CN107299139A - A kind of composition and its application for being used to detect microsatellite instability - Google Patents

A kind of composition and its application for being used to detect microsatellite instability Download PDF

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CN107299139A
CN107299139A CN201710612301.3A CN201710612301A CN107299139A CN 107299139 A CN107299139 A CN 107299139A CN 201710612301 A CN201710612301 A CN 201710612301A CN 107299139 A CN107299139 A CN 107299139A
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刘沛
王林海
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BEIJING SINO-MDGENE TECHNOLOGY Co Ltd
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Abstract

The invention discloses a kind of composition and its application for being used to detect microsatellite instability, for detecting that the composition of microsatellite instability includes being directed to the primer pair for being used for detecting specific target gene related to microsatellite instability in measuring samples respectively, for detecting the kit of microsatellite instability using specific multiple fluorescence PCR fragment analysis capillary electrophoresis detection Bat 26, Bat 25, NR 21, NR 24, NR 27, Mono 27, D5S346, this 9 sites of D2S123 and D17S250 and Penta C, Penta D, the microsatellite distribution situation in this 3 sites of Amel, for judging that microsatellite is in stable or unstable state.Proportioning, the consumption of Taq enzyme, the consumption of magnesium ion and PCR system by adjusting 12 pairs of primers etc., improve the Sensitivity and Specificity of whole kit.

Description

A kind of composition and its application for being used to detect microsatellite instability
Technical field
The invention belongs to technical field of gene detection, and in particular to a kind of technology for being used to detect microsatellite instability.
Background technology
The inherent cause that colorectal cancer (Colorectal Cancer, CRC) occurs includes chromosome instability (Chromosome Instability, CIN) and microsatellite instability (Microsatellite Instability, MSI). About 80-85% CRC is caused by CIN, including familial adenomatous polyposis (Familial adenomatous Polyposis, FAP) (apc gene germline mutation) and sporadic CRC (gene mutation such as APC, P53, DCC, KRAS)；And in addition 15-20% CRC is then mainly caused by MSI, including hereditary nonpolyposis colorectal cancer (Hereditary Nonpolyposis Colorectal Cancer, HNPCC, also known as Lynch syndromes) (mismatch repair gene germline mutation) and Sporadic MSI (+) CRC (mismatch repair gene MLH1 gene promoter methylations).
Microsatellite instability (Microsatallite Instability, MSI) shows as same microsatellite locus not Between same individual and between same individual normal structure and some abnormal structures, the number of the recurring unit of microsatellite locus It is different.Numerous studies in recent years show：With tumour closely related, such as colorectal cancer, stomach cancer, carcinoma of endometrium occur for MSI. Especially colorectal cancer, clinically regard MSI as colorectal cancer prognosis and the important molecule mark of formulation supplemental treatment regimens Thing.According to the literature, there is MSI phenomenons in about 15% colorectal cancer, compared with the colorectal cancer without MSI, carry MSI-H II phase colorectal cancer patients prognosis preferably, but benefits less from 5-FU NACTs.
Lynch syndromes, also known as hereditary nonpolyposis colorectal cancer (HNPCC), are to be mutated institute by MMR germlines The dominant hereditary disease of cause.More than 90% Lynch syndromes have MSI features, and there was only about 15% in Sporadic Colorectal Carcinoma, So clinically the examination of Lynch syndromes can be carried out with MSI detections.
The NCT01876511 results of study delivered in New England Journal of Medicine for 2015 show, the treatment pair of PD-1 monoclonal antibodies MSI-H mCRC shows high remission rate, therefore PEMBRO monoclonal antibodies treatment MSI-H mCRC obtains FDA breakthrough sex therapy identifications. ASCO in 2016 can go up the CheckMate-142 of issue, and the as shown by data of II phase research, MSI-H mCRC patient not only may be used Benefit from PD-1 immunization therapies, and clinical activity to PD-1 and CTLA-4 therapeutic alliances and life cycle are all considerably beyond list Solely using PD-1 curative effect.On May 23rd, 2017, U.S. FDA have approved " star " PD-1 antibody of MSD Corp. Pembrolizumab (trade names：Keytruda) it is used to treat and carries a kind of any solid tumor of specific gene feature, tool For body, Keytruda is approved for the adult and childhood solid tumor patient that treatment carries " MSI-H/dMMR hypotypes ".
1997, NCI recommended Bat 26, Bat 25,5 site primers of D5S346, D2S123 and D17S250 and swollen Knurl, is high-frequency MSI (MSI-H) if wherein 2 or more than 2 sites have change, and it is then low frequency to have 1 site to change MSI (MSI-L), does not change and is then referred to as microsatellite stabilization (MSS).In NCI seminars in 2002, due to dinucleotide The sensitivity and specificity detection in D5S346, D2S123 and D17S250 site is limited, is below mononucleotide, have modified MSI inspections Guide is surveyed, more mononucleotides is mainly recommended and is detected (in the case of dinucleotide is unstable).Research is reported at present There are Bat 26, Bat 25, NR-21, NR-24, NR-27, Mono-27 in road, the mononucleotide site of detection, and dinucleotide site has D5S346, D2S123 and D17S250.
Therefore, microsatellite instability detection is significant for colorectal cancer prognostic evaluation and chemotherapy side effect.And Detection MSI nucleotide primer and method Sensitivity and Specificity are relatively low at present, it is difficult in wide clinical application.So exploring one The focus that the fast and reliable nucleotide primer group of kind detection microsatellite instability and method have become clinical and experimental study is asked Topic.
The content of the invention
It is an object of the invention to provide a kind of composition and its application for being used to detect microsatellite instability, pass through adjustment Consumption of the proportioning of 12 pairs of primers, the consumption of Taq enzyme and magnesium ion etc., 12 pairs of primers are placed into a pipe and detected, only It need to detect once and can be while detecting the distribution situation in 12 sites.Further by the adjustment of above-mentioned system, improve whole The Sensitivity and Specificity of kit, Sensitivity and Specificity is able to reach 95% and 100%, solves and detects micro- in the past The problem of Sensitivity and Specificity is relatively low during satellite unstability.
To achieve these goals, a kind of composition for being used to detect microsatellite instability that the present invention is provided, it is described Composition includes being directed to respectively and is used to detect drawing for specific target gene related to microsatellite instability in measuring samples Thing pair, including：
NR-21 gene forward primers F (NR-21-F)：FAM-GAAGACACATCCCTTT,
NR-21 gene reverse primer R (NR-21-R)：CGCATTCACACTTTC；
NR-27 gene forward primers F (NR-27-F)：FAM-AGGGGGTGGAGATTGC,
NR-27 gene reverse primer R (NR-27-R)：CATAGGGCAAGCAGAAA；
Penta D gene forward primers F (Penta D-F)：FAM-AGTTCCTGTCTCTCTGCCA,
Penta D gene reverse primers R (Penta D-R)：GCTGTAGCCTTTTGCC；
D5S346 gene forward primers F (D5S346-F)：FAM-GCAGTTAGTTAGTTAGTT,
D5S346 gene reverse primer R (D5S346-R)：AGTTTATAGCAGATAAGACAG；
BAT-25 gene forward primers F (BAT-25-F)：HEX-GCAAAGGGCATGGC,
BAT-25 gene reverse primer R (BAT-25-R)：GCTTTTAACTATGGCTCT；
BAT-26 gene forward primers F (BAT-26-F)：HEX-TCAGCCAGTATATGAAATTG,
BAT-26 gene reverse primer R (BAT-26-R)：AGCTCCTTTCTAAGCCTTCT；
Penta C gene forward primers F (Penta C-F)：HEX-AGCAGAGGTTTTCTAAATA,
Penta C gene reverse primers R (Penta C-R)：AAGTCGTCTTTGGACAGG；
D2S123 gene forward primers F (D2S123-F)：HEX-GACAAAAACAGGATGCCT,
D2S123 gene reverse primer R (D2S123-R)：GGGACTTTCCACCTATGGGAC；
MONO-27 gene forward primers F (MONO-27-F)：TMR-GTGGGAGACAGAGCA,
MONO-27 gene reverse primer R (MONO-27-R)：TGGCCAACTAAAAAAGAA；
NR-24 gene forward primers F (NR-24-F)：TMR-AATTTTACCTCCTGACTCCAA,
NR-24 gene reverse primer R (NR-24-R)：CAACCTGGGTGACAGAGT；
Amel gene forward primers F (Amel-F)：TMR-GGCGCGCGACACCGCAG,
Amel gene reverse primer R (Amel-R)：CCCTTTCTCAAGGGAACC；
D17S250 gene forward primers F (D17S250-F)：TMR-CATACATAAACTTTCAAAT,
D17S250 gene reverse primer R (D17S250-R)：GGTTTAAATATATATATGGC.
Preferably, to 5 ' ends of the forward primer of NR-21 genes, NR-27 genes, Penta D genes and D5S346 genes Carry out FAM modifications；Held to the 5 ' of the forward primer of BAT-25 genes, BAT-26 genes, Penta C genes and D2S123 genes into Row HEX is modified；5 ' ends of the forward primer of MONO-27 genes, NR-24 genes, Amel genes and D17S250 genes are carried out TMR is modified；Realize the detection in multi-fluorescence site in a pipe.
Preferably, when carrying out one-time detection, NR-21-F addition 0.1-0.3 μ L, NR-21-R addition 0.1-0.3 μ L, NR-27-F addition 0.01-0.2 μ L, NR-27-R addition 0.01-0.2 μ L, Penta D-F addition 0.1-0.3 μ L, Penta D-R addition 0.1-0.3 μ L, D5S346-F addition 0.01-0.2 μ L, D5S346-R addition 0.01-0.2 μ L, BAT-25-F addition 0.1-0.3 μ L, BAT-25-R addition 0.1-0.3 μ L, BAT-26-F addition 0.1-0.3 μ L, BAT- 26-R addition 0.1-0.3 μ L, Penta C-F addition 0.01-0.2 μ L, Penta C-R addition 0.01-0.2 μ L, D2S123-F addition 0.1-0.3 μ L, D2S123-R addition 0.1-0.3 μ L, MONO-27-F addition 0.01-0.2 μ L, MONO-27-R addition 0.01-0.2 μ L, NR-24-F addition 0.01-0.2 μ L, NR-24-R addition 0.01-0.2 μ L, Amel-F addition 0.1-0.3 μ L, Amel-R addition 0.1-0.3 μ L, D17S250-F addition 0.01-0.2 μ L, D17S250-R addition 0.01-0.2 μ L.
A kind of kit for being used to detect microsatellite instability that the present invention is provided, the kit includes described above Be used for detect the composition of microsatellite instability.
Preferably, the kit includes archaeal dna polymerase (Taq enzyme), dNTPs, 10 × archaeal dna polymerase buffer, UDG enzyme And Mg2+, when carrying out one-time detection, archaeal dna polymerase addition 0.1-0.4 μ L, dNTPs addition 1-3 μ L, 10 × archaeal dna polymerase Buffer addition 1-4 μ L, UDG enzyme addition 0.01-0.2 μ L, Mg2+Addition 2-5 μ L.
A kind of sample processing method for being used to detect the kit of microsatellite instability that the present invention is provided, the sample Processing method includes：
(1) sample is extracted, check sample and detection sample is extracted, wherein check sample can select whole blood sample, detection Sample can select FFPE tumor tissues sample genomic dna；
(2) sample after extraction is subjected to concentration mensuration, and determined fluorescence after concentration dilution to 10-20ng/ μ L, is carried out Measure PCR reactions；
(3) interpretation of result after being reacted according to PCR：Read 12 target gene sites in check sample and detection sample Fragment distribution situation, and then judge detection sample whether there is microsatellite instability state.
Preferably, PCR courses of reaction are：UDG enzyme reactions 2min at 37 DEG C, 95 DEG C of pre-degenerations 3min, 94 DEG C of denaturation 15s, 60 DEG C of annealing extension 45s, 45 circulations.
A kind of composition and its application for being used to detect microsatellite instability that the present invention is provided, with following beneficial effect Really：
Comprehensive detection mononucleotide of the present invention repeats site Bat 26, Bat 25, NR-21, NR-24, NR-27, Mono-27 Site D5S346, D2S123, D17S250 are repeated with dinucleotide, detection specificity and the sensitivity of kit is improved.And again this On the basis of detection, this kit increase pentanucleotide Repeat Polymorphism Penta C and Penta D and Sex Determination site Amel Detection, it is intended to whether there is pollution condition in monitor sample detection process.This kit improves the comprehensive of detection site, Monitor sample pollution course is added simultaneously, kit detection stability and reliability is further improved.
The present invention carries out related gene detection using this technology, and easy to operate, be easy to interpretation, the requirement to instrument is not Height, and whole PCR processes use totally-enclosed form, it is to avoid the possibility of cross pollution, makes result more accurate.
One tube method detection is carried out based on multiple fluorescence PCR fragment analysis capillary electrophoresis, i.e., can be by a pipe simultaneously Several or even more than ten of site is detected, the simplicity of detection is improved, and then reaches and precisely effectively detects in purpose site.
Requirement of the present invention to detecting sample in detection process is strict, and each detection process is required for check sample Participate in, i.e. check sample and detection sample is detected simultaneously.So increase the accuracy and uniformity of subsequent result interpretation.
The consumption of proportioning, the consumption of Taq enzyme and magnesium ion of the present invention by adjusting 12 pairs of primers etc., improves whole reagent The Sensitivity and Specificity of box, Sensitivity and Specificity is able to reach 95% and 100%.
Brief description of the drawings
Fig. 1 is the gene peak of 12 detection sites of check sample in the present embodiment 1.
Embodiment
In order that those skilled in the art more fully understand the present invention program, with reference to embodiment to this hair It is bright to be described in further detail.
Related locus proposed by the present invention：6 mononucleotides repeat site Bat 26, Bat 25, NR-21, NR-24, NR- 27、Mono-27；3 dinucleotides repeat site D5S346, D2S123, D17S250；2 pentanucleotide Repeat Polymorphisms Penta C, Penta D and Sex Determination site Amel.
Embodiment 1：The present invention is configured to pre- mixed 12 detecting positions of detection on the basis of multiple fluorescence PCR The Primer composition of point.It can directly be detected to extracting the sample completed, so make detecting step more easy.
Comprise the following steps that：
1) check sample (such as whole blood sample) and FFPE tumor tissues sample (5-10 pieces, 5um or so) gene is extracted Group DNA (using commercialized peripheral blood genomic DNA and paraffin-embedded tissue genome DNA extracting reagent kit).
2) genomic DNA after extraction is subjected to concentration mensuration, and concentration dilution to 10-20ng/ μ L is carried out subsequently PCR courses of reaction.
3) the composition composition of reaction system：dNTPs、Mg2+, archaeal dna polymerase buffer, reverse transcription buffer, 12 genes Primer pair, archaeal dna polymerase etc..
4) PCR reaction conditions：UDG enzyme reactions 2min at 37 DEG C；95 DEG C of pre-degeneration 3min；94 DEG C of denaturation 15s, are moved back at 60 DEG C Fire extension 45s, 45 circulations.
5) configuration of PCR reaction systems：
PCR amplification MIX (12.5 μ L, remaining uses purified water polishing)
PCR primer mixture system (6.5 μ L, remaining uses purified water polishing)
PCR amplification system
PCR amplification programs are as follows：
First amplification stage：37 DEG C of UDG enzyme reactions 2min；
Second amplification stage：95 DEG C of pre-degeneration 3min；
3rd amplification stage：94 DEG C of denaturation 15s, 60 DEG C of annealing extension 45s, 45 circulations.
6) the μ L of 1 μ L, LIZ-500 molecular weight internal standard of amplified production, 1 μ L and Hi-Di Formamide (high-purity formamide) 8 are taken It is placed in 0.2mL PCR single tubes mesoscale eddies to vibrate 10 seconds, 2000rpm is centrifuged 15 seconds.Illustrate according to the loading of Capillary Electrophoresis machine, Detected on suitable capillary electrophoresis detection machine.
7) interpretation of result
1. first analysis sex site Amel, Penta C, Penta D, Amel site is regular at 206bp or 212bp Allele peak, Penta C and Penta D has well-regulated allele peak, and this three is used to confirm tumor tissues and right Whether the normal sample answered is same source, then carries out subsequent analysis, otherwise there may be sample and mixes or pollute, it is proposed that weight It is new to extract sample row detection again.
2. microsatellite stable type (MSS)：Do not changed in 6 mononucleotide repetition flag things, be rule etc. Position gene peak.
3. microsatellite minuent instability mode (MSI-L)：Only have 1 in 6 mononucleotide repetition flag things to change, its Remaining is the allele peak of rule.
4. the highly unstable type of microsatellite (MSI-H)：There are >=2 to change in 6 mononucleotide repetition flag things, remaining It is the allele peak of rule.
5. check sample (such as whole blood sample) belongs to microsatellite stable type (MSS).
6. detection sample (FFPE tumor tissues) compares mononucleotide repetition flag thing according to check sample and changed The number of change, carries out result interpretation.
8) performance verification of finished product kit
The finished product kit completed using configuration carries out product specificity and sensitivity technique.
40, the colorectal cancer sample of known MSI types is collected, specific detection, knot are carried out using above-mentioned finished product kit Fruit is as follows：
It can draw from the above, the detection accuracy of this kit reaches 100%, and specificity also reaches 100%.
Detected using this kit occur about 1%, 5%, 20%, 50%, 100%MSI-H sample detected, this examination Agent box lowest detection distribution proportion is to 5%, and therefore, the detection sensitivity of this kit can reach 95% or so.
Embodiment 2：
Collect 400 random colorectal cancer samples and 400 corresponding peripheral blood control samples from Wuhan hospital of Tongji University This, is detected using above-mentioned finished product kit.Concrete operation step is as described in case study on implementation 1.400 random colorectal cancers The sample of 60 MSI-H types, the sample of 13 MSI-L types are detected in sample, remaining sample is MSS types.According to The above results can show that the ratio for showing colorectal cancer of microsatellite instability accounts for 18%, with corresponding document report It is consistent.
Specific case used herein is elaborated to inventive concept, and the explanation of above example is only intended to Help to understand core concept of the invention.It should be pointed out that for those skilled in the art, not departing from this On the premise of inventive concept, any obvious modification, equivalent substitution or other improvement made should be included in the present invention Protection domain within.
SEQUENCE LISTING
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<120>A kind of composition and its application for being used to detect microsatellite instability
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Claims (8)

1. a kind of composition for being used to detect microsatellite instability, it is characterised in that the composition is included respectively for using The primer pair of the specific target gene related to microsatellite instability in detection measuring samples, including：
NR-21 gene forward primers F (NR-21-F)：SEQ ID NO.1,
NR-21 gene reverse primer R (NR-21-R)：SEQ ID NO.2；
NR-27 gene forward primers F (NR-27-F)：SEQ ID NO.3,
NR-27 gene reverse primer R (NR-27-R)：SEQ ID NO.4；
Penta D gene forward primers F (Penta D-F)：SEQ ID NO.5,
Penta D gene reverse primers R (Penta D-R)：SEQ ID NO.6；
D5S346 gene forward primers F (D5S346-F)：SEQ ID NO.7,
D5S346 gene reverse primer R (D5S346-R)：SEQ ID NO.8；
BAT-25 gene forward primers F (BAT-25-F)：SEQ ID NO.9,
BAT-25 gene reverse primer R (BAT-25-R)：SEQ ID NO.10；
BAT-26 gene forward primers F (BAT-26-F)：SEQ ID NO.11,
BAT-26 gene reverse primer R (BAT-26-R)：SEQ ID NO.12；
Penta C gene forward primers F (Penta C-F)：SEQ ID NO.13,
Penta C gene reverse primers R (Penta C-R)：SEQ ID NO.14；
D2S123 gene forward primers F (D2S123-F)：SEQ ID NO.15,
D2S123 gene reverse primer R (D2S123-R)：SEQ ID NO.16；
MONO-27 gene forward primers F (MONO-27-F)：SEQ ID NO.17,
MONO-27 gene reverse primer R (MONO-27-R)：SEQ ID NO.18；
NR-24 gene forward primers F (NR-24-F)：SEQ ID NO.19,
NR-24 gene reverse primer R (NR-24-R)：SEQ ID NO.20；
Amel gene forward primers F (Amel-F)：SEQ ID NO.21,
Amel gene reverse primer R (Amel-R)：SEQ ID NO.22；
D17S250 gene forward primers F (D17S250-F)：SEQ ID NO.23,
D17S250 gene reverse primer R (D17S250-R)：SEQ ID NO.24.
2. the composition according to claim 1 for being used to detect microsatellite instability, it is characterised in that to NR-21 bases 5 ' ends of the forward primer of cause, NR-27 genes, Penta D genes and D5S346 genes carry out FAM modifications；To BAT-25 genes, 5 ' ends of the forward primer of BAT-26 genes, Penta C genes and D2S123 genes carry out HEX modifications；To MONO-27 genes, 5 ' ends of the forward primer of NR-24 genes, Amel genes and D17S250 genes carry out TMR modifications；Realize multi-fluorescence in a pipe The detection in site.
3. the composition according to claim 1 for being used to detect microsatellite instability, it is characterised in that once examined During survey, NR-21-F addition 0.1-0.3 μ L, NR-21-R addition 0.1-0.3 μ L, NR-27-F addition 0.01-0.2 μ L, NR-27-R addition 0.01-0.2 μ L, Penta D-F addition 0.1-0.3 μ L, Penta D-R addition 0.1-0.3 μ L, D5S346-F addition 0.01-0.2 μ L, D5S346-R addition 0.01-0.2 μ L, BAT-25-F addition 0.1-0.3 μ L, BAT-25-R addition 0.1-0.3 μ L, BAT-26-F addition 0.1-0.3 μ L, BAT-26-R addition 0.1-0.3 μ L, Penta C-F addition 0.01-0.2 μ L, Penta C-R addition 0.01-0.2 μ L, D2S123-F addition 0.1-0.3 μ L, D2S123- R addition 0.1-0.3 μ L, MONO-27-F addition 0.01-0.2 μ L, MONO-27-R addition 0.01-0.2 μ L, NR-24-F Addition 0.01-0.2 μ L, NR-24-R addition 0.01-0.2 μ L, Amel-F addition 0.1-0.3 μ L, Amel-R additions 0.1-0.3 μ L, D17S250-F addition 0.01-0.2 μ L, D17S250-R addition 0.01-0.2 μ L.
4. being used for according to any one of claim 1-3 detects that the composition of microsatellite instability is being prepared for examining Application in the instable reagent of micrometer satellite.
5. a kind of kit for being used to detect microsatellite instability, it is characterised in that the kit includes claim 1-3 Any one of be used for detect the composition of microsatellite instability.
6. the kit according to claim 5 for being used to detect microsatellite instability, it is characterised in that the kit Including archaeal dna polymerase (Taq enzyme), dNTPs, 10 × archaeal dna polymerase buffer, UDG enzyme and Mg2+, when carrying out one-time detection, DNA Polymerase addition 0.1-0.4 μ L, dNTPs addition 1-3 μ L, 10 × archaeal dna polymerase buffer addition 1-4 μ L, UDG enzymes add Enter amount 0.01-0.2 μ L, Mg2+Addition 2-5 μ L.
7. a kind of sample processing method according to claim 6 for being used to detect the kit of microsatellite instability, its It is characterised by, the sample processing method includes：
(1) sample is extracted, check sample and detection sample is extracted, wherein check sample can select whole blood sample, detect sample FFPE tumor tissues sample genomic dna can be selected；
(2) sample after extraction is subjected to concentration mensuration, and by after concentration dilution to 10-20ng/ μ L, carries out fluorescent quantitation PCR reacts；
(3) interpretation of result after being reacted according to PCR：Read the piece in 12 target gene sites in check sample and detection sample Section distribution situation, and then judge whether detection sample microsatellite instability state occurs.
8. the sample processing method according to claim 7 for being used to detect the kit of microsatellite instability, its feature It is, PCR courses of reaction are：UDG enzyme reactions 2min at 37 DEG C, 95 DEG C of pre-degeneration 3min, 94 DEG C of denaturation 15s, 60 DEG C of annealing are prolonged Stretch 45s, 45 circulations.
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