CN110218772A - A kind of Primer composition, kit, detection method and its application detecting microsatellite instability - Google Patents
A kind of Primer composition, kit, detection method and its application detecting microsatellite instability Download PDFInfo
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Abstract
The present invention provides a kind of Primer composition, kit, detection method and its application for detecting microsatellite instability, the Primer composition includes NO:1~18 primer pair SEQ ID for NR21, NR24, NR27, BAT25, BAT26, MONO-27, Penta D, Penta E and Amel.The present invention is directed to six SNP site design primers of NR21, NR24, NR27, BAT25, BAT26 and MONO-27, according to the state of six SNP sites, evaluate microsatellite instability, simultaneously according to Penta D, Penta E and tri- control polymorphic sites of Amel, the consistency of test serum sample and its control is assessed, accuracy is high, the sample size used is few, amplification efficiency is high, and band is clear, high sensitivity.
Description
Technical field
The invention belongs to technical field of gene detection, are related to Primer composition, the examination of a kind of detection microsatellite instability
Agent box, detection method and its application.
Background technique
Microsatellite (Microsatallite) refers to the tandem repetitive sequence being made of the short chain DNA of 1~6 base,
It is widely present in genome.Compared with normal tissue, insertion or missing of the microsatellite due to repetitive sequence in tumor tissues are led
The change of microsatellite length is caused, is formed microsatellite instability (Microsatallite Instability, MSI).Study table
Bright, the reason of causing MSI is the mutation or supermethylation due to DNA mismatch reparation (MMR) gene (such as MLH1, MSH2 and MSH6)
Modification, error when gene DNA being caused to replicate in cell.
The generation of MSI and tumour is closely related, can be in a variety of cancer kinds such as colorectal cancer, gastric cancer, carcinoma of endometrium
Detect the MSI positive.MSI occurs the most frequently in colorectal cancer, nearly 15% sporadic colorectal cancer and hereditary nonpolyposis
Characteristic of disease colorectal cancer patients contain MSI feature.For sporadic colorectal cancer, the hyper-methylation of MLH1 promoter causes MMR function
Can be abnormal, cause MSI, such patient accounts for about 12%, Yi Fasheng of all colorectal cancer patients without the old of obvious household heredity factors
In ageization individual, MMR defect is occurred directly in colorectal cancer body cell.In contrast to this, hereditary nonpolyposis characteristic of disease knot
The carcinoma of the rectum (HNPCC), also known as Lynch syndrome lead to MMR functional defect, draw because carrying MMR gene mutation in reproduction cell
MSI is played, which is a kind of Familial Occurrence disease, the 2%-5% of Zhan Suoyou colorectal cancer patients.
Colorectal cancer is that cancer the most frequent, the earliest biomarker as colorectal cancer of MSI detection occur for MSI.
From 2013, the 2013v.1 version colorectal cancer guide of US National comprehensive cancer network (NCCN) publication was recommended to carry out for the first time
MSI detection.Newest 2019v.1 editions NCCN guide recommends the patient for having colorectal cancer medical history to carry out MMR or MSI screening；High frequency
The colorectal cancer II phase patient of MSI (MSI-H) has preferable prognosis, but cannot benefit in 5-FU treatment；MSI may be used also simultaneously
To be applied to the diagnosis of Jessica Lynch syndrome patient.In addition, MSI is also good immunization therapy marker.FDA has been approved by three kinds
For the immunotherapy medicaments of MSI symptom.For example, NCCN recommend PD-1 immunization therapy monoclonal antibody pembrolizumab and
Nivolumab is used for dMMR/MSI-H colorectal cancer treatment of late stage.
CN108998536A discloses a kind of mankind's microsatellite instability state MSI detection kit and its detection side
Method, primer, probe including detecting mankind's microsatellite instability state MSI.The primer, probe are able to detect mankind's microsatellite
Unstability state MSI, high sensitivity, high specificity；The present invention also provides a kind of mankind's microsatellite instability state MSI
Detection kit, the kit being capable of quick, efficient, accurate detection mankind's microsatellite instability states；In addition, the present invention mentions
For detecting the detection method of mankind's microsatellite instability state, the operation letter of this method detection method using mentioned reagent box
Just, the advantages that high sensitivity, specificity is high, and Fit Models are wide, and testing result is true and reliable and easy to spread.However, on a variety of
The 5 ' of trip primer are terminal modified identical fluorophor, impacts to interpretation of result.
Therefore it provides a kind of high sensitivity, accuracy are high, the inspection of easy, the lower-cost microsatellite instability of step
Primer composition, kit and detection method are surveyed, is of great significance in field of cancer treatment.
Summary of the invention
In view of the deficiencies of the prior art, the present invention provides a kind of Primer compositions for detecting microsatellite instability, examination
Agent box, detection method and its application, the method are based on multiple fluorescence PCR and capillary electrophoresis technique, examine to MSI state
It surveys, is conducive to provide clinical treatment and medication guide to colorectal cancer patients and other cancer patients.
To achieve this purpose, the present invention adopts the following technical scheme:
In a first aspect, the present invention provides a kind of Primer composition for detecting microsatellite instability, the primer combination
Object includes the primer pair SEQ for NR21, NR24, NR27, BAT25, BAT26, MONO-27, Penta D, Penta E and Amel
NO:1~18 ID；
Nucleotide sequence shown in SEQ ID NO:1 are as follows: 5 '-CCTACTCCGCATTCAC-3 '；
Nucleotide sequence shown in SEQ ID NO:2 are as follows: 5 '-CACATCCCTTTCAGCA-3 '；
Nucleotide sequence shown in SEQ ID NO:3 are as follows: 5 '-TACCTCCTGACTCCAAAA-3 '；
Nucleotide sequence shown in SEQ ID NO:4 are as follows: 5 '-AGTGAGCGGAGATTGTG-3 '；
Nucleotide sequence shown in SEQ ID NO:5 are as follows: 5 '-CTGTGAGATCCAGGAAAC-3 '；
Nucleotide sequence shown in SEQ ID NO:6 are as follows: 5 '-CTAGCAATGACCAATAAGC-3 '；
Nucleotide sequence shown in SEQ ID NO:7 are as follows: 5 '-AAGTGGGAGTGATTCT-3 '；
Nucleotide sequence shown in SEQ ID NO:8 are as follows: 5 '-TCCTCTGCTTTTGGTTAC-3 '；
Nucleotide sequence shown in SEQ ID NO:9 are as follows: 5 '-AACTTTGGACAGTTTGAACT-3 '；
Nucleotide sequence shown in SEQ ID NO:10 are as follows: 5 '-CTCCTTTATAAGCTTCTTCAG-3 '；
Nucleotide sequence shown in SEQ ID NO:11 are as follows: 5 '-CAAGTTTAAGCAACAGAC-3 '；
Nucleotide sequence shown in SEQ ID NO:12 are as follows: 5 '-GTGACATAATGAGACCCT-3 '；
Nucleotide sequence shown in SEQ ID NO:13 are as follows: 5 '-GAGCCTGGAAGGTCGAA-3 '；
Nucleotide sequence shown in SEQ ID NO:14 are as follows:
5'-GAAATATTTGCCTAACCTATGGTC-3'；
Nucleotide sequence shown in SEQ ID NO:15 are as follows: 5 '-GACTGAGCAAGACTCAGTCT-3 '；
Nucleotide sequence shown in SEQ ID NO:16 are as follows:
5'-GATTACATAACATACATGTGTGTAAA-3'；
Nucleotide sequence shown in SEQ ID NO:17 are as follows: 5 '-CCCTGGGCTCTGTAAAGAA-3 '；
Nucleotide sequence shown in SEQ ID NO:18 are as follows:
5’-ATCAGAGCTTAAACTGGGAAGCTG-3’。
In the present invention, for NR21, NR24, NR27, BAT25, BAT26, MONO-27, Penta D, Penta E and Amel
Nine SNP site design primers, according to the state of six SNP sites of NR21, NR24, NR27, BAT25, BAT26 and MONO-27,
Microsatellite instability is evaluated, accuracy is high；According to Penta D, Penta E and tri- sites Amel control site the most, use
To evaluate the consistency of detection sample and its check sample.
Preferably, the upstream primer of NR21 is as shown in SEQ ID NO:1, and downstream primer is as shown in SEQ ID NO:2.
Preferably, the upstream primer of NR24 is as shown in SEQ ID NO:3, and downstream primer is as shown in SEQ ID NO:4.
Preferably, the upstream primer of NR27 is as shown in SEQ ID NO:5, and downstream primer is as shown in SEQ ID NO:6.
Preferably, the upstream primer of BAT25 is as shown in SEQ ID NO:7, and downstream primer is as shown in SEQ ID NO:8.
Preferably, the upstream primer of BAT26 is as shown in SEQ ID NO:9, and downstream primer is as shown in SEQ ID NO:10.
Preferably, the upstream primer of MONO-27 is as shown in SEQ ID NO:11, downstream primer such as SEQ ID NO:12 institute
Show.
Preferably, the upstream primer of Penta D is as shown in SEQ ID NO:13, downstream primer such as SEQ ID NO:14 institute
Show.
Preferably, the upstream primer of Penta E is as shown in SEQ ID NO:15, downstream primer such as SEQ ID NO:16 institute
Show.
Preferably, the upstream primer of Amel is as shown in SEQ ID NO:17, and downstream primer is as shown in SEQ ID NO:18.
Preferably, 5 ' of the upstream primer in the Primer composition terminal modified have fluorophor.
Preferably, the fluorophor includes any one in HEX, FAM, ROX, TET, JOE, CY3 or CY5.
Preferably, the 5 ' of the upstream primer of the NR21 terminal modified have HEX.
Preferably, the 5 ' of the upstream primer of the NR24 terminal modified have FAM.
Preferably, the 5 ' of the upstream primer of the NR27 terminal modified have FAM.
Preferably, the 5 ' of the upstream primer of the BAT25 terminal modified have ROX.
Preferably, the 5 ' of the upstream primer of the BAT26 terminal modified have ROX.
Preferably, the 5 ' of the upstream primer of the MONO-27 terminal modified have HEX.
Preferably, the 5 ' of the upstream primer of the Penta D terminal modified have FAM.
Preferably, the 5 ' of the upstream primer of the Penta E terminal modified have HEX.
Preferably, the 5 ' of the upstream primer of the Amel terminal modified have ROX.
In the present invention, 5 ' ends of 9 polymorphic sites are connected with identical fluorophor, as NR-24 and NR-27,
BAT-25 and BAT-26, PCR product length differ by more than 30bp, PCR product are allowed to carry out area by Capillary Electrophoresis
Point.
Second aspect, the present invention provides a kind of kit, the kit includes primer sets as described in relation to the first aspect
Close object.
Preferably, the kit further includes any one in enzyme mixation, PCR reaction solution or dNTP or at least two
Combination.
In the present invention, archaeal dna polymerase is contained in the enzyme mixation, PCR reaction solution contains 500mM Tris-HCl (pH=
9.0)、200mM(NH4)2SO4、20mM MgSO4With 10% glycerol.
The third aspect, the present invention provides a kind of methods for detecting microsatellite instability, and the method includes following steps
It is rapid:
Kit using Primer composition as described in relation to the first aspect, or as described in second aspect carries out DNA to be measured
Quantitative fluorescent PCR and Capillary Electrophoresis judge the state of microsatellite instability according to result.
Preferably, DNA in paraffin embedding pathological section tissue, tumour cell or the peripheral blood any one or extremely
Few two kinds of combination.
Preferably, final concentration of 1~10ng/ μ L of the DNA, for example, can be 1ng/ μ L, 2ng/ μ L, 3ng/ μ L,
4ng/ μ L, 5ng/ μ L, 6ng/ μ L, 7ng/ μ L, 8ng/ μ L, 9ng/ μ L or 10ng/ μ L.
Preferably, the NR21 primer, NR24 primer, NR27 primer, BAT25 primer, BAT26 primer, MONO-27 draw
Object, Penta D primer, Penta E primer and Amel primer molar ratio be (15~20): (8~12): (14~17): (12~
16): (15~20): (10~15): (14~18): (13~17): (18~22).
Preferably, the NR21 primer, NR24 primer, NR27 primer, BAT25 primer, BAT26 primer, MONO-27 draw
Object, Penta D primer, Penta E primer and Amel primer molar ratio be 19:10.5:16.5:14:18:12.5:16:
15.5:20。
In the present invention, the proportion can balance nine pairs of primers, avoid because of G/C content difference, and TM value is different, caused by expand
The difference of Increasing Efficiency, while it is optimal the specificity of nine pairs of primers also；If will cause certain using the ratio other than this ratio
The abnormal of the amplification efficiency of a little primer pairs increases, or abnormal reduction, so that the result of moiety site can not differentiate；It also will cause certain
The specificity of a little primer pairs reduces, so that its result can not differentiate.
Preferably, final concentration of 0.1~0.2 μM of the NR21 primer, preferably 0.1~0.122 μM.
Preferably, final concentration of 0.05~0.08 μM of the NR24 primer, preferably 0.05~0.067 μM.
Preferably, final concentration of 0.1~0.15 μM of the NR27 primer, preferably 0.1~0.106 μM.
Preferably, final concentration of 0.05~0.1 μM of the BAT25 primer, preferably 0.09~0.1 μM.
Preferably, final concentration of 0.1~0.15 μM of the BAT26 primer, preferably 0.1~0.115 μM.
Preferably, final concentration of 0.05~0.1 μM of the MONO-27 primer, preferably 0.07~0.08 μM.
Preferably, final concentration of 0.08~0.12 μM of the Penta D primer, preferably 0.09~0.102 μM.
Preferably, final concentration of 0.05~0.1 μM of the Penta E primer, preferably 0.09~0.1 μM.
Preferably, final concentration of 0.1~0.2 μM of the Amel primer, preferably 0.1~0.128 μM.
Preferably, the reaction condition of the quantitative fluorescent PCR are as follows:
First stage: 90~96 DEG C of 3~8min of denaturation, 1 circulation；
Second stage: 90~96 DEG C of denaturation 20~50s, 55~60 DEG C of annealing 30~60s, 60~70 DEG C of 50~80s of extension,
10 circulations；
Phase III: 90~96 DEG C of denaturation 20~50s, 50~55 DEG C of annealing 30~60s, 60~70 DEG C of 50~80s of extension,
20 circulations；
Fourth stage: 60~70 DEG C of 3~8min of extension, 1 circulation；
It saves: 4~12 DEG C.
Preferably, the reaction condition of the quantitative fluorescent PCR are as follows:
First stage: 94 DEG C of denaturation 5min, 1 circulation；
Second stage: 94 DEG C of denaturation 30s, 58 DEG C of annealing 40s, 65 DEG C of extension 60s, 10 recycle；
Phase III: 94 DEG C of denaturation 30s, 53 DEG C of annealing 40s, 65 DEG C of extension 60s, 20 recycle；
Fourth stage: 65 DEG C of extension 5min, 1 circulation；
It saves: 12 DEG C.
Preferably, the condition of the Capillary Electrophoresis is 95~99 DEG C of denaturation 3~10min, 0~4 DEG C of 1~5min of holding.
Preferably, the condition of the Capillary Electrophoresis is 98 DEG C of denaturation 5min, 0~4 DEG C of holding 2min.
In the present invention, using the matched Data Analysis Software of Genetic Analyser, Capillary Electrophoresis result is analyzed, it is right
Than tumor sample and check sample as a result, determine the state of each mononucleotide polymorphism site, the mark of MSI state interpretation
It is quasi- are as follows: 6 mononucleotide polymorphism sites are stablized, and interpretation is that microsatellite stablizes (MSS)；1 mononucleotide polymorphism site is not
Stablize, interpretation is low frequency MSI (MSI-L)；2 and the above mononucleotide polymorphism site it is unstable, interpretation be high frequency MSI
(MSI-H)。
Fourth aspect, the present invention provides a kind of Primer composition as described in relation to the first aspect, as described in second aspect
Kit or application of the method in preparation tumor therapeutic agent or building detection device as described in the third aspect.
Compared with prior art, the invention has the following beneficial effects:
(1) present invention is directed to six SNP site design primers of NR21, NR24, NR27, BAT25, BAT26 and MONO-27,
According to the state of six SNP sites, microsatellite instability is evaluated, accuracy is high；
(2) present invention is directed to Penta D, Penta E and tri- SNP site design primers of Amel simultaneously, according to three SNP
The state in site, evaluation detection sample and its check sample match situation, avoid false positive caused by incorrect pairing or vacation
It is negative；
(3) kit of the invention includes the primer pair of design, and high sensitivity, required detection sample size is few, it is only necessary to
The detection of 9 polymorphic sites can be completed in 10ng or so, meets the needs of to patient's MSI state-detection；
(4) in detection method of the invention, PCR amplification system is high-efficient, and a PCR system can be detected simultaneously more than 9
State property site, avoids multiple PCR from detecting, and testing result can satisfy the needs of analysis.
Detailed description of the invention
Fig. 1 is that poba gene group compares peak figure in embodiment 3；
Fig. 2 is tumor tissues peak figure in embodiment 3.
Specific embodiment
The technological means and its effect taken for the present invention is further explained, with reference to embodiments with attached drawing to this hair
It is bright to be further described.It is understood that the specific embodiments described herein are used only for explaining the present invention, rather than
Limitation of the invention.
In the examples where no specific technique or condition is specified, described technology or conditions according to the literature in the art,
Or it is carried out according to product description.Reagents or instruments used without specified manufacturer, be can be by regular channel commercially available from
The conventional products of acquisition.
The preparation of 1 design of primers of embodiment and primer combination mixed liquor
The standard and " Revised Bethesda that the present embodiment is recommended according to National Cancer research institute
Guidelines " suggest, determine 6 mononucleotide polymorphism site NR-21, NR-24, NR-27, BAT-25, MONO-27,
BAT-26 and three control polymorphic site Penta D, Penta E and Amel, according to the GenBank sequence information in 9 sites
Specific primer design is carried out using Primer Premier 5.0, and artificial synthesized according to existing method progress, obtained such as SEQ
Primer composition shown in NO:1~18 ID；
Seedless sour water is added in 9 pairs of primers of synthesis, is dissolved to 100 μM: by taking NR-21 as an example, taking 5 μ L forward primers, 5 μ L are anti-
To primer and the 40 seedless sour waters of μ L, it is configured to NR-21 primer working solution, concentration is 10 μM；Other 8 pairs of primers are using same side
Method configures primer working solution；9 kinds of primer working solutions are mixed according to ratio as shown in Table 1, are prepared primer and are combined mixed liquor,
The detection of 30 samples can be met.
Table 1
| Primer | Ratio |
| NR-21 working solution (10 μM) | 19μL |
| NR-24 working solution (10 μM) | 10.5μL |
| NR-27 working solution (10 μM) | 16.5μL |
| BAT-25 working solution (10 μM) | 14.0μL |
| BAT-26 working solution (10 μM) | 18.0μL |
| MONO-27 working solution (10 μM) | 12.5μL |
| Penta D working solution (10 μM) | 16.0μL |
| Penta E working solution (10 μM) | 15.5μL |
| Amel working solution (10 μM) | 20.0μL |
| Total volume | 142μL |
The present embodiment can balance nine pairs of primers using proportion described in table 1, avoid because of G/C content difference, and TM value is different, makes
At amplification efficiency difference, while make nine pairs of primers specificity be also optimal.
The assembling of 2 kit of embodiment
The kit of the present embodiment includes 4 kinds of components: enzyme mixation, PCR reaction solution, dNTP and including 9 polymorphisms
The primer in site combines mixed liquor, and concrete component is as shown in table 2:
Table 2
3 pattern detection of embodiment
The present embodiment is by taking the paraffin embedding tumor tissues of 1 colorectal cancer patients (patient code HP001) as an example, using reality
Apply microsatellite instability state in the kit detection tumor tissues of example 2, comprising the following steps:
(1) DNA and blood sample leucocyte gDNA of patient HP001 paraffin embedding tumor tissues sample are extracted as control
Sample detects concentration of specimens using Qubit 2.0, and tissue samples DNA concentration is 20.4ng/ μ L, and leucocyte gDNA concentration is
56.4ng/μL；
(2) PCR reaction system is prepared according to table 3, prepared 47 μ L PCR reaction system is added in each detection architecture；
Table 3
| Component | Volume/μ L |
| Enzyme mixation | 1.05 |
| PCR reaction solution | 2.1 |
| dNTPs | 8.4 |
| Primer combines mixed liquor | 5.25 |
| Nuclease-free water | 73.5 |
(3) template is diluted according to the concentration for extracting obtained DNA, patient's HP001 tumor tissues Sample Dilution 7
Times, leucocyte gDNA dilutes 19 times, so that DNA concentration is 3ng/ μ L or so；Concentration of specimens, tissue are detected using Qubit 2.0
Sample DNA concentration is 2.94ng/ μ L, and leucocyte gDNA concentration is 3.02ng/ μ L；After adding 3 μ L dilution into each PCR pipe
Tumour DNA or comparison DNA；
(4) pcr amplification reaction program is set, PCR reaction system is detected, response procedures are as shown in table 4；
Table 4
(5) 1 μ L PCR product is taken, 8.5 μ L formamide (Hi-Di are addedTM) and 0.5 μ L GeneScan Formamide
600LIZ size standard, 98 DEG C of denaturation 5min, put 2min on ice immediately after mixing, are carried out using ABI 3500Dx instrument
Capillary electrophoresis detection；
(6) software is analyzed using ABI 3500Genetic Analyzer, Capillary Electrophoresis result is analyzed.
The standard of MSI state interpretation are as follows: 6 mononucleotide polymorphism sites are stablized, and interpretation is that microsatellite stablizes (MSS)；1
A mononucleotide polymorphism site is unstable, and interpretation is low frequency MSI (MSI-L)；2 and the above mononucleotide polymorphism site are not
Stablize, interpretation is high frequency MSI (MSI-H)；3 control polymorphic sites, test serum and its results of comparison are consistent.
Patient's HP001 tissue samples result and leucocyte gDNA sample results are shown in Fig. 1 and Fig. 2, patient NR-21, the NR-
24, unstable state is presented in six sites NR-27, BAT-25, BAT-26, Mono-27, according to interpretation standard, is judged as
MSI-H。
In conclusion the present invention is for six SNP site designs of NR21, NR24, NR27, BAT25, BAT26 and MONO-27
Primer evaluates microsatellite instability according to the state of six SNP sites, and accuracy is high；It simultaneously include three control polymorphisms
Site evaluates the consistency of test serum sample and its control, avoids false positive or false negative caused by obscuring because of control, improves
The accuracy and sensibility of judgement；Kit includes nine pairs of primers of design, and high sensitivity, required detection sample size is few,
It only needs 10ng or so that the detection of nine mononucleotide polymorphism sites can be completed, meets the needs of to patient's MSI state-detection；
In detection method, PCR amplification system is high-efficient, and a PCR system can detect nine mononucleotide polymorphism sites simultaneously, keeps away
Exempt from multiple PCR detection, testing result can satisfy the needs of analysis.
The Applicant declares that the present invention is explained by the above embodiments method detailed of the invention, but the present invention not office
Be limited to above-mentioned method detailed, that is, do not mean that the invention must rely on the above detailed methods to implement.Technical field
Technical staff it will be clearly understood that any improvement in the present invention, equivalence replacement and auxiliary element to each raw material of product of the present invention
Addition, selection of concrete mode etc., all of which fall within the scope of protection and disclosure of the present invention.
SEQUENCE LISTING
<110>Shenzhen sea Prologis Laboratory of medical test
<120>a kind of Primer composition, kit, detection method and its application for detecting microsatellite instability
<130> 20190610
<160> 18
<170> PatentIn version 3.3
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Claims (10)
1. a kind of Primer composition for detecting microsatellite instability, which is characterized in that the Primer composition includes being directed to
NR21, NR24, NR27, BAT25, BAT26, MONO-27, Penta D, Penta E and Amel primer pair SEQ ID NO:1~
18。
2. Primer composition according to claim 1, which is characterized in that the upstream primer of NR21 such as SEQ ID NO:1 institute
Show, downstream primer is as shown in SEQ ID NO:2；
Preferably, the upstream primer of NR24 is as shown in SEQ ID NO:3, and downstream primer is as shown in SEQ ID NO:4；
Preferably, the upstream primer of NR27 is as shown in SEQ ID NO:5, and downstream primer is as shown in SEQ ID NO:6；
Preferably, the upstream primer of BAT25 is as shown in SEQ ID NO:7, and downstream primer is as shown in SEQ ID NO:8；
Preferably, the upstream primer of BAT26 is as shown in SEQ ID NO:9, and downstream primer is as shown in SEQ ID NO:10.
Preferably, the upstream primer of MONO-27 is as shown in SEQ ID NO:11, and downstream primer is as shown in SEQ ID NO:12；
Preferably, the upstream primer of Penta D is as shown in SEQ ID NO:13, and downstream primer is as shown in SEQ ID NO:14；
Preferably, the upstream primer of Penta E is as shown in SEQ ID NO:15, and downstream primer is as shown in SEQ ID NO:16；
Preferably, the upstream primer of Amel is as shown in SEQ ID NO:17, and downstream primer is as shown in SEQ ID NO:18.
3. Primer composition according to claim 1 or 2, which is characterized in that the upstream primer in the Primer composition
5 ' terminal modified have fluorophor；
Preferably, the fluorophor includes any one in HEX, FAM, ROX, TET, JOE, CY3 or CY5；
Preferably, the 5 ' of the upstream primer of the NR21 terminal modified have HEX；
Preferably, the 5 ' of the upstream primer of the NR24 terminal modified have FAM；
Preferably, the 5 ' of the upstream primer of the NR27 terminal modified have FAM；
Preferably, the 5 ' of the upstream primer of the BAT25 terminal modified have ROX；
Preferably, the 5 ' of the upstream primer of the BAT26 terminal modified have ROX；
Preferably, the 5 ' of the upstream primer of the MONO-27 terminal modified have HEX；
Preferably, the 5 ' of the upstream primer of the Penta D terminal modified have FAM；
Preferably, the 5 ' of the upstream primer of the Penta E terminal modified have HEX；
Preferably, the 5 ' of the upstream primer of the Amel terminal modified have ROX.
4. a kind of kit, which is characterized in that the kit includes primer combination as described in any one of claims 1-3
Object；
Preferably, the kit further include in enzyme mixation, PCR reaction solution or dNTP any one or at least two group
It closes.
5. a kind of method for detecting microsatellite instability, which is characterized in that the described method comprises the following steps:
Using Primer composition as described in any one of claims 1-3 or kit as claimed in claim 4, to be measured
DNA carries out quantitative fluorescent PCR and Capillary Electrophoresis, and the state of microsatellite instability is judged according to result.
6. according to the method described in claim 5, it is characterized in that, DNA is thin from paraffin embedding pathological section tissue, tumour
In born of the same parents or peripheral blood any one or at least two combination；
Preferably, final concentration of 1~10ng/ μ L of the DNA.
7. method according to claim 5 or 6, which is characterized in that the NR21 primer, NR24 primer, NR27 primer,
BAT25 primer, BAT26 primer, MONO-27 primer, Penta D primer, Penta E primer and Amel primer molar ratio be
(15~20): (8~12): (14~17): (12~16): (15~20): (10~15): (14~18): (13~17): (18~
22)；
Preferably, the NR21 primer, NR24 primer, NR27 primer, BAT25 primer, BAT26 primer, MONO-27 primer,
The molar ratio of Penta D primer, Penta E primer and Amel primer is 19:10.5:16.5:14:18:12.5:16:15.5:
20；
Preferably, final concentration of 0.1~0.2 μM of the NR21 primer, preferably 0.1~0.122 μM；
Preferably, final concentration of 0.05~0.08 μM of the NR24 primer, preferably 0.05~0.067 μM；
Preferably, final concentration of 0.1~0.15 μM of the NR27 primer, preferably 0.1~0.106 μM；
Preferably, final concentration of 0.05~0.1 μM of the BAT25 primer, preferably 0.09~0.1 μM；
Preferably, final concentration of 0.1~0.15 μM of the BAT26 primer, preferably 0.1~0.115 μM；
Preferably, final concentration of 0.05~0.1 μM of the MONO-27 primer, preferably 0.07~0.08 μM；
Preferably, final concentration of 0.08~0.12 μM of the Penta D primer, preferably 0.09~0.102 μM；
Preferably, final concentration of 0.05~0.1 μM of the Penta E primer, preferably 0.09~0.1 μM；
Preferably, final concentration of 0.1~0.2 μM of the Amel primer, preferably 0.1~0.128 μM.
8. according to the described in any item methods of claim 5-7, which is characterized in that the reaction condition of the quantitative fluorescent PCR are as follows:
First stage: 90~96 DEG C of 3~8min of denaturation, 1 circulation；
Second stage: 90~96 DEG C of 20~50s of denaturation, 55~60 DEG C of 30~60s of annealing, 60~70 DEG C of 50~80s of extension, 10 follow
Ring；
Phase III: 90~96 DEG C of 20~50s of denaturation, 50~55 DEG C of 30~60s of annealing, 60~70 DEG C of 50~80s of extension, 20 follow
Ring；
Fourth stage: 60~70 DEG C of 3~8min of extension, 1 circulation；
It saves: 4~12 DEG C；
Preferably, the reaction condition of the quantitative fluorescent PCR are as follows:
First stage: 94 DEG C of denaturation 5min, 1 circulation；
Second stage: 94 DEG C of denaturation 30s, 58 DEG C of annealing 40s, 65 DEG C of extension 60s, 10 recycle；
Phase III: 94 DEG C of denaturation 30s, 53 DEG C of annealing 40s, 65 DEG C of extension 60s, 20 recycle；
Fourth stage: 65 DEG C of extension 5min, 1 circulation；
It saves: 12 DEG C.
9. according to the described in any item methods of claim 5-8, which is characterized in that the condition of the Capillary Electrophoresis is 95~99
DEG C denaturation 3~10min, 0~4 DEG C of 1~5min of holding；
Preferably, the condition of the Capillary Electrophoresis is 98 DEG C of denaturation 5min, 0~4 DEG C of holding 2min.
10. a kind of Primer composition as described in any one of claims 1-3, kit as claimed in claim 4 are such as weighed
Benefit requires application of the described in any item methods of 5-9 in preparation tumor therapeutic agent or building detection device.
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| CN111850129A (en) * | 2020-08-17 | 2020-10-30 | 中山大学附属第六医院 | Primer pair, kit and method for detecting stability of NR21 locus of microsatellite |
| CN112481381A (en) * | 2020-12-10 | 2021-03-12 | 江苏吉诺思美精准医学科技有限公司 | Primer, probe, kit and detection method for detecting instability of microsatellite |
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