CN108315426A

CN108315426A - Marker combination, primer sets and the kit of microsatellite sequence Detection of Stability

Info

Publication number: CN108315426A
Application number: CN201810323302.0A
Authority: CN
Inventors: 王明旭; 王冬; 石旺
Original assignee: Beijing Cigna One Hundred Physicians From A Family For Generations Learn Co Ltd Of Inspection Institute
Current assignee: Beijing Cigna One Hundred Physicians From A Family For Generations Learn Co Ltd Of Inspection Institute
Priority date: 2018-04-12
Filing date: 2018-04-12
Publication date: 2018-07-24

Abstract

The invention belongs to biology and medical fields, marker combination, primer sets and kit for microsatellite sequence Detection of Stability is provided, wherein marker combination is made of marker BAT 25, BAT 26, NR21, NR24, NR27 and MONO27, and primer sets are for expanding these markers.The marker combination of the present invention has higher sensitivity, and effect is visual and clear, and more accurately analysis can be provided for the medication guide and prognosis prediction of patient and is suggested.

Description

Marker combination, primer sets and the kit of microsatellite sequence Detection of Stability

Technical field

The invention belongs to biology and medical fields, are related to the auxiliary detection of colorectal cancer.

Background technology

Colorectal cancer (CRC) is worldwide one of the death rate and the higher tumour of incidence, about 25% CRC and Genetic correlation.Hereditary nonpolyposis colorectal cancer (HNPCC) is most important type in heredity colorectal cancer, be it is a kind of by Dominant heredity disease caused by DNA mismatch revision points (MMR) mutation, accounts for about the 3%~5% of CRC.

HNPCC morbidities are early (~45 years old), and disease progression is rapid, but the more other type colorectal cancers of prognosis are good.HNPCC is most heavy The feature wanted is exactly that patient carries MMR gene mutations, and the crowd for carrying MMR gene mutations suffers from the risk of CRC and is up to throughout one's life 80%, while being also the High risk group of the related neoplasms such as carcinoma of endometrium, oophoroma and gastric cancer.Therefore HNPCC early stages Diagnosis is extremely important to patient and its first degree relative, and the diagnosing and treating of early stage can reduce the family member influenced by HNPCC 65% death rate.

The diagnosis of HNPCC can directly be diagnosed by clinical and family investigation and with MMR detection in Gene Mutation. MMR gene mutations are the most important characterization of molecules of HNPCC patient, and MMR defects can cause DNA of tumor cell that microsatellite sequence occurs Unstability (Microsatellite Instability, MSI), microsatellite sequence unstability include highly unstable property (MSI-H) and low unstability (MSI-L), and if microsatellite sequence is stablized, referred to as MSS.HNPCC or HNPCC is related In tumour>90% patient and 15% sporadic CRC in had been found that MSI.

MSI states are also related to CRC prognosis, the more other type patient good prognosis of CRC patient of MSI-H, meanwhile, MSI shapes State is also related to the 5-FU class drug therapy prognosis of CRC patient.It is pointed out in Chinese colorectal cancer diagnosis and treatment specification (version in 2010)：Ⅱ Phase colorectal cancer, there is patients with high risk factors, it is proposed that carries out adjuvant chemotherapy.But before adjuvant chemotherapy, it is proposed that detection MMR gene mutations Or MSI, it is such as MMR defects or MSI-H, does not recommend single medicine adjuvant chemotherapy of fluorouracil drug.

Newest clinical data shows：Metastatic colorectal carcinoma (mCRC) patient of MSI-H is to PD-1 immunization therapies The mCRC patient for showing high remission rate rather than MSI-H does not show curative effect to PD-1 treatments.Therefore, FDA (U.S.'s food And Drug Administration management board) approval " PD-1 immunotherapies " can be used as possess MSI-H metastatic colorectal carcinoma (mCRC) trouble The potential treatment scheme of person.With the ardent development of immunotherapy of tumors (PD-1/PD-L1), as it with one of diagnosis MSI detections are also the necessary detection needed for other solid tumor patients with terminal.It is cheap with examining compared with high immune drug The disconnected possibility that can quickly and accurately assess its drug income, has very high market acceptance.With drawing for external drug Into the success of the clinical test with domestic drug, immunization therapy has become the critical treatment means that these patients continue life, and To be important and necessary for these patients as the MSI with diagnosis.

Invention content

The purpose of the present invention is to provide a kind of new MSI detection modes, can accurately, delicately detect MSI.

The present invention is by being detected BAT 25, BAT 26, NR21, NR24, NR27, MONO27 marker, to auxiliary Help HNPCC diagnosis and CRC patient prognosis evaluation.

The present invention means be：Suitable gene marker is selected, designs specific primer, clip size is between 60- Between 500, when Capillary Electrophoresis, can distinguish different segments.

Therefore, in the first aspect, the present invention provides a kind of combination of marker, by marker BAT 25, BAT 26, NR21, NR24, NR27 and MONO27 are formed.The marker combination is for detecting microsatellite sequence stability.

Wherein, the sequence of each marker is respectively：

BAT25:

Ggctcatacatagaaagagatgtgactcccgccatcatggaggatgacgagttggccctagacttagaagacttgct gagcttttcttaccaggtggcaaagggcatggctttcctcgcctccaagaatgtaagtgggagtgattctctaaaga gttttgtgttttgtttttttgatttttttttttttttttttttttttgagaacagagcattttagagccatagttaa aatgcagaatgtcattttgaagtgtggtaaccaaaagcagaggaaatttagtttcttcatgttccaactgctgtctc tttggaattcctgttctaatttataagctgtaaagtacaagcctgtctaaatgagtttttctatgaatattctttta tatgcagtgaaattcttttaaaactttt(SEQ ID NO:13)；

BAT26:

Tggtatagaaatcttcgatttttaaattcttaattttaggttgcagtttcatcactgtctgcggtaatcaagttttt agaactcttatcagatgattccaactttggacagtttgaactgactacttttgacttcagccagtatatgaaattgg atattgcagcagtcagagcccttaacctttttcaggtaaaaaaaaaaaaaaaaaaaaaaaaaaagggttaaaaatgt tgaatggttaaaaaatgttttcattgacatatactgaagaagcttataaaggagctaaaatattttgaaatattatt atacttggattagataactagctttaaatggctgtatttttctctcccctcctccactccactttttaacttttttt tttttaagtcagagtctcacttgttccct(SEQ ID NO:14)；

NR21:

Gaggtaaaggcagtctcctgttttattagggggagaggtgaagggaaatccaggctcactttctgaataagccactg cctggtgcacagagcagaaccatcctggtttctgaagacacatccctttcagcagaattccagccggagtcgctggc acagttctatttttatatttaaatgtatgtctcccctggcctttttttttttttttttttttagcaacacttttctt gtttgtaaacgcgagtgaccagaaagtgtgaatgcggagtaggaatatttttcgtgttctcttttatctgcttgcct tttttagagagtagcagtggttcctatttcggaaaaggacgttctaattcaaagctctctcccaatatatttacacg aatacgcatttagaaagggaggcagct(SEQ ID NO:15)；

NR24:

Ttcctgcccatcactgccttcctcaagacctaaaatagctccctatttagtgaaaaattatctgaatatttaaggtc tgccttaacgtgatccccattgctgaattttacctcctgactccaaaaactcttctcttccctgggcccagtcctat ttttttttttttttttttttttgtgagacagagtctcactctgtcacccaggttggaatgcaatggcacaatctccg ctcactgcaagctccgcctcccgggttcacgccattctcctgcctcagcctcccgaatagctgggactacaggtgcc cgccaacacgcccggct(SEQ ID NO:16)；

NR27:

Aataattgttggtaatgagatgtgatgtttctcctgccacctggaaacaaagcattgaagtctgcagttgaaaagcc caacgtctgtgagatccaggaaaccatgcttgcaaaccactggtaaaaaaaaaaaaaaaaaaaaaaaaaagccacag tgacttgcttattggtcattgctagtattatcgactcagaacctctttactaatggctagtaaatcataattgagaa attctgaattttgacaaggtctctgctgttgaaatggtaaatttattattttttttgtcatgataaattc(SEQ ID NO:17)；

MONO27：

Gctgtatgcttcaatttcattgttatgtgaaccacctatgaattgcagatcctggatggcttcagatttattttggg cttcactctttttttttttttttttttttttttttgagacagagtctagctctgtcactcaggctggagtgcagtgg aacgatctctgctcactgcaagctccgcctcccgggttcatgc(SEQ ID NO:18)。

In the second aspect, the primer sets of detection microsatellite sequence stability are provided, the primer is for expanding the present invention Marker combination.

Preferably, the primer sets are：

BAT 25F, sequence are CAGGTGGCAAAGGGCATGGCT (SEQ ID NO:1)；

BAT 25R, sequence are TCTGCATTTTAACTATGGCT (SEQ ID NO:2)；

BAT 26F, sequence are ACTGACTACTTTTGACTTCAG (SEQ ID NO:3)；

BAT 26R, sequence are AACCATTCAACATTTTTAACC (SEQ ID NO:4)；

NR21F, sequence are GAGTCGCTGGCACAGTTCTATTT (SEQ ID NO:5)；

NR21R, sequence are GGTCACTCGCGTTTACAAACAAGA (SEQ ID NO:6)；

NR24F, sequence are CCATTGCTGAATTTTACCTCCT (SEQ ID NO:7)；

NR24R, sequence are GTGCCATTGCATTCCAACC (SEQ ID NO:8)；

NR27F, sequence are ACCATGCTTGCAAACCACTGGT (SEQ ID NO:9)；

NR27R, sequence are AGTCGATAATACTAGCAATG (SEQ ID NO:10)；

MONO27F, sequence are AACCACCTATGAATTGCAGATCCT (SEQ ID NO:11)；With

MONO27R, sequence are GCTTGCAGTGAGCAGAGATCGT (SEQ ID NO:12).

From the name of these primers as can be seen that these primers correspond to above-mentioned marker, and each marker respectively It is R and F primers.If MONO27F and MONO27R are F the and R primers for expanding MONO27 respectively.

It will be appreciated by those skilled in the art that the corresponding marker of the primer amplification, it is complete not need to amplification marker Whole sequence.The frequency occurred according to nucleotide is it is found that primer can amplify a portion, especially core sequence, i.e. table The marker can be expanded by showing.Therefore, in the present invention, the corresponding marker sequence of the primer amplification and the one of amplification marker Partial sequence meaning is equivalent, as long as this partial sequence is more than 10 nucleotide.

It is further preferred that above-mentioned primer has modification.Specifically, BAT25F is modified with VIC, and BAT26F is repaiied with NED Decorations, NR21F are modified with HEX, and NR24F is modified with FAM, and NR27F is modified with FAM, and MONO27 is modified with FAM.

Above-mentioned modification is in the saccharide ring at 5 ends of primer, to add corresponding fluorescein base group after primer synthesis.It is different Fluorescein base group electrophoresis when can detect the fluorescence signal of corresponding color.

FAM:6- Fluoresceincarboxylic acids, signal display color blue；

HEX:5- chlordene fluoresceins, signal display color green；

NED:NED amidates, signal display color black；

VIC:VIC amidates, signal display color green.

In terms of third, application of the primer sets of the present invention in the marker group of the amplification present invention is provided.

4th aspect, provides a kind of kit for detecting microsatellite sequence stability, and the kit includes this The primer sets of invention.

Preferably, the kit further includes Taq enzyme, dNTP, PCR buffer solution.

It is further preferred that the Taq enzyme is Es Taq archaeal dna polymerases.

5th aspect, provides a kind of system for detecting microsatellite sequence stability, the system comprises the present invention Primer sets and ABI 3130XL sequenators.

The marker that the present invention detects is that 6 singlet nucleotide repeat site, is that applicant is true after repeatedly selection, experiment Fixed efficient combination, relatively before detection panel in 1997 and other combinations have higher sensitivity.Also, it will be different Fluorescence separate detection, has the effect of more visual and clear, can be provided more accurately for the medication guide and prognosis prediction of patient Analysis and suggestion.

Description of the drawings

Fig. 1 is the peak figure of NR21, MONO27 of A samples；

Fig. 2 is the peak figure of NR24, BAT26 of A samples；

Fig. 3 is the peak figure of NR27, BAT25 of A samples；

Fig. 4 is the peak figure of NR21, MONO27 of B samples；

Fig. 5 is the peak figure of NR24, BAT26 of B samples；

Fig. 6 is the peak figure of NR27, BAT25 of B samples；

Fig. 7 is the peak figure of NR21, MONO27 of C sample；

Fig. 8 is the peak figure of NR24, BAT26 of C sample；

Fig. 9 is the peak figure of NR27, BAT25 of C sample；

Figure 10 is the peak figure of the NR21 and MONO27 of D patient tumors cells；

Figure 11 is the peak figure of NR24, BAT26 of D patient tumors cells；

Figure 12 is the peak figure of NR27, BAT25 of D patient tumors cells；

Figure 13 is the peak figure of the NR21 and MONO27 of D patient's normal cell；

Figure 14 is the peak figure of NR24, BAT26 of D patient's normal cell；

Figure 15 is the peak figure of NR27, BAT25 of D patient's normal cell.

Specific implementation mode

1,1997 American National institute of oncology (National Cancer Institute, NCI) formulated MSI inspections Mark is accurate, it is determined that detect reference the Panel of MSI, it include 32 nucleotide repetition sites (D5S346, D17S250, D2S123) and two mononucleotides repeat site (BAT 25, BAT 26) (Genetic/Familial High- RiskAssessment:Colorectal Version 1.2016,NCCN Clinical Practice Guidelines in Oncology).If provide this 5 sites detect 2 it is unstable, be height microsatellite instability (MSI-H)；One site Unstable rule is low microsatellite instability (MSI-L)；Otherwise it is that microsatellite is stablized.This detection panle has certain limitation Property, because the specificity of the repetition of 2 nucleotide and sensitivity are relatively low, and also have high polymorphism, it is unfavorable for sample Detection.Therefore 2002, NCI suggestion mononucleotides repeated to detect, to improve the sensitivity of detection.

Therefore, the present invention, by thousands of experiments, detects the MSI of tumour cell on the basis of the above, final to select Monomorphism mononucleotide is taken to repeat site：The combination of BAT 25, BAT 26, NR21, NR24, NR27 and MONO27 are marked as detection Will object.

2, design of primers

To expand these markers, the primer of detection site is designed, and modified：

Primer	Sequence	Modified types *
			BAT25F	CAGGTGGCAAAGGGCATGGCT	VIC
BAT25R	TCTGCATTTTAACTATGGCT	No
			BAT26F	ACTGACTACTTTTGACTTCAG	NED
BAT26R	AACCATTCAACATTTTTAACC	No
			NR21F	GAGTCGCTGGCACAGTTCTATTT	HEX
NR21R	TCTTGTTTGTAAACGCGAGTGACC	No
			NR24F	CCATTGCTGAATTTTACCTCCT	FAM
NR24R	GTGCCATTGCATTCCAACC	No
			NR27F	ACCATGCTTGCAAACCACTGGT	FAM
NR27R	AGTCGATAATACTAGCAATG	No
			MONO27F	AACCACCTATGAATTGCAGATCCT	FAM
MONO27R	GCTTGCAGTGAGCAGAGATCGT	No

* modified types explanation：These types modification is after primer synthesis, and in the saccharide ring at 5 ends of primer, addition is corresponding Fluorescein modification group.The fluorescence signal of corresponding color can be detected when different fluorescein base group electrophoresis.

FAM:6- Fluoresceincarboxylic acids, signal display color blue；

HEX:5- chlordene fluoresceins, signal display color green；

NED:NED amidates, signal display color black；

VIC:VIC amidates, signal display color green.

The above primer is designed by Primer Premier and NCBI Primer Blast, and primer TM values exist as possible when design Within the scope of 62 ± 3 DEG C, amplified production length difference 12bp or more.

3, gDNA extracts (by taking blood sample as an example, universal method)

200 μ L whole bloods, mixing is added to 1.5mL centrifuge tubes in 3.1 400 μ L erythrocyte cracked liquids of packing.

3.256 DEG C of incubation 10min, mixing is for several times therebetween.

3.313000g centrifuges 1min, abandons supernatant, and 200 μ L cell pyrolysis liquids, mixing is added.

3.4 are added 10 μ L Proteinase Ks, mixing.

3.513000g centrifuges 1min, supernatant is transferred in clean 1.5mL centrifuge tubes, and 400 μ L isopropanols are added, and mixes It is even, it can be seen that DNA Precipitations after mixing.

3.613000g centrifuges 1min, abandons supernatant, and 400 μ L, 70% ethyl alcohol rinsing precipitation is added.

3.713000g centrifuges 1min, abandons supernatant, and repeated washing is primary.

3.8, which uncap, which dries, makes ethyl alcohol volatilize completely, and 100 μ L are added keeps precipitation completely molten without DNA enzymatic/RNA enzyme deionized water Solution.

3.9 detect DNA concentration using spectrophotometer, and are diluted to 50ng/ μ l.

4, PCR system

4.1Taq enzymes are important one of the ingredients in amplification system the inside.Es Taq archaeal dna polymerases are chosen in this experiment (CWBIO) PCR amplification is carried out.The enzyme is a kind of mixed enzyme, and compared with Taq archaeal dna polymerases, Es Taq archaeal dna polymerases have Higher amplification efficiency, the low excellent performance of mismatch rate.

4.2PCR systems are chosen 25 μ l and 50 μ l and are expanded, and effect is substantially suitable.

6 markers prepare PCR reaction systems according to upper table respectively, and carry out PCR reactions according to following program.

5, PCR programs

95 DEG C, 4-10min：1 cycle；

94 DEG C of 30s, 56-60 DEG C of 30-45s, 72 DEG C of 30-60s：25 cycles；

72 DEG C, 5-10min：1 cycle；

It 15 DEG C, preserves.

6, capillary electrophoresis detection

Above-mentioned 6 pipe PCR product is divided into three groups by 6.1：Mono- group of NR21, MONO27；Mono- group of NR24, BAT26；BAT25、NR27 One group.Every group often pipe PCR product take 0.5 μ l, mixed, be added to containing in 9 μ l formamides of target in 0.2 μ l ROX500.

After 6.2 mixings, 95 DEG C of denaturation 4-10min are put into ABI 3130XL sequenators and are detected.

Basis for estimation

Each sample has 6 significant detection sites, is judged according to the peak figure result in each site：The each position of sample Only there are one Five Fingers Peaks (characteristic peak) for point, then are MSS；If 1 significant detection site more than one Five Fingers Peak in sample, For MSI-L；If being no less than 2 significant detection sites in sample there is more than one Five Fingers Peak, for MSI-H.

The present invention design in, every group of two different colors are easily distinguishable, and also have between clip size it is certain away from From, will not be overlapped and generate mistake interpretation.

For sample in following embodiment both from patient in hospital, each patient has signed informed consent form.

Embodiment 1

According to above-mentioned gDNA extracting methods, the DNA (abbreviation A samples, B samples and C sample) of patient A, B and C are extracted respectively, Wherein B and C have been diagnosed as tumor patient.Capillary electrophoresis detection is carried out after being expanded according to PCR flows.

From electrophoresis result Fig. 1-3 as it can be seen that A sample detection results are MSS.From electrophoresis result Fig. 4-9 as it can be seen that B samples are MSI-H, C sample MSI-H.The testing result is consistent with the truth of three patients, and according to the testing result of B and C Medication adjustment is carried out respectively.

Embodiment 2

According to above-mentioned gDNA extracting methods, the DNA of the tumour cell and normal cell of patient D is extracted respectively, is flowed according to PCR Capillary electrophoresis detection is carried out after Cheng Jinhang amplifications.

Electrophoretogram 10-12 is the MSI testing results of patient tumors cell, and electrophoretogram 13-15 is the MSI of patient's normal cell Testing result, so as to show that the patient is MSI-H.The testing result is consistent with the truth of patient, is examined also according to MSI It surveys result and has carried out medication adjustment.

This method repeats site relative to NCI panel in 1997, using detection monomorphism nucleotide, meets 2002 editions NCI recommends examination criteria.Monomorphism nucleotide is repeated into site grouping when electrophoresis simultaneously and carries out electrophoresis, it is easier to which peak figure is carried out Interpretation.

<110>One hundred physicians from a family for generations of Beijing Cigna learn Co., Ltd of inspection institute

<120>Marker combination, primer sets and the kit of microsatellite sequence Detection of Stability

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tctgcatttt aactatggct 20

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actgactact tttgacttca g 21

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aaccattcaa catttttaac c 21

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<213>Artificial sequence

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gagtcgctgg cacagttcta ttt 23

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ggtcactcgc gtttacaaac aaga 24

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gtgccattgc attccaacc 19

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gcttgcagtg agcagagatc gt 22

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actacttttg acttcagcca gtatatgaaa ttggatattg cagcagtcag agcccttaac 180

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gcttgccttt tttagagagt agcagtggtt cctatttcgg aaaaggacgt tctaattcaa 360

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taaaaaaaaa aaaaaaaaaa aaaaaaagcc acagtgactt gcttattggt cattgctagt 180

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c 301

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gcctcccggg ttcatgc 197

Claims

1. a kind of marker combination, is made of marker BAT 25, BAT 26, NR21, NR24, NR27 and MONO27, wherein respectively The sequence of marker is respectively such as SEQ ID NO:Shown in 13-18.

2. detecting the primer sets of microsatellite sequence stability, the primer sets are for expanding marker group described in claim 1 It closes.

3. primer sets according to claim 2, are：

BAT 25F, sequence are CAGGTGGCAAAGGGCATGGCT (SEQ ID NO:1)；

BAT 25R, sequence are TCTGCATTTTAACTATGGCT (SEQ ID NO:2)；

BAT 26F, sequence are ACTGACTACTTTTGACTTCAG (SEQ ID NO:3)；

BAT 26R, sequence are AACCATTCAACATTTTTAACC (SEQ ID NO:4)；

NR21F, sequence are GAGTCGCTGGCACAGTTCTATTT (SEQ ID NO:5)；

NR21R, sequence are GGTCACTCGCGTTTACAAACAAGA (SEQ IDNO:6)；

NR24F, sequence are CCATTGCTGAATTTTACCTCCT (SEQ ID NO:7)；

NR24R, sequence are GTGCCATTGCATTCCAACC (SEQ ID NO:8)；

NR27F, sequence are ACCATGCTTGCAAACCACTGGT (SEQ ID NO:9)；

NR27R, sequence are AGTCGATAATACTAGCAATG (SEQ ID NO:10)；

MONO27F, sequence are AACCACCTATGAATTGCAGATCCT (SEQ ID NO:11)；With

MONO27R, sequence are GCTTGCAGTGAGCAGAGATCGT (SEQ ID NO:12).

4. application of the primer sets according to claim 2 or 3 in expanding marker combination described in claim 1.

5. a kind of kit for detecting microsatellite sequence stability, including primer sets according to claim 2 or 3.

6. kit according to claim 5, wherein the kit further includes Taq enzyme, dNTP, PCR buffer solution.

7. kit according to claim 6, wherein the Taq enzyme is Es Taq archaeal dna polymerases.

8. a kind of system for detecting microsatellite sequence stability, the system comprises primer sets according to claim 2 or 3 With ABI 3130XL sequenators.