample ERCC1 Gene mutation ratio (%)
| Sample number | T354C | G262T | G197T | T931G | A1187G |
| 1 | 2％ | 1％ | 1％ | 2％ | 1％ |
| 2 | 3％ | 2％ | 3％ | 2％ | 1％ |
| 3 | 2％ | 2％ | 2％ | 2％ | 2％ |
| 4 | 53％ | 1％ | 2％ | 2％ | 2％ |
| 5 | 2％ | 1％ | 1％ | 2％ | 2％ |
| 6 | 2％ | 98％ | 2％ | 2％ | 1％ |
| 7 | 2％ | 1％ | 2％ | 2％ | 1％ |
| 8 | 3％ | 1％ | 2％ | 1％ | 52％ |
| 9 | 2％ | 2％ | 2％ | 2％ | 1％ |
| 10 | 2％ | 1％ | 2％ | 1％ | 2％ |
| 11 | 1％ | 1％ | 98％ | 2％ | 2％ |
| 12 | 2％ | 1％ | 2％ | 2％ | 3％ |
| 13 | 2％ | 3％ | 1％ | 1％ | 1％ |
| 14 | 2％ | 2％ | 2％ | 3％ | 2％ |
| 15 | 1％ | 2％ | 2％ | 1％ | 2％ |
| 16 | 2％ | 2％ | 2％ | 1％ | 2％ |
| 17 | 2％ | 2％ | 1％ | 2％ | 2％ |
| 18 | 1％ | 2％ | 2％ | 93％ | 2％ |
| 19 | 2％ | 2％ | 2％ | 3％ | 2％ |
| 20 | 1％ | 1％ | 2％ | 2％ | 2％ |
TABLE 6 results of analysis of ERCC1 Gene mutation types
| Sample number | Liquid phase chip detection result | Sequencing results |
| 1 | Wild type | Wild type |
| 2 | Wild type | Wild type |
| 3 | Wild type | Wild type |
| 4 | 354TC | 354TC |
| 5 | Wild type | Wild type |
| 6 | 262TT | 262TT |
| 7 | Wild type | Wild type |
| 8 | 1187AG | 1187AG |
| 9 | Wild type | Wild type |
| 10 | Wild type | Wild type |
| 11 | 197TT | 197TT |
| 12 | Wild type | Wild type |
| 13 | Wild type | Wild type |
| 14 | Wild type | Wild type |
| 15 | Wild type | Wild type |
| 16 | Wild type | Wild type |
| 17 | Wild type | Wild type |
| 18 | 931GG | 931GG |
| 19 | Wild type | Wild type |
| 20 | Wild type | Wild type |
Example 3 detection of SNP site of ERCC1 Gene by different liquid chip kits of ASPE primers
Design of liquid phase chip preparation (selection of Tag sequence and Anti-Tag sequence)
Taking ERCC1 gene T354C, G262T, G197T and T931G site mutation detection liquid phase chip as an example, specific primer sequences at the 3 'end of ASPE primers are designed aiming at wild type and mutant type of T354C, G262T, G197T and T931G respectively, Tag sequences at the 5' end of the ASPE primers are selected from SEQ ID NO.1-SEQ ID NO.10, and correspondingly, anti-Tag sequences which are coated on microspheres and are complementary to corresponding Tag sequences are selected from SEQ ID NO.21-SEQ ID NO. 30. The specific design is shown in the following table (table 7). The synthesis of ASPE primers, the coating of microspheres with anti-tag sequences, the amplification of primers, the detection method and the like are as described in examples 1 and 2.
TABLE 7 design of liquid phase chip preparation
Third, sample detection
The liquid chip kit prepared by the design is adopted to detect the samples 21-40 according to the detection process and the method described in the embodiment 2, and the detection results are as follows:
TABLE 8 sample ERCC1 Gene T354C test results and analysis of Gene polymorphisms
Table 9 sample ERCC1 Gene G262T test results and Gene polymorphism analysis
TABLE 10 samples ERCC1 Gene G197T test results and analysis of Gene polymorphisms
Table 11 sample ERCC1 Gene T931G test results and Gene polymorphism analysis
As can be seen from the above examples, in other liquid phase chips for different mutation sites, the ASPE primers use different tag sequences, and the results are still stable and reliable. When the tag sequence in the embodiment 1 is selected as the ASPE primer to match with the specific primer sequence, the effect is better (the signal to noise ratio is better), and the test group1, the test group 5, the test group 7 and the test group 11 are referred to in the embodiment. Other different tag sequences are matched with the specific primer sequences, the results are the same as those of the example 2 and the example, and specific data are omitted.
Example 4 selection of primer sequences specific for the detection of ERCC1 Gene mutations
Design of liquid phase chip preparation (selection of wild type and mutant type specific primer sequences)
Taking the polymorphic site detection liquid phase chip of ERCC1 gene T354C and G262T as an example, taking the forward or reverse complementary sequence of the target sequence of the mutation site as a template, designing specific primer sequences at the 3' end of ASPE primers aiming at the wild type and the mutant type of T354C and G262T respectively, wherein the specific primer sequences comprise the preferred specific primer sequence in the embodiment 1 of the invention and 2 selected alternative specific primer sequences, and the sequences are shown in Table 12. Wherein, the in-frame base is a polymorphic site.
TABLE 12 specific primer sequences
Taking the liquid chip kit for detecting the polymorphic sites of ERCC1 genes T354C and G262T as an example, different specific primer sequences are selected for T354C and G262T, and the tag sequence at the 5' end of the ASPE primer is fixed as the optimal effect sequence in example 1, and an anti-tag sequence corresponding to the optimal effect sequence is selected, and the specific design is shown in the following table (Table 13). The synthesis of ASPE primers, the coating of microspheres with anti-tag sequences, the amplification of primers, the detection method and the like are as described in examples 1 and 2.
TABLE 13 design of liquid phase chip preparation
Second, sample detection
The liquid chip kit prepared by the design is adopted to detect the samples 41-60 according to the detection process and method described in the embodiment 2, and the detection results are as follows:
TABLE 14 samples ERCC1 Gene T354C test results and analysis of Gene polymorphisms
Table 15 sample ERCC1 Gene G262T test results and Gene polymorphism analysis
As can be seen from this example, when the specific primer sequence in example 1 is selected as the ASPE primer to match with the tag sequence, the effect is better (the signal to noise ratio is better), see test group 13 and test group 16 in this example. Other different specific primer sequences derived from the forward or reverse complementary sequence of the target detection site are matched with the tag sequence, which is the same as the results of the embodiment 2 and the embodiment, i.e., the matching effect of the specific primer sequence and the different tag sequences in the embodiment 2 is still better, and the specific data are omitted.
Other specific primer sequences aiming at different SNP sites are matched with tag sequences, and the results are the same as those of the embodiment 2 and the embodiment, namely, the specific primer selected in the embodiment 1 has better signal to noise ratio, better detection effect and specific data are omitted.
The technical features of the embodiments described above may be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the embodiments described above are not described, but should be considered as being within the scope of the present specification as long as there is no contradiction between the combinations of the technical features.
The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the present invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.
Claims (6)
1. A liquid chip kit for detecting ERCC1 gene mutation is characterized by comprising:
(A) wild type and mutant ASPE primer pairs designed for different mutation sites of ERCC1 gene, respectively: each ASPE primer consists of a tag sequence at the 5 'end and a specific primer sequence at the 3' end aiming at the mutation site of the target gene, wherein the specific primer sequence is selected from at least one pair of the following: SEQ ID NO.11 and SEQ ID NO.12 for the T354C site, SEQ ID NO.13 and SEQ ID NO.14 for the G262T site, SEQ ID NO.15 and SEQ ID NO.16 for the G197T site, SEQ ID NO.17 and SEQ ID NO.18 for the T931G site, and SEQ ID NO.19 and SEQ ID NO.20 for the A1187G site; the tag sequence is selected from SEQ ID NO.1-SEQ ID NO. 10;
(B) microspheres coated by the anti-tag sequence and having different color codes, wherein a spacer arm sequence is further arranged between the anti-tag sequence and the microspheres; the anti-tag sequence is selected from SEQ ID NO.21-SEQ ID NO.30, and the anti-tag sequence can be complementarily paired with the tag sequence selected in the step (A) correspondingly;
(C) a primer for amplifying a target sequence to be detected having a mutation site corresponding to the ASPE primer.
2. The ERCC1 gene mutation detection liquid chip kit as claimed in claim 1, wherein the amplification primers are selected from at least one pair of: SEQ ID NO.31 and SEQ ID NO.32 for the T354C site, SEQ ID NO.33 and SEQ ID NO.34 for the G262T site, SEQ ID NO.35 and SEQ ID NO.36 for the G197T site, SEQ ID NO.37 and SEQ ID NO.38 for the T931G site, and SEQ ID NO.39 and SEQ ID NO.40 for the A1187G site.
3. The ERCC1 gene mutation detection liquid chip kit as claimed in claim 1, wherein the ASPE primers are selected from at least one pair of the following: a sequence consisting of SEQ ID NO.1 and SEQ ID NO.11 and a sequence consisting of SEQ ID NO.2 and SEQ ID NO.12 for the T354C site; the sequence consisting of SEQ ID NO.3 and SEQ ID NO.13 and the sequence consisting of SEQ ID NO.4 and SEQ ID NO.14 for position G262T; a sequence consisting of SEQ ID NO.5 and SEQ ID NO.15 and a sequence consisting of SEQ ID NO.6 and SEQ ID NO.16 for the G197T site; a sequence consisting of SEQ ID NO.7 and SEQ ID NO.17 and a sequence consisting of SEQ ID NO.8 and SEQ ID NO.18 for the G197T site; and a sequence consisting of SEQ ID NO.9 and SEQ ID NO.19 and a sequence consisting of SEQ ID NO.10 and SEQ ID NO.20 to the G197T site.
4. The ERCC1 gene mutation detection liquid chip kit of claim 1,
(A) the ASPE primers are: a sequence consisting of SEQ ID NO.1 and SEQ ID NO.11 and a sequence consisting of SEQ ID NO.2 and SEQ ID NO.12 for the T354C site; the sequence consisting of SEQ ID NO.3 and SEQ ID NO.13 and the sequence consisting of SEQ ID NO.4 and SEQ ID NO.14 for position G262T; a sequence consisting of SEQ ID NO.5 and SEQ ID NO.15 and a sequence consisting of SEQ ID NO.6 and SEQ ID NO.16 for the G197T site; a sequence consisting of SEQ ID NO.7 and SEQ ID NO.17 and a sequence consisting of SEQ ID NO.8 and SEQ ID NO.18 for the G197T site; and a sequence consisting of SEQ ID NO.9 and SEQ ID NO.19 and a sequence consisting of SEQ ID NO.10 and SEQ ID NO.20 directed to position G197T;
(B) microspheres coated by the anti-tag sequence and having different color codes, wherein a spacer arm sequence is further arranged between the anti-tag sequence and the microspheres; the anti-tag sequence is selected from SEQ ID NO.21-SEQ ID NO.30, and the anti-tag sequence can be complementarily paired with the tag sequence selected in the step (A) correspondingly;
(C) a primer for amplifying a target sequence to be detected, which has a mutation site corresponding to the ASPE primer, wherein the amplification primer is: SEQ ID NO.31 and SEQ ID NO.32 for the T354C site, SEQ ID NO.33 and SEQ ID NO.34 for the G262T site, SEQ ID NO.35 and SEQ ID NO.36 for the G197T site, SEQ ID NO.37 and SEQ ID NO.38 for the T931G site, and SEQ ID NO.39 and SEQ ID NO.40 for the A1187G site.
5. The ERCC1 gene mutation detection liquid chip kit of any one of claims 1-4, wherein the spacer arm is 5-10T.
6. Specific primers for detecting ERCC1 gene mutation, wherein the specific primer sequence is selected from at least one pair of the following: SEQ ID NO.11 and SEQ ID NO.12 for the T354C site, SEQ ID NO.13 and SEQ ID NO.14 for the G262T site, SEQ ID NO.15 and SEQ ID NO.16 for the G197T site, SEQ ID NO.17 and SEQ ID NO.18 for the T931G site, and SEQ ID NO.19 and SEQ ID NO.20 for the A1187G site.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510151622.9A CN106148488B (en) | 2015-03-31 | 2015-03-31 | ERCC1 gene mutation detection specific primer and liquid chip kit |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510151622.9A CN106148488B (en) | 2015-03-31 | 2015-03-31 | ERCC1 gene mutation detection specific primer and liquid chip kit |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN106148488A CN106148488A (en) | 2016-11-23 |
| CN106148488B true CN106148488B (en) | 2020-03-31 |
Family
ID=57338686
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201510151622.9A Active CN106148488B (en) | 2015-03-31 | 2015-03-31 | ERCC1 gene mutation detection specific primer and liquid chip kit |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN106148488B (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107227363A (en) * | 2017-07-05 | 2017-10-03 | 上海赛安生物医药科技股份有限公司 | ERCC1 genetic polymorphism detections system and its kit |
Family Cites Families (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2011109440A1 (en) * | 2010-03-01 | 2011-09-09 | Caris Life Sciences Luxembourg Holdings | Biomarkers for theranostics |
| CN202519261U (en) * | 2012-04-17 | 2012-11-07 | 马蓉 | ERCC1 (excision repair cross complementing 1) gene polymorphism and enzyme digestion detection kit |
| CN103451271B (en) * | 2012-05-31 | 2015-02-04 | 益善生物技术股份有限公司 | THADA gene mutation detection specific primer and liquid phase chip |
| CN103310105A (en) * | 2013-06-13 | 2013-09-18 | 浙江加州国际纳米技术研究院绍兴分院 | Method for screening non-small-cell lung cancer curative effect biomarker |
-
2015
- 2015-03-31 CN CN201510151622.9A patent/CN106148488B/en active Active
Also Published As
| Publication number | Publication date |
|---|---|
| CN106148488A (en) | 2016-11-23 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN101805798B (en) | Specific primmer, liquid phase chip and detection method for CYP2E1 (Cytochrome P450 2E1) gene SNP (Single Nucleotide Polymorphism) detection | |
| CN111826426A (en) | Method for detecting molecular marker based on KASP technology | |
| CN101671740B (en) | Method for detecting gene polymorphism of UGT1A1 and liquid phase chip | |
| CN106148488B (en) | ERCC1 gene mutation detection specific primer and liquid chip kit | |
| CN103451271B (en) | THADA gene mutation detection specific primer and liquid phase chip | |
| CN112592995B (en) | Universal primer for detecting target gene expression in transgenic plant and detection method | |
| CN106148486B (en) | XRCC1 gene mutation detection specific primer and liquid chip kit | |
| CN103451267B (en) | TERT detection in Gene Mutation Auele Specific Primer and liquid-phase chip | |
| CN106148487B (en) | ERCC2 gene mutation detection specific primer and liquid chip kit | |
| CN108315396B (en) | Novel method for simply and conveniently detecting SNP | |
| CN103849942B (en) | TOX3 detection in Gene Mutation specific primer and liquid-phase chip | |
| CN103849943B (en) | MC1R detection in Gene Mutation specific primer and liquid-phase chip | |
| CN103451272A (en) | BAT3 gene mutation detection specific primer and liquid phase chip | |
| CN103031367A (en) | VHL (Von Hippel Lindau) genetic mutation detection specific primer and liquid phase chip | |
| CN102952866B (en) | Specific primer and liquid chip for detecting polymorphism of DPYD (dihydropyrimidine dehydrogenase) gene | |
| CN103865987B (en) | C6ORF97 detection in Gene Mutation Auele Specific Primer and liquid-phase chip | |
| CN103571925B (en) | Specific detection primers and detection liquid phase chip for BIM gene mutation | |
| CN103571919A (en) | Specific detection primers and detection liquid phase chip for HNF1B gene mutation | |
| CN102952869B (en) | Specific primer and liquid chip for detecting polymorphism of SLCO1B1 gene | |
| CN103451266B (en) | NKX3.1 gene mutation detection specific primers and liquid chip | |
| CN103849938B (en) | MAP3K1 detection in Gene Mutation specific primer and liquid-phase chip | |
| CN103849940A (en) | Specific primer and liquid phase chip for detecting BARD1 gene mutation | |
| CN103374611B (en) | LIG3 (DNA (deoxyribonucleic acid) Ligase III) gene mutation detection specific primers and liquid chip | |
| CN102952873B (en) | Specific primer and liquid chip for detecting polymorphism of NAT1 (N-acetyltransferase 1) gene | |
| CN103571922B (en) | SLC22A3 detection in Gene Mutation Auele Specific Primer and liquid-phase chip |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| TR01 | Transfer of patent right |
Effective date of registration: 20231116 Address after: 510700, 8th floor, No. 29 Helix 3 Road, Guangzhou International Biological Island, Huangpu District, Guangzhou City, Guangdong Province Patentee after: GUANGZHOU SUREXAM MEDICAL LABORATORY Co.,Ltd. Address before: Five 510663 Guangdong city of Guangzhou province Guangzhou Science City Moon Road No. 80, Guangzhou technology innovation base B, C Patentee before: SUREXAM BIO-TECH Co.,Ltd. |
|
| TR01 | Transfer of patent right |