CN109097478A - A kind of mankind's microsatellite instability state MSI detection kit and its detection method - Google Patents
A kind of mankind's microsatellite instability state MSI detection kit and its detection method Download PDFInfo
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Abstract
The present invention provides a kind of mankind's microsatellite instability state MSI detection kit and its detection methods；Primer, probe including detecting mankind's microsatellite instability state MSI.The primer, probe are able to detect mankind microsatellite instability state MSI, high sensitivity, high specificity；The present invention also provides a kind of mankind's microsatellite instability state MSI detection kit, which being capable of quick, efficient, accurate detection mankind's microsatellite instability state；In addition, the advantages that this method detection method is easy to operate, high sensitivity the present invention provides the detection method for using mentioned reagent box detection mankind's microsatellite instability state, and specificity is high, and Fit Models are wide, and testing result is true and reliable and easy to spread.
Description
Technical field
The present invention relates to genetic test fields, and in particular to arrives mankind's microsatellite instability state MSI detection kit
And its detection method.
Background technique
Currently, tumour has become Chinese major causes of death and tumor load is still in lasting rising.According to China in 2018
" the Chinese treatment and prevention of tumour progress " of medical association's publication reports display, and China is average, and there are about 10,000 people to make a definite diagnosis cancer, 7500 cancers daily
Disease death, therefore tumour medicine is developed, the research for the treatment of therapy is increasingly important.In recent years, with accurate medical treatment and individual
Change the development of medical treatment, new tumor-targeting drug research and development and the research that deepens continuously, personalized medicine is predicted by genetic test
Become current development trend therewith.
Microsatellite instability (microsatellite instability, MSI) is one kind due to gene duplication mistake
Caused by simple repeated sequence change.The presence of MSI implies that cell exists in terms of the DNA sequence dna of errors repair and lacks
It falls into, this is closely related with the formation of malignant tumour (especially alimentary system malignant tumour and gynecological tumor).MSI is according to its change
Degree is divided into high frequency MSI (MSI-H), low frequency MSI (MSI-L) and microsatellite stablizes (MSS), research shows that the MSI of detection patient
Parting has important clinical significance.It is reported that MSI detection is considered that the adjuvant treatment of colorectal cancer and the carcinoma of the rectum can be provided
Foundation, the colon cancer and carcinoma of the rectum clinical practice guideline of NCCN publication are recommended all with colorectal cancer and carcinoma of the rectum illness history
Patient carries out MSI detection, to instruct clinical application and immunization therapy, diagnosis woods Cotard (Lynch syndrome).Have big
Quantifier elimination show MSI state for determine colorectal cancer patients be using 5-FU treatment or irinotecan it is critically important,
MSI colorectal cancer and other types MSI Advanced Carcinoma Patient are benefited larger using the immunization therapy of PD-1 monoclonal antibody.NCCN recommends
PD-1 immunization therapy monoclonal antibody pembrolizumab and nivolumab are used for dMMR/MSI-H colorectal cancer treatment of late stage.Thus may be used
See, as the research mechanism of drug is made clear further, MSI detection there will be a very big market prospect.
At present detection tumour cell in MSI when, can both rely on molecular level (including multiplex PCR-Capillary Electrophoresis and
NGS MSI phenomenon) is detected, immunohistochemistry can also be passed through and detect MMR gene defect.
Summary of the invention
The present invention provides primer, the probes of detection mankind's microsatellite instability state MSI.The primer, probe can
Detect mankind's microsatellite instability state MSI, high sensitivity, high specificity.
The present invention also provides a kind of mankind's microsatellite instability state MSI detection kit, the kit energy
Enough quick, efficient, accurate detection mankind's microsatellite instability states.
The present invention also provides the detection method for using mentioned reagent box detection mankind's microsatellite instability state, the party
Method detection method is easy to operate, high sensitivity, and specificity is high, and Fit Models are wide, and testing result is true and reliable and easy to spread etc.
Advantage.
The present invention includes following content:
A kind of mankind's microsatellite instability state MSI detection kit, the detection kit include being directed to
Six mononucleotides repeat the primer in site and the detection of three control sites, and the detection primer is as follows:
BAT-25-Fo:FAM-CTCGCCTCCAAGAATGTAAGT
BAT-25-Re:ctatggctctaaaatgctctgttc
BAT-26-Fo:TAMRA-TGACTACTTTTGACTTCAGCC
BAT-26-Re:AACCATTCAACATTTTTAACCC
NR-21-Fo:VIC-CTTTCTGGTCACTCGCGTTTACAA
NR-21-Re:AAGGGGAGGTAAAGGCAGTCTC
NR-24-Fo:VIC-GCTGAATTTTACCTCCTGAC
NR-24-Re:GGAGATTGTGCCATTGCA
NR-27-Fo:FAM-AACCATGCTTGCAAACCACT
NR-27-Re:TGAGTCGATAATACTAGCAATGACC
MONO-27-Fo:TAMRA-AAGGGTGGATCAAATTTCACTTG
MONO-27-Re:TGGGAGACAGAGCAAGACTCTG
PentaC-Fo:VIC-gaacacactttgcacctgtcag
PentaC-Re:CTGAGCGCTTCTAGGGACTTCT
PentaD-Fo:TAMRA-atttgcctaacctatggtCATAACG
PentaD-Re:ACACTCCAGCCTAGGTGACAGA
Amel-Fo:FAM-ccctgggctctgtaaagaatag
Amel-Re:TTGAGGCCAACCATCAGAGCTTA.
Preferably, mankind's microsatellite instability state MSI detection kit includes MSI primer mixed liquor, PCR
Premixed liquid, negative controls, positive reference substance；
The MSI primer mixed liquor includes to repeat site and 2 pentanucleotide repeats bits for detecting 6 mononucleotides
The primer of point and 1 sex identification gene, 6 mononucleotides repeat sites be respectively BAT-25, BAT-26, NR-21, NR-24,
NR-27, MONO-27, it is PentaC, PentaD that 2 pentanucleotides, which repeat site, and sex identification gene is Amel；
The PCR premixed liquid includes UDG enzyme, archaeal dna polymerase, PCR buffer, MgCl2, dNTPs and dUTP；
The negative controls are sterilizing ultrapure water；
The positive reference substance is cell line HT-29 genomic DNA.
A kind of detection method of mankind's microsatellite instability state MSI, the detection method include micro- using the mankind
The primer of satellite unstability state MSI detects the kit detection of the mankind's microsatellite instability state MSI mankind is micro- defends
The step of star unstability state；
The detection method carries out sequence fragment using multiple fluorescence quantitative PCR technology combination Capillary Electrophoresis and analyzes the mankind
Microsatellite instability state in cell, the specific steps are as follows:
Step 1: the tumor sample and check sample DNA of same individual to be checked are extracted, as detection template；
Step 2: fluorescent PCR expands
According to sample number to be detected (containing 1 positive control and 1 negative control), reaction mixture is calculated and prepares, it will
Prepared detection reaction solution is dispensed into eight PCR reaction tubes respectively；
Step 3: sample to be examined, positive reference substance, negative controls are added in corresponding PCR reaction tube respectively；
Step 4: carry out multiple fluorescence quantitative PCR detection, multiplexed PCR amplification program are as follows: 37 DEG C 2 minutes；94 DEG C 10 points
Clock；94 DEG C 30 seconds, 60 DEG C 1 minute, 70 DEG C 1 minute, 35 circulation；60 DEG C 45 minutes；4℃∞；
Step 5: fluorescence PCR products Capillary Electrophoresis；
Capillary sample is prepared, capillary electrophoresis detection is carried out with Genetic Analyser；
Step 6: detection data is analyzed
Detection data carries out data analysis using analysis software, determines microsatellite instability state according to criterion.
To sum up, a kind of specific primer detecting mankind's microsatellite instability state provided by the invention, the primer
The primer in site and the detection of three control sites is respectively repeated for six mononucleotides.Detection primer is as follows:
BAT-25-Fo:FAM-CTCGCCTCCAAGAATGTAAGT
BAT-25-Re:ctatggctctaaaatgctctgttc
BAT-26-Fo:TAMRA-TGACTACTTTTGACTTCAGCC
BAT-26-Re:AACCATTCAACATTTTTAACCC
NR-21-Fo:VIC-CTTTCTGGTCACTCGCGTTTACAA
NR-21-Re:AAGGGGAGGTAAAGGCAGTCTC
NR-24-Fo:VIC-GCTGAATTTTACCTCCTGAC
NR-24-Re:GGAGATTGTGCCATTGCA
NR-27-Fo:FAM-AACCATGCTTGCAAACCACT
NR-27-Re:TGAGTCGATAATACTAGCAATGACC
MONO-27-Fo:TAMRA-AAGGGTGGATCAAATTTCACTTG
MONO-27-Re:TGGGAGACAGAGCAAGACTCTG
PentaC-Fo:VIC-gaacacactttgcacctgtcag
PentaC-Re:CTGAGCGCTTCTAGGGACTTCT
PentaD-Fo:TAMRA-atttgcctaacctatggtCATAACG
PentaD-Re:ACACTCCAGCCTAGGTGACAGA
Amel-Fo:FAM-ccctgggctctgtaaagaatag
Amel-Re:TTGAGGCCAACCATCAGAGCTTA
A kind of mankind's microsatellite instability state MSI detection kit, component see the table below 1, and the kit is with multiple glimmering
Based on light round pcr, fragment analysis is carried out in conjunction with Capillary Electrophoresis, detects microsatellite instability state in human cell.
Kit provided by the invention detects 6 mononucleotides and repeats site and 2 pentanucleotides repetition sites and 1 sex identification base
Cause, specific detection site see the table below 2.
Table 1: reagent constituents
2 kit detection site of table
The present invention also provides the detection method of mankind's microsatellite instability state MSI a kind of, this method includes using
The reagent of the primer of above-mentioned mankind's microsatellite instability state MSI or above-mentioned detection mankind microsatellite instability state MSI
Box detects the step of mankind's microsatellite instability state.
Detection method carries out sequence fragment using multiple fluorescence quantitative PCR technology combination Capillary Electrophoresis and analyzes people
Microsatellite instability state in class cell, the specific steps are as follows:
(1) tumor sample and check sample DNA for extracting same individual to be checked, as detection template；
(2) fluorescent PCR expands
According to sample number to be detected (containing 1 positive control and 1 negative control), reaction mixture is calculated and prepares, it will
Prepared detection reaction solution is dispensed into eight PCR reaction tubes respectively.
(3) sample to be examined, positive reference substance, negative controls are added in corresponding PCR reaction tube respectively.
(4) carry out multiple fluorescence quantitative PCR detection, multiplexed PCR amplification program are as follows: 37 DEG C 2 minutes；94 DEG C 10 minutes；94
DEG C 30 seconds, 60 DEG C 1 minute, 70 DEG C 1 minute, 35 circulations；60 DEG C 45 minutes；4℃∞.
(5) fluorescence PCR products Capillary Electrophoresis
Capillary sample is prepared, capillary electrophoresis detection is carried out with Genetic Analyser.
(6) detection data is analyzed
Detection data carries out data analysis using analysis software, determines microsatellite instability state according to criterion
2.3 technical solution of the present invention bring beneficial effects
Compared with prior art, the present invention has the following advantages:
(1) sample size needed for is few, is detected using multiple fluorescence PCR technology, and decontamination system and internal control system are added,
More accurate, steadily sample can be detected, it is ensured that real result is credible；
(2) the control test site containing 2 fermentation by five tubes and 1 sex identification control site, avoid sample
Originally obscure mistake.
(3) easy to operate quick, and result interpretation is simply objective, convenient for analysis.
Detailed description of the invention
In order to more clearly explain the embodiment of the invention or the technical proposal in the existing technology, to embodiment or will show below
There is attached drawing needed in technical description to be briefly described, it should be apparent that, the accompanying drawings in the following description is only this
Some embodiments of invention without any creative labor, may be used also for those of ordinary skill in the art
To obtain other drawings based on these drawings.
Fig. 1 is cancer side normal control tissue peak figure in the embodiment of the present invention 3；
Fig. 2 is tumor tissues peak figure in the embodiment of the present invention 3.
Specific embodiment
Below in conjunction with the drawings and specific embodiments, the present invention will be described in detail, in following embodiment unless otherwise specified, institute
Using raw material sources in conventional practices commercially available, that used method is well known to those skilled in the art.
A kind of embodiment 1: mankind's microsatellite instability state MSI detection kit
Specific primer is designed according to target gene sequence, the primer and probe of design can carry out people according to existing method
Work synthesis.Specific primer is as follows:
BAT-25-Fo:FAM-CTCGCCTCCAAGAATGTAAGT
BAT-25-Re:ctatggctctaaaatgctctgttc
BAT-26-Fo:TAMRA-TGACTACTTTTGACTTCAGCC
BAT-26-Re:AACCATTCAACATTTTTAACCC
NR-21-Fo:VIC-CTTTCTGGTCACTCGCGTTTACAA
NR-21-Re:AAGGGGAGGTAAAGGCAGTCTC
NR-24-Fo:VIC-GCTGAATTTTACCTCCTGAC
NR-24-Re:GGAGATTGTGCCATTGCA
NR-27-Fo:FAM-AACCATGCTTGCAAACCACT
NR-27-Re:TGAGTCGATAATACTAGCAATGACC
MONO-27-Fo:TAMRA-AAGGGTGGATCAAATTTCACTTG
MONO-27-Re:TGGGAGACAGAGCAAGACTCTG
PentaC-Fo:VIC-gaacacactttgcacctgtcag
PentaC-Re:CTGAGCGCTTCTAGGGACTTCT
PentaD-Fo:TAMRA-atttgcctaacctatggtCATAACG
PentaD-Re:ACACTCCAGCCTAGGTGACAGA
Amel-Fo:FAM-ccctgggctctgtaaagaatag
Amel-Re:TTGAGGCCAACCATCAGAGCTTA
A kind of mankind's microsatellite instability state MSI detection kit of the invention includes following three component, is respectively as follows:
MSI primer mixed liquor: comprising 6 mononucleotides repeat site and 2 pentanucleotides repeat site and 1 for detecting
The primer of a sex identification gene, it is respectively BAT-25, BAT-26, NR-21, NR-24, NR- that 6 mononucleotides, which repeat site,
27, MONO-27, it is PentaC, PentaD that 2 pentanucleotides, which repeat site, and sex identification gene is Amel.Primer is institute as above
State sequence.
PCR premixed liquid includes UDG enzyme, archaeal dna polymerase, PCR buffer, MgCl2, dNTPs and dUTP.
Positive reference substance is cell line HT-29 genomic DNA；Negative controls are sterilizing ultrapure water.
Embodiment 2: mankind's microsatellite instability condition detection method
Mankind's microsatellite instability state is detected using the kit of the embodiment of the present invention 1, comprising the following steps:
(1) sample to be tested processing and template extraction；
Same individual tumor sample (paraffin embedding tumor tissues) and check sample (paraffin embedding cancer beside organism) are according to mentioning
Kit specification is taken to extract DNA.The DNA of extraction is diluted to after 20ng-100ng/ μ L as PCR amplification template.
(2) system is prepared
All components in kit are melted on ice, according to sample number to be detected, calculates and prepares PCR reaction mixture
(being shown in Table 3).
3 PCR reaction solution of table prepares formula
PCR reaction working solution is added to PCR reaction tube by 20 holes μ L/, carries out PCR amplification.
(3) PCR amplification
PCR amplification program is shown in Table 4
4 PCR amplification program of table
(4) Capillary Electrophoresis
Capillary electrophoresis detection sample is prepared according to table 5, Capillary Electrophoresis inspection is carried out with seqstudio Genetic Analyser
It surveys, is selected " Fragment Analysis " to detect according to Genetic Analyser operating instruction.Export data GeneMapper
Software analysis.
5 Capillary Electrophoresis sample preparation of table
| Component | Additional amount (μ L) |
| Deionized formamide HiDi | 8.5 |
| ROX500 | 0.3 |
| PCR product | 1 |
(5) interpretation of result
Endogenous control ROX500 clip size mark is accurate, be followed successively by 70,80,100,120,140,160,180,200,
240,280,320,360,400,450,490 and 500nt.
There is not superthreshold phenomenon in detection peak height, otherwise should dilute PCR product and carry out capillary electrophoresis detection.
Negative controls are without product amplification, and 9 biomarkers of positive reference substance have product amplification, and clip size exists
In zone of reasonableness.
The two pentanucleotide marker Penta C and Penta D and gender of same individual tumor tissues and control tissue
It identifies that Gene A mel amplified fragments size is consistent, shows that tumor tissues and cancer side compare and derive from same patient.With normal
Tissue peak figure compares, comparison of tumor tissue 6 mononucleotide markers BAT-26, NR-21, BAT-25, MONO-27, NR-27
Change situation with NR-24 clip size, the variation of product segment determines that the mrna instability is fixed more than 2.5nt, otherwise stablizes.According to
Same tissue tumor sample and the unstable mononucleotide of check sample repeat the number in site, determine the sample microsatellite state,
Microsatellite instability judgment criteria part table 6.
6 microsatellite instability judgment criteria of table
The practical application of 3 detection method of embodiment
Using kit of the present invention and detection method detection tumor patient microsatellite instability detection, the method is as follows: (1)
The processing of sample to be examined
FFPE tissue uses QIAamp DNA FFPE Tissue Kit by an example sample tumour and cancer, says according to kit
Sample DNA is extracted in bright book operation.It is spare that DNA concentration is diluted to 20ng/ μ L respectively.
(2) system is prepared
All components in kit are melted on ice, according to sample number to be detected, calculates and prepares PCR reaction mixture
(being shown in Table 7).
7 PCR reaction solution of table is with tabulation
| Serial number | Component | Additional amount (μ L) |
| 1 | MSI primer mixed liquor | 3×4 |
| 2 | PCR premixed liquid | 12.5×4 |
| 3 | Nuclease free pure water | 4.5×4 |
| 4 | Template | 2.5μL |
PCR reaction working solution is added to PCR reaction tube by 20 holes μ L/, carries out PCR amplification.
(3) PCR amplification
PCR amplification program is shown in Table 4
4 PCR amplification program of table
(5) Capillary Electrophoresis
Capillary electrophoresis detection sample is prepared according to table 5, Capillary Electrophoresis inspection is carried out with seqstudio Genetic Analyser
It surveys, is selected " Fragment Analysis " to detect according to Genetic Analyser operating instruction.Export data GeneMapper
Software analysis.
5 Capillary Electrophoresis sample preparation of table
| Component | Additional amount (μ L) |
| Deionized formamide HiDi | 8.5 |
| ROX500 | 0.3 |
| PCR product | 1 |
(6) interpretation of result
In Capillary Electrophoresis figure, Fig. 1 is normal control tissue peak figure by cancer, and Fig. 2 is tumor tissues peak figure, and abscissa represents
Amplified production clip size, ordinate represent amplified production amount (fluorescence intensity).Negative controls go out without product amplification, positive right
There is product amplification to go out according to 9 biomarkers of product, and clip size is in reasonable range.Tumor tissues and normal tissue
Two pentanucleotide marker Penta C and Penta D and sex identification gene magnification Amle clip size it is consistent, show
It is compareed by tumor tissues and cancer and derives from same patient.It is compared with normal tissue peak figure, 2 mononucleotide marks of tumor tissues
Will object NR-21 occurs 5bp clip size and changes, and NR-25 occurs 8bp clip size and changes, the microsatellite instability of sample
MSI state is the high unstability of microsatellite (MSI-H).
In addition, it should be noted that, the specific embodiments described in this specification, source chemicals title etc. can not
Together.The equivalent or simple change that all principles described according to the invention patent design are done, is included in the protection of the invention patent
In range.Those skilled in the art described specific embodiment can be made it is various modification or
It supplements or is substituted in a similar manner, without departing from structure of the invention or surmount model defined in the claims
It encloses, is within the scope of protection of the invention.
Claims (3)
1. a kind of mankind's microsatellite instability state MSI detection kit, it is characterised in that: the detection kit includes
The primer in site and the detection of three control sites is repeated for six mononucleotides, the detection primer is as follows:
BAT-25-Fo:FAM-CTCGCCTCCAAGAATGTAAGT
BAT-25-Re:ctatggctctaaaatgctctgttc
BAT-26-Fo:TAMRA-TGACTACTTTTGACTTCAGCC
BAT-26-Re:AACCATTCAACATTTTTAACCC
NR-21-Fo:VIC-CTTTCTGGTCACTCGCGTTTACAA
NR-21-Re:AAGGGGAGGTAAAGGCAGTCTC
NR-24-Fo:VIC-GCTGAATTTTACCTCCTGAC
NR-24-Re:GGAGATTGTGCCATTGCA
NR-27-Fo:FAM-AACCATGCTTGCAAACCACT
NR-27-Re:TGAGTCGATAATACTAGCAATGACC
MONO-27-Fo:TAMRA-AAGGGTGGATCAAATTTCACTTG
MONO-27-Re:TGGGAGACAGAGCAAGACTCTG
PentaC-Fo:VIC-gaacacactttgcacctgtcag
PentaC-Re:CTGAGCGCTTCTAGGGACTTCT
PentaD-Fo:TAMRA-atttgcctaacctatggtCATAACG
PentaD-Re:ACACTCCAGCCTAGGTGACAGA
Amel-Fo:FAM-ccctgggctctgtaaagaatag
Amel-Re:TTGAGGCCAACCATCAGAGCTTA.
2. mankind's microsatellite instability state MSI detection kit according to claim 1, it is characterised in that: described
Detection kit include MSI primer mixed liquor, PCR premixed liquid, negative controls, positive reference substance；
The MSI primer mixed liquor include for detect 6 mononucleotides repeat sites and 2 pentanucleotides repeat sites and
The primer of 1 sex identification gene, it is respectively BAT-25, BAT-26, NR-21, NR-24, NR- that 6 mononucleotides, which repeat site,
27, MONO-27, it is PentaC, PentaD that 2 pentanucleotides, which repeat site, and sex identification gene is Amel；
The PCR premixed liquid includes UDG enzyme, archaeal dna polymerase, PCR buffer, MgCl2, dNTPs and dUTP；
The negative controls are sterilizing ultrapure water；
The positive reference substance is cell line HT-29 genomic DNA.
3. a kind of detection method of mankind's microsatellite instability state MSI, it is characterised in that: the detection method includes making
With the kit detection of the primer or detection mankind's microsatellite instability state MSI of mankind's microsatellite instability state MSI
The step of mankind's microsatellite instability state；
The detection method carries out sequence fragment using multiple fluorescence quantitative PCR technology combination Capillary Electrophoresis and analyzes human cell
Middle microsatellite instability state, the specific steps are as follows:
Step 1: the tumor sample and check sample DNA of same individual to be checked are extracted, as detection template；
Step 2: fluorescent PCR expands
According to sample number to be detected, reaction mixture is calculated and prepared, prepared detection reaction solution is dispensed into eight respectively
PCR reaction tube；
Step 3: sample to be examined, positive reference substance, negative controls are added in corresponding PCR reaction tube respectively；
Step 4: carry out multiple fluorescence quantitative PCR detection, multiplexed PCR amplification program are as follows: 37 DEG C 2 minutes；94 DEG C 10 minutes；94
DEG C 30 seconds, 60 DEG C 1 minute, 70 DEG C 1 minute, 35 circulations；60 DEG C 45 minutes；4℃∞；
Step 5: fluorescence PCR products Capillary Electrophoresis；
Capillary sample is prepared, capillary electrophoresis detection is carried out with Genetic Analyser；
Step 6: detection data is analyzed
Detection data carries out data analysis using analysis software, determines microsatellite instability state according to criterion.
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| CN109830265A (en) * | 2019-01-18 | 2019-05-31 | 臻悦生物科技江苏有限公司 | Detect kit, reference database, preparation method and the application of MSI |
| CN110106241A (en) * | 2019-06-27 | 2019-08-09 | 南京科佰生物科技有限公司 | The standard items and its preparation method and application of MSI microsatellite instability detection |
| CN110218772A (en) * | 2019-06-10 | 2019-09-10 | 深圳海普洛斯医学检验实验室 | A kind of Primer composition, kit, detection method and its application detecting microsatellite instability |
| CN114085895A (en) * | 2021-11-30 | 2022-02-25 | 普瑞斯新(上海)生物医疗科技有限公司 | Detection primer for rapidly detecting MSI and kit thereof |
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