CN105256057A - Colon cancer microsatellite instability detection kit based on next generation sequencing platform - Google Patents
Colon cancer microsatellite instability detection kit based on next generation sequencing platform Download PDFInfo
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Abstract
The invention discloses a colon cancer microsatellite instability detection kit based on a next generation sequencing platform. The colon cancer microsatellite instability detection kit comprises 4.5-6.5mu l of a primer panel solution, wherein the concentration of solute in the primer panel solution is 45-55 mmole/L; the primer panel solution comprises primers of 10 microsatellite loci; the kit further comprises 8-10mu l of Master Mix and 1.3-1.7ml of non-enzyme water; Master Mix comprises 3-7mu l of 10*LA Taq Buffer and 3-5mu l of 2-4mmol/L dNTPs, and 0.5-1mu l of LA Taq enzyme. By adopting the kit disclosed by the invention, all MSI loci of multiple samples can be simultaneously detected by sequencing once, influence caused by artificial operation can be reduced, and the detection result reliability can be improved.
Description
Technical field
The present invention relates to a kind of colorectal carcinoma microsatellite instability detection kit, especially relate to a kind of detection kit colorectal cancer patients microsatellite instability detected based on two generations order-checking platform.
Background technology
Colorectal cancer incidence rate is in the 3rd of all kinds of cancer morbidity in China, accounts for the 5th of the cancer cause of the death, and after its radical excision, 5 years survival rates are about 50%, and postoperative recurrence and transfer are the major reasons of its death.Early stage colorectal cancer patients can borrow excision usually, once transfer, the method for its treatment is also few, and patient's five year survival rate is also undesirable.Research finds, the pathogeny of genomic unstable and colorectal cancer is closely related, genomic unstable comprises chromosome instability (chromosomalinstability, and microsatellite instability (microsatelliteinstability, MSI) CI).
Micro-satellite refers on gene containing the short and small sequence of DNA repeated or mononucleotide region.In tumour cell, when DNA methylates or transgenation causes mismatch repair gene disappearance, micro-satellite repetitive sequence mispairing (micro-satellite sudden change) can be caused, cause its sequence to shorten or extend, thus cause microsatellite instability (microsatelliteinstability, MSI).According to the instable degree of MSI, high unstable (MSI-H) and low unstable (MSI-L) can be divided into, be called that micro-satellite stablizes (microsatellitestability, MSS) under normal circumstances.
Large quantity research shows, MSI participates in the generation evolution of malignant tumour, occurs closely related with colorectal carcinoma, cancer of the stomach, carcinoma of endometrium etc.There is MSI phenomenon in the colorectal cancer patients of about 15%, wherein typical hereditary nonpolyposis colorectal cancer (hereditarynonpolyposiscolerectalcancer, HNPCC) patient more than 90% is MSI knurl, shows that MSI can be used as the important symbol thing whether detection is HNPCC patient; Compared with the colorectal carcinoma of MSS (namely micro-satellite is stablized), its prognosis of colorectal cancer patients carrying MSI is better, and the two drug reaction is also different, and prompting MSI can be used as the independentpredictor of colorectal cancer prognosis, therefore, MSI detects significant to colorectal cancer patients.
American National institute of oncology (NationalCancerInstitute, NCI) unified standard (i.e. BethesdaGuidelines) that MSI detects has been formulated in the International Workshop meeting once detected by a microsatellite instability for tumour and replication error phenotype, as relating to the reference Panel (i.e. NCIPanel) detecting MSI, it comprises the site (BAT-25, BAT-26) of 2 mononucleotides repetitions and the site (D2S123, D5S346, D17S250) of 32 Nucleotide repetitions.When detecting tumour MSI state accordingly, if 5 STR sites detect that there is MSI in the site of more than 2 or 2, be then that MSI-H, MSI-H Tumors display goes out distinctive clinical pathology phenotype; If only detect, there is MSI in 1 site, be then MSI-L, is MSS without any site change person.But the tumor phenotypes difference of MSI-L and MSS is little, distinguish the tumor phenotypes of MSI-L and MSS, need to select when more multidigit point detects and could realize.
Second time in 2002 is correlated with in special meeting, dispute has been there is in the reference Panel for the detection MSI adopted before to detect MSI, because the site that 32 Nucleotide that NCIPanel uses repeat has the feature of high polymorphism, must make comparisons by the healthy tissues matched with tumor tissues when detecting and just can obtain a result, make it show lower specificity and sensitivity when detecting and there is the tumour of mis-match repair deficient.Therefore adopt NCIPanel to detect MSI to have some limitations, conference suggestion increases mononucleotide repetition site and detects MSI to improve the sensitivity detected.
The drug metabolism enzyme of up-to-date issue in 2015 and the middle clear stipulaties of drug target technique of gene detection guide (trying), the detection of MSI can be used for the auxiliary curative effect prediction of fluorouracil drug.Recent studies have found that, compared with the colorectal cancer patients of MSI-L and MSS, accepting PD-1 antibody, (i.e. two kinds of immunologic test points of FDA approval have better clinical responsiveness after suppressing medicines (Nivolumab (MDX1106) and Pembrolizumab)) treatment to MSI-H, show that the MSI level of colorectal cancer patients can be used as one and predicts to the independentpredictor of PD-1/PD-L1 antibody response the colorectal cancer patients colony that screening benefits from the treatment of PD-1 antibody mediated immunity.To the MSI level detecting colorectal cancer patients, can be used for the auxiliary curative effect prediction of immunologic test point inhibitor class medicine.
Therefore, MSI detection system is set up to find high sensitivity and specific being used for detects the focus that the related locus of MSI has become Recent study MSI detection field.
The method of existing detection MSI is mainly:
1) ImmunohistochemistryMethods Methods, the method, only for MLH1, MSH2, MSH6 and PMS2 protein binding in tumour cell, if not in conjunction with any albumen, then proves to there occurs MSI; Although the sensitivity of the method is close to 90%, specificity and repeatability lower, operation more complicated;
2) capillary electrophoresis, the method, mainly for simple repeating structure in the relevant gene order of MSI, can be judged the repetition number of repetition base, thus carry out MSI gene type by fragment analysis; The method use relative maturity, sample size is few, resolving power and level of automation higher, but due to sample size few, thus preparative capacibility is poor; And the little light path that causes of capillary diameter is too short, during with some detection methods (as ultraviolet absorption spectroscopy), sensitivity is lower; In addition, electric osmose can change because of sample composition, and then impact is separated circulation ratio;
3) PCR method, the method, by selected special primer, with self healthy tissues for contrast, carries out pcr amplification to microsatellite locus in vitro, and the product after amplification, through polyacrylamide gel electrophoresis, analyzes the change with or without mobility through radioautograph etc.; The method is detection method conventional at present and has been proved to be the most effective primary dcreening operation means, but PCR method only detects five micro-satellite genes, and product does the method for gel electrophoresis, there is complex operation, the many defects of high in cost of production;
4) multiple fluorescence PCR, increase in the site that the method is advised mainly for NCI, to determine MSI state; Adopt the method to detect micro-satellite, the competition between its primer and interfering factors are comparatively complicated, and amplified production is not easy to stagger, and directly can cause the uncertainty of detected result, therefore the method has very high requirement to primer specificity and primer concentration;
5) direct sequencing, the method is carried out the order-checking of sanger method for the amplified production in each region of MMR gene (MLH1, MSH2, MSH6 and PMS2 etc.) and is determined its MSI state; The sense cycle of the method is longer, and cost is relatively high.
Therefore urgent need develops the MSI detection system gesture of more multidigit point and better sensitivity to meet the active demand of market and medical treatment.
Summary of the invention
The technical problem to be solved in the present invention is, overcome the deficiencies in the prior art, a kind of colorectal carcinoma microsatellite instability detection kit based on two generations order-checking platform is provided, this test kit is used to detect colorectal carcinoma microsatellite instability, detection flux is high, sensitivity is strong, and high specificity, can disposable detection all sites.
The technical scheme that the present invention solves the employing of its technical problem is, based on the colorectal carcinoma microsatellite instability detection kit of two generations order-checking platform, comprise the primer panel solution of 4.5-6.5 μ l (preferably 5 μ l), in described primer panel solution, the concentration of each solute is 45-55mmol/L (preferred 50mmol/L).
Described primer panel comprises the primer of 10 microsatellite locus, its forward primer and reverse primer sequences as follows:
| Site | Forward primer | Reverse primer |
| NR-21 | TCGCTGGCACAGTTCTATTT | TGTTTGTAAACGCGAGTGAC |
| NR-22 | TAATCGAGGCTTGTCAAGGA | GTCTGGAAGTTTTGTCTTGG |
| NR-24 | TGCTGAATTTTACCTCCTGA | AGGTTGGAATGCAATGGCAC |
| NR-27 | CCATGCTTGCAAACCACTGG | TTGGTCATTGCTAGTATTAT |
| BAT-25 | TCGAACTGTCACCTCGGCTTT | ACTTCAAAATGACATTCTG |
| BAT-26 | TGACACTTTTGACTTCAGCC | AACCATTCAACATTTTTAACC |
| MONO-27 | GATTGCAGTGAGCTGAG | TAGCCTTAGAATGTTAGC |
| D2S123 | AAACAGGATGCCTGCCTTTA | GGACCTTCCACCTATGGGAC |
| D5S346 | CGTGAACAGGAGCTTCATCG | ATCTGCAGTCTCTTTGGCCT |
| D17S250 | AGCTTCCAAACTAGTAGAGG | TAAATATATATATGGCCAGCT |
In described test kit, also comprise MasterMix and 1.3-1.7mL (preferred 1.5mL) of 8-10 μ l (preferably 9.5 μ l) without enzyme water;
Described MasterMix comprises the dNTPs of 3 ~ 7 μ l (preferably 5 μ l) 10*LATaqBuffer and 3 ~ 5 μ l (preferably 4 μ l) 2 ~ 4mmol/L, and 0.5 ~ 1 μ lLATaqenzyme.
The multi-PRC reaction condition of the described colorectal carcinoma microsatellite instability detection kit based on two generations order-checking platform is: 93 DEG C ~ 98 DEG C denaturation 2 ~ 3min, then the polymerase chain reaction (PCR) amplification stage is entered: 93 DEG C ~ 96 DEG C sex change 20 ~ 30s, 55 DEG C ~ 65 DEG C annealing 20 ~ 30s, 70 DEG C ~ 75 DEG C extend 20 ~ 45s; Last 70 DEG C ~ 74 DEG C extend 10min, and carry out 10 ~ 12 circulations, and 4 DEG C leave standstill.
Multiplex PCR process carries out negative control experiment simultaneously, and check sample used is normal people's genomic dna.
The detection gene of the described colorectal carcinoma microsatellite instability detection kit based on two generations order-checking platform comprises NR-21, NR-22, NR-24, NR-27, BAT-25, BAT-26, MONO-27, D2S123, D5S346 and D17S250.
Consisting of of two generation sequencing reaction systems: DNA sample multi-PRC reaction, template library construction, upper machine template prepare and enrichment reaction, upper machine sequencing reaction and test data Treatment Analysis.
The present invention MiSeq bis-generation based on Illumina company order-checking platform to the using method of the detection kit of colorectal cancer patients microsatellite instability, comprise carry out 10 marker gene that MSI is correlated with multi-PRC reaction, template library construction, upper machine order-checking and based on sequencing result, the result of micro-satellite status is judged.Particular content is as follows:
(1) select 10 relevant marker gene of MSI and carry out multi-PRC reaction:
1. according to Besthestaguideline, 10 best MSI marker gene site sequences of Sensitivity and Specificity (repeating site and 3 dinucleotides repetition sites comprising 7 mononucleotides) are transferred, in table 1:
A table 110 microsatellite locus information
Table 2 microsatellite locus sequencing primer
| Site | Forward primer (F) | Reverse primer (R) |
| NR-21 | TCGCTGGCACAGTTCTATTT | TGTTTGTAAACGCGAGTGAC |
| NR-22 | TAATCGAGGCTTGTCAAGGA | GTCTGGAAGTTTTGTCTTGG |
| NR-24 | TGCTGAATTTTACCTCCTGA | AGGTTGGAATGCAATGGCAC |
| NR-27 | CCATGCTTGCAAACCACTGG | TTGGTCATTGCTAGTATTAT |
| BAT-25 | TCGAACTGTCACCTCGGCTTT | ACTTCAAAATGACATTCTG |
| BAT-26 | TGACACTTTTGACTTCAGCC | AACCATTCAACATTTTTAACC |
| MONO-27 | GATTGCAGTGAGCTGAG | TAGCCTTAGAATGTTAGC |
| D2S123 | AAACAGGATGCCTGCCTTTA | GGACCTTCCACCTATGGGAC |
| D5S346 | CGTGAACAGGAGCTTCATCG | ATCTGCAGTCTCTTTGGCCT |
| D17S250 | AGCTTCCAAACTAGTAGAGG | TAAATATATATATGGCCAGCT |
2. relevant to these 10 MSI gene locus carries out multiplexed PCR amplification reaction by design primer, and described amplimer is in table 2.
(2) library construction and sequencing reaction is carried out
Template library construction process comprises that DNA interrupts at random, end-filling reparation and purifying, end connect enrichment after sequence measuring joints, template amplification and purifying, purifying, comprises end-filling enzyme mixture 20 μ l, 0.4-1U ligase enzyme, 0.4-1U increases the reagent such as Taq enzyme, 5-10mM sequence measuring joints A and P1,5-10mM sequence measuring joints A and P1 universal primer, 10-15 × Buffer and 2-5 × Ampure purifying magnetic bead in described reaction system.
Above-mentioned sequencing reaction comprises MiSeq sequencing reaction process, its principle adopts reversibility distal edge synthesis limit sequencing reaction, first the universal joint structure library that sequence is known is added at DNA fragmentation two ends, library is loaded on sequence testing chip Flowcell, the known array at two ends, library and the suprabasil Oligo complementary of Flowcell, every bar library fragments all forms one bunch through bridge type pcr amplification, synthesis limit, limit sequencing reaction is adopted during order-checking, namely in base extension process, each circulating reaction can only extend a correct complementary base, base kind is confirmed according to the fluorescent signal that four kinds different, ensure final nucleotide sequence quality, after multiple circulation, complete reading nucleotide sequence.The data results that the reaction of upper machine obtains by sequence screening, splicing and than counterpart method and bioinformatic analysis, the final SNP site information obtaining test sample book.
The present invention adopt two generation sequencing technologies to detect MSI gene locus, namely, first the multi-PRC reaction of 10 marker gene that MSI is correlated with is carried out, then build template library, prepared by a series of upper machine template and obtain sequencing library after enrichment reaction, then the MiSeq bis-generation order-checking platform based on Illumina company checks order to containing MSI mark of correlation gene locus, can the variation information of all microsatellite locus of disposable acquisition, the advantages such as it is high that detected result has flux, and sensitivity is strong, specificity is good.This method successfully can be produced in batches along with kit developing, and working conditions can medelling, and all processes has automatic equipment.In addition, detect the MSI level of colorectal cancer patients with this test kit after, also can be applicable to the colorectal cancer patients crowd screening applicable PD-L1 Antybody therapy.
The microsatellite instability site of MiSeq bis-generation based on Illumina company order-checking platform to the higher colorectal cancer patients of domestic and international sickness rate of the present invention (comprises NR-21, NR-22, NR-24, NR-27, BAT-25, BAT-26, MONO-27, D2S123, D5S346 and D17S250) carry out the test kit that detects, this test kit can all MSI sites of disposable detection, it is more accurate that its result detects than 5 sites, specificity is better, artificial interference factor is little, this method successfully can be produced in batches along with kit developing, working conditions can medelling, all processes has automatic equipment, greatly can improve the detection level of the microsatellite instability that tumour is correlated with.In addition, detect the MSI level of colorectal cancer patients with this test kit after, also can be applicable to screen applicable PD ?the colorectal cancer patients crowd of L1 Antybody therapy.
The present invention is that order-checking of a kind of MiSeq bis-generation based on Illumina company platform carries out colorectal cancer patients microsatellite instability detecting a kind of detection kit used.It is the sequence information according to existing or acquired tumour microsatellite instability related locus, design the primer in these sites, and be made into primer panel, carry out multi-PRC reaction and obtain target sequence, then build template library, prepared and obtain sequencing library after enrichment reaction by a series of upper machine template, the MiSeq bis-generation order-checking platform then based on Illumina company checks order to containing MSI mark of correlation gene locus.By carrying out high-flux sequence to catching the fragment obtained, and based on the Given information of microsatellite instability related locus of Besthestaguideline suggestion, bioinformatics method is utilized to carry out data analysis, obtain the heritable variation information with the nucleotide sequence in colorectal cancer patients microsatellite instability site in sample to be tested, micro-satellite status of final judgement patient, thus anticipation is made to the diagnosis and treatment of colorectal cancer patients.
Test kit of the present invention to the large advantage that colorectal cancer patients microsatellite instability detects is, all MSI sites of multiple sample can be detected by once sequencing simultaneously, reduce the impact that manual operation brings, the reliability of detected result can be improved, detect the MSI level of colorectal cancer patients with this test kit after, also can be applicable to the colorectal cancer patients crowd screening applicable PD-L1 Antybody therapy; Simple operation, is applicable to batch operation; Along with the continuous progress of new-generation sequencing technology and the reduction greatly of order-checking cost, high-throughput genome-based technologies based on DNA greatly can accelerate the process of gene test, while raising nucleic acid sequencing speed, specific inaccuracy and single base order-checking cost can also be reduced; And can all gene orders of disposable detection all MSI related locus, high specificity, can meet that to detect sample size at present large well, the testing requirements such as detection site is many, distribution is wide, detection accuracy is high.
Embodiment
Below in conjunction with specific embodiment, the present invention is described in further detail.
Embodiment
Based on the colorectal carcinoma microsatellite instability detection kit of two generations order-checking platform, comprise the primer panel solution of 5 μ l, the concentration of primer panel solution is 50mmol/L.
Described primer panel comprises the primer of 10 microsatellite locus, its forward primer and reverse primer sequences as follows:
| Site | Forward primer (F) | Reverse primer (R) |
| NR-21 | TCGCTGGCACAGTTCTATTT | TGTTTGTAAACGCGAGTGAC |
| NR-22 | TAATCGAGGCTTGTCAAGGA | GTCTGGAAGTTTTGTCTTGG |
| NR-24 | TGCTGAATTTTACCTCCTGA | AGGTTGGAATGCAATGGCAC |
| NR-27 | CCATGCTTGCAAACCACTGG | TTGGTCATTGCTAGTATTAT |
| BAT-25 | TCGAACTGTCACCTCGGCTTT | ACTTCAAAATGACATTCTG |
| BAT-26 | TGACACTTTTGACTTCAGCC | AACCATTCAACATTTTTAACC |
| MONO-27 | GATTGCAGTGAGCTGAG | TAGCCTTAGAATGTTAGC |
| D2S123 | AAACAGGATGCCTGCCTTTA | GGACCTTCCACCTATGGGAC |
| D5S346 | CGTGAACAGGAGCTTCATCG | ATCTGCAGTCTCTTTGGCCT |
| D17S250 | AGCTTCCAAACTAGTAGAGG | TAAATATATATATGGCCAGCT |
In described test kit, also comprise MasterMix and 1.5ml of 9.5 μ l without enzyme water;
Described MasterMix comprises the dNTPs of 5 μ l10*LATaqBuffer and 4 μ l3mmol/L, and 1 μ lLATaqenzyme.
The detection gene of the described colorectal carcinoma microsatellite instability detection kit based on two generations order-checking platform comprises NR-21, NR-22, NR-24, NR-27, BAT-25, BAT-26, MONO-27, D2S123, D5S346 and D17S250.
The primer panel (protection point) in 10 sites of being correlated with by design MSI; carry out the object fragment needed for multiplex PCR acquisition; then through library construction and the order-checking of upper machine; the variant structure of micro-satellite is obtained finally by bioinformatic analysis; after using this test kit based on two generations order-checking detection colorectal carcinoma microsatellite instability to detect the MSI level of colorectal cancer patients, also can be applicable to the colorectal cancer patients crowd screening applicable PD-L1 Antybody therapy.
The present embodiment specifically comprises the following steps:
1, relate in this experiment 30 routine clinical blood sample standard deviations are from ×× hospital.After sample collection, immediately in 2700xg, 10min, collect upper serum in clean tube pipe,-80 DEG C save backup, and adopt QIAGENDNeasyBlood & TissueKit (QIAGEN, Hilden, Germany) extracting peripheral blood DNA, QIAampCirculatingNucleicAcidKit extraction cycle Tumour DNA.
2, multiplexed PCR amplification reaction is carried out
(1) according to multi-PRC reaction system below: MasterMix9.5 μ l, the primer panel of 5 μ l, DNA sample 4 μ l, 5 μ lddH to be measured
2o.Wherein MasterMix comprises 5 μ l10*LATaqBuffer, 4 μ l2.5mMdNTPs, 0.5 μ lLATaqenzyme.
(2) carry out multiplexed PCR amplification response procedures according to above reaction system: pcr amplification condition is: 95 DEG C of denaturation 3min, 95 DEG C of sex change 30s, 60 DEG C of annealing 30s, 72 DEG C extend 45s; Last 72 DEG C extend 10min, and 4 DEG C leave standstill.After having tested, carry out library construction and examination with computer.
3, library construction (preparation method in library is well known to those skilled in the art (IIIuminaHiseq or Miseq official products specification sheets)), includes but not limited to following steps:
1. after adopting CovarisE-210 (Covaris, Inc., Woburn, MA) to be interrupted at random by the DNA in step 2;
2. end-filling reparation and purification step: end-filling enzyme mixture 20 μ l and DNA profiling 50 μ l mixes latter 20 DEG C and hatches 30min; Add 120 μ lAmpure purifying magnetic bead adsorption of DNA fragments, dry after 80% ethanol washes twice and be dissolved in water;
3. each 10 μ l of sequence measuring joints Connection Step: sequence measuring joints A and P1 fill with 20 μ l after product mix, hatch 15min for 20 DEG C, hatch 5min for 65 DEG C subsequently;
4. template amplification: carry out the PCR of 10 ~ 12 circulations and use the gel electrophoresis of 2% to run glue and glue recovery.Bioanalyzer2100 (AgilentTechnologies, SantaClara, CA) is used to carry out the concentrated of DNA library.(in system, add the product 20 μ l after purifying, amplification Taq enzyme mixture 25 μ l, universal primer 5 μ l carries out pcr amplification.Response procedures is 95 DEG C of denaturation 3min, 95 DEG C of sex change 30s, 60 DEG C of annealing 30s, and 72 DEG C extend 45s; Last 72 DEG C extend 10min, and 4 DEG C leave standstill.)；
5. purifying after amplification: because the sample after step 4. middle hybrid capture may comprise impurity, need eluting further, concrete grammar can carry out with reference to the commercialization kit specification sheets of RocheNimbleGen company.With certain density acetic acid (18%) neutralization after alkaline eluant reclaims, the neutralizer obtained can utilize the PCRpurification test kit of Qiagen to carry out purifying, finally obtains the DNA sample be dissolved in pure water;
6. the sequence enrichment after purifying: the fragment that purifying gets off is carried out the pcr amplification of 10 circulations and generated the library of checking order.Response procedures is 95 DEG C of denaturation 3min, 95 DEG C of sex change 30s, 60 DEG C of annealing 30s, and 72 DEG C extend 45s; Last 72 DEG C extend 10min, and 4 DEG C leave standstill.
4, upper machine order-checking: adopt IlluminaMiSeq sequenator to check order according to operation steps DNA obtained above.
5, interpretation of result: the data results that upper machine reaction obtains by sequence screening, splicing and than counterpart method and bioinformatic analysis, the final Mutation information obtaining test sample book, and determine micro-satellite status according to Mutation information.
6, verify two generation sequencing result: for determining further and the assessment sequence capturing effect of probe and high-flux sequence situation, classical generation order-checking platform (ABI3730Sanger sequenator) is adopted to check order, to above-mentioned obtained variation detected result by carrying out Sanger method sequence verification after pcr amplification, sequencing primer is in table 2.Both sequencing results are consistent, show that the real result detected MSI based on the order-checking of two generations is reliable.
Bioinformatic analysis is carried out to the sequencing data obtained, according to the result of software analysis, filter out normal peripheral blood DNA and Tumour DNA microsatellite markers respectively and compare with reference to genome hg19, thus obtain the abrupt information of the microsatellite marker gene in Tumour DNA and contrast DNA, further the abrupt information of 10 of Tumour DNA microsatellite markers and normal DNA microsatellite markers are compared, obtain the variation information of the MSI gene locus of sample to be tested.According to Besthestaguideline, if there is the site more than 20% to have its neoplastic state of tumor-necrosis factor glycoproteins length variations person to be judged to be MSI-H in 10 sites, be then MSI-L when at least 1 site but the site being less than 20% exist MSI, be MSS without any site change person, concrete outcome is shown in (table 3).
The interpretation of table 3MSI result
| MSI | Unstable mark ratio | Positive mark's number | Detected result |
| MSI-H | ≥20％ | ≥2 | 2(n＝11),3(n＝5)，3(n＝2)，4(n＝1) |
| MSI-L | 0～20％ | 1 | 1(n＝7) |
| MSS | 0 | 0 | 0(n＝4) |
Embodiment recited above is only be described the preferred embodiment of the present invention; not scope of the present invention is limited; do not departing under the present invention designs spiritual prerequisite; the various distortion that the common engineering technical personnel in this area make technical solution of the present invention and improvement, all should fall in protection domain that claims of the present invention determine.
Sequence table
<110> Hunan Hong Ya gene engineering company limited
<120> is based on the colorectal carcinoma microsatellite instability detection kit of two generations order-checking platform
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TTGGTCATTG CTAGTATTAT 20
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TCGAACTGTC ACCTCGGCTT T 21
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Claims (10)
1., based on the colorectal carcinoma microsatellite instability detection kit of two generations order-checking platform, it is characterized in that, comprise the primer panel solution of 4.5-6.5 μ l, in primer panel solution, the concentration of each solute is 45-55mmol/L;
Described primer panel comprises the primer of 10 microsatellite locus, its forward primer and reverse primer sequences as follows:
。
2. the colorectal carcinoma microsatellite instability detection kit based on two generations order-checking platform according to claim 1, is characterized in that, also comprise MasterMix and 1.3-1.7mL of 8-10 μ l without enzyme water; Described MasterMix comprises the dNTPs of 3 ~ 7 μ l 10*LATaqBuffer and 3 ~ 5 μ l 2 ~ 4mmol/L, and 0.5 ~ 1 μ lLATaqenzyme.
3. the colorectal carcinoma microsatellite instability detection kit based on two generations order-checking platform according to claim 1 and 2, it is characterized in that, the quantity of described primer panel solution is 5 μ l.
4. the colorectal carcinoma microsatellite instability detection kit based on two generations order-checking platform according to claim 1 and 2, it is characterized in that, in described primer panel solution, the concentration of each solute is 50mmol/L.
5. the colorectal carcinoma microsatellite instability detection kit based on two generations order-checking platform according to claim 1 and 2, it is characterized in that, the quantity of MasterMix is 9.5 μ l.
6. the colorectal carcinoma microsatellite instability detection kit based on two generations order-checking platform according to claim 3, it is characterized in that, the quantity of MasterMix is 9.5 μ l.
7. the colorectal carcinoma microsatellite instability detection kit based on two generations order-checking platform according to claim 1 and 2, it is characterized in that, described MasterMix comprises the dNTPs of 5 μ l 10*LATaqBuffer and 4 μ l 2 ~ 4mmol/L, and 0.5 ~ 1 μ lLATaqenzyme.
8. the colorectal carcinoma microsatellite instability detection kit based on two generations order-checking platform according to claim 3, it is characterized in that, described MasterMix comprises the dNTPs of 5 μ l 10*LATaqBuffer and 4 μ l 2 ~ 4mmol/L, and 0.5 ~ 1 μ lLATaqenzyme.
9. the colorectal carcinoma microsatellite instability detection kit based on two generations order-checking platform according to claim 4, it is characterized in that, described MasterMix comprises the dNTPs of 5 μ l 10*LATaqBuffer and 4 μ l 2 ~ 4mmol/L, and 0.5 ~ 1 μ lLATaqenzyme.
10. the colorectal carcinoma microsatellite instability detection kit based on two generations order-checking platform according to claim 1 and 2, it is characterized in that, the multi-PRC reaction condition of the described colorectal carcinoma microsatellite instability detection kit based on two generations order-checking platform is: 93 DEG C ~ 98 DEG C denaturation 2 ~ 3min, then the polymerase chain reaction (PCR) amplification stage is entered: 93 DEG C ~ 96 DEG C sex change 20 ~ 30s, 55 DEG C ~ 65 DEG C annealing 20 ~ 30s, 70 DEG C ~ 75 DEG C extend 20 ~ 45s; Last 70 DEG C ~ 74 DEG C extend 10min, and carry out 10 ~ 12 circulations, and 4 DEG C leave standstill.
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