CN110004224A - A kind of quick detection kit of K-ras gene mutation - Google Patents
A kind of quick detection kit of K-ras gene mutation Download PDFInfo
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Abstract
The present invention relates to a kind of quick detection kits of K-ras gene mutation, belong to technical field of molecular biology.Kit includes for detecting the ARMS upstream primer in 10 sensitizing mutation sites, downstream primer, triggering primer, fluorescence probe.K-ras gene detecting kit can detect 10 main mutation simultaneously under the conditions of single tube, the design of triggering primer is utilized, in the presence of any one mutation, reaction system can be made fluorescence occur, realize quickly detection determine K-ras mutation effect, the kit have the advantages that detect speed fastly, sensitivity can reach 0.001%.
Description
Technical field
The present invention relates to a kind of quick detection kits of K-ras gene mutation, belong to technical field of molecular biology.
Background technique
KRAS is EGFR(EGF-R ELISA) a crucial downstream regulatory factor in signal transduction pathway, ginseng
With the adjusting of cell growth, play an important role in canceration generating process.KRAS gene product takes part in control genetic transcription
Kinase signal transduction path, to be adjustable the growth and differentiation of cell.The change of KRAS gene becomes KRAS albumen
Change, result KRAS albumen no longer has cracking GTP molecule and discharges the ability of GTP molecule.Such change causes this to believe
Number conducting path is in the logical state of "ON" always.The signal of this "ON" leads to the growth and hyperplasia of cell.Therefore KRAS
Overexpression leads to lasting cell Proliferation with amplification, this is a tumorigenic crucial step.
Primary lung cancer (hereinafter referred to as lung cancer) is one of most common malignant tumour in China.WHO statistics display in 2012,
Lung cancer is the current morbidity and mortality in China the 1st malignant tumour.Biological characteristics, treatment and prognosis based on lung cancer,
WHO is classified as two major classes: non-small cell lung cancer NSCLC and Small Cell Lung Cancer SCLC.Wherein NSCLC accounts for 85% of lung cancer or more.
Although treatment lung cancer technology is greatly improved, survival rate is not obviously improved within 5 years, and most of patients with lung cancer is died of
Uncontrollable DISTANT METASTASES IN, traditional chemotherapy and radiation also tend to while obtaining curative effect to patient due to lacking specificity
Bring biggish side effect.Therefore, the special molecular target of selection lung carcinoma cell, is controlled using the drug for the target spot
It treats, avoids the treatment mode of the injury to normal cell again while obtaining obvious curative effects, by tumour academia and extensively
Big patient accepts and uses.
Three kinds of EGFR-TKI(Gefitinib/Iressas, Erlotinib/Erlotinib and Conmana) it is currently used for NSCLC
The key agents of molecular targeted therapy, but the Different therapeutical effect of EGFR-TKI is very big in different patients.A large amount of clinical trial hairs
Existing, in addition to EGFR is mutated, KRAS mutation can help patient NSCLC preferably to predict predicating curative effect of gefitinib；The NSCLC of KRAS mutation
Clinical prognosis is poor after patient's Tarceva Combined Treatment.
" US National cancer integrated network (NCCN) non-small cell lung cancer clinical practice guideline " pathology assessment is former within 2012
Clear in then: KRAS mutation is related with endogenous TKI drug resistance, determines that KRAS gene appearance helps to select to be suitble to TKI treatment
Patient.It points out simultaneously, KRAS gene appearance is that the prognostic indicator of NSCLC existence and the outcome prediction of EGFR-TKI drug refer to
Mark；It is also related to cis-platinum/vinorelbine chemotherapeutic efficacy.
K-ras gene is one of RAS gene family, is located on No. 12 chromosomes of the mankind, is a kind of former cancer base
Cause influences human cancer very big.K-ras gene coding molecule amount is the ras albumen also known as p21 albumen of 21kD, p21 albumen position
In the inner surface of cell membrane, there is GTP enzymatic activity, participate in intracellular signal transduction, is activated state when in conjunction with GTP, in conjunction with GDP
When for inactivation state.K-ras gene seems molecular switch in body, it is in the processes such as growth of tumour cell and angiogenesis
Important regulating and controlling effect is played in signal transduction pathway, normal K-ras gene can inhibit growth of tumour cell, but when being abnormal
Can induced gene permanently activate, make Cellular Signaling Transduction Mediated disorder, uncontrolled cellular proliferation and canceration.K-ras gene is activated
Mode has 3 kinds: point mutation, gene great expression, gene is inserted into and indexing.Its point mutation is most common, mostly occurs at No. 2
On No. 12 codons and No. 13 codons of exon, codon 61,63,117,119 and 146 are more rare.
Patent CN101608241A discloses a kind of multiple real time fluorescence PCR that can quickly detect K-ras gene mutation
Method simultaneously devises relevant primer, and the mutant primers being respectively adopted for its different mutational formats design carry out sample to be tested
Detection, only mutant sample can smoothly be amplified double stranded DNA product, which could issue in conjunction with double chain DNA probe
Fluorescence signal is to be detected.Patent CN102041313A design is directed to the PNA and phase of 12 codon of Wild-Type K-ras Genes
The abrupt climatic change probe K-ras-FAMTagman MGB probe and K-ras-VIC Tagman MGB probe answered；Utilize PNA and spy
Needle carries out real-time quantitative PCR detection to the plasmid standard of known mutations amount, obtains standard curve and fluorescence types；It extracts, is pure
Change sample DNA, measure concentration, carry out real-time quantitative PCR detection, according to standard curve and fluorescence types, so as to differentiating K-ras base
Because of Sudden Changing Rate and mutation type.But since K-ras includes multiple sensitizing mutation sites, above-mentioned fluorescence PCR method exists
The problem of needing a point multiple PCR pipes to be detected, existing fluorescence PCR detection reagent kit on the market generally uses 7 PCR pipes
It is detected, analytic process is cumbersome and is not necessarily to, while entire testing process is slower, at least needs could go out result within 1 day.
Summary of the invention
The purpose of the present invention is: a kind of quantitative fluorescent PCR inspection is devised for 8 sensitizing mutation sites of K-ras gene
Test agent box is utilized triggering primer as the bridge between the primer and fluorescence probe of detection catastrophe point, realizes to 8 positions
The quick detection of point.
Technical solution is:
A kind of quick detection kit of K-ras gene mutation includes: for detecting the ARMS in 10 sensitizing mutation sites
Swim primer, downstream primer, triggering primer, fluorescence probe；
The sequence of the upstream primer is as shown in NO.1~10 SEQ ID；
The sequence of the downstream primer is as shown in SEQ ID NO. 11~13；
The sequence of the triggering primer is as shown in SEQ ID NO. 14~16；
The sequence of the fluorescence probe is as shown in SEQ ID NO. 17.
It in one embodiment, further include quenching probes, sequence is as shown in SEQ ID NO. 18.
In one embodiment, 5 ' ends of fluorescence probe are modified by FAM.
In one embodiment, 5 ' ends of quenching probes are modified by BHQ.
It in one embodiment, further include having retardance primer, sequence is as shown in SEQ ID NO. 19~21.
In one embodiment, the 3 ' ends for blocking primer carry out double deoxidation base modification, and 5 ' ends carry out dephosphorylation
Modification.
In one embodiment, the composition of the kit is: 2.5 μ l of 10x buffer, Mg2+3.5μl、dNTP2.5
μ l, 1.2 μ l of upstream primer, 1.2 μ l of downstream primer, 0.5 μ l of fluorescence probe, 0.2 μ l of Taq enzyme, 3 μ l of sample DNA, ddH2O is mended
Enough to 25 μ l.
The rapid detection method of K-ras gene mutation, includes the following steps:
Sample DNA is extracted, Fluorescence PCR is carried out using kit, collects fluorescence；According to the fluorescence curve and standard items of sample
Fluorescence curve comparison, judgement sample with the presence or absence of mutation.
In one embodiment, PCR response procedures are as follows: 95 DEG C 5 minutes；40 circulation: 95 DEG C 15 seconds, 60 DEG C 45 seconds.
Beneficial effect
K-ras gene detecting kit provided by the invention can detect 10 main mutation, benefit simultaneously under the conditions of single tube
Reaction system can be made fluorescence occur in the presence of any one mutation with the design of triggering primer, realize quick detection
Determine K-ras mutation effect, the kit have the advantages that detect speed fastly, sensitivity can reach 0.001%.
Detailed description of the invention
Fig. 1 is the mutant plasmids of 12GGT > GAT and the fluorescent PCR curve comparison figure of wild plasmid；
Fig. 2 is the mutant plasmids of 12GGT > AGT and the fluorescent PCR curve comparison figure of wild plasmid；
Fig. 3 is the mutant plasmids of 12GGT > GTT and the fluorescent PCR curve comparison figure of wild plasmid；
Fig. 4 is the mutant plasmids of 12GGT > GCT and the fluorescent PCR curve comparison figure of wild plasmid；
Fig. 5 is the mutant plasmids of 12GGT > TGT and the fluorescent PCR curve comparison figure of wild plasmid；
Fig. 6 is the mutant plasmids of 12GGT > CGT and the fluorescent PCR curve comparison figure of wild plasmid；
Fig. 7 is the mutant plasmids of 13GGC > GAC and the fluorescent PCR curve comparison figure of wild plasmid；
Fig. 8 is the mutant plasmids of 13GGC > CGC and the fluorescent PCR curve comparison figure of wild plasmid；
Fig. 9 is the mutant plasmids of 61AAG > ATG and the fluorescent PCR curve comparison figure of wild plasmid；
Figure 10 is the mutant plasmids of 61AAG > ACG and the fluorescent PCR song comparison line chart of wild plasmid；
Figure 11 is Δ CT comparison diagram of the retardance primer of different length in fluorescent PCR amplification.
Specific embodiment
Below by specific embodiment combination attached drawing, invention is further described in detail.But those skilled in the art
It will be understood that the following example is merely to illustrate the present invention, and should not be taken as limiting the scope of the invention.It is not specified in embodiment
Particular technique or condition person, described technology or conditions are (such as with reference to J. Pehanorm cloth Shandong according to the literature in the art
It is gram equal to write, " Molecular Cloning:A Laboratory guide " that Huang Peitang etc. is translated, the third edition, Science Press) or according to product description
It carries out.Reagents or instruments used without specified manufacturer, being can be with conventional products that are commercially available.
Term in the present invention:
As used herein, term " refer to genome " or " reference sequences " refer to any specific of any organism or virus
Known group sequence (either part or complete), it can be used for carrying out the sequence of the identification from subject
Reference.For example, the reference genome for human experimenter and many other organisms is found in American National biotechnology
Information centre (the National Center for Biotechnology Information),
ncbi.nlm.nih.gov." genome " refers to the entire genetic information of organism or virus, by table in nucleic acid sequence
It reaches.
" allele " generally refers to DNA section, in the same, physical on homologue, controls relativity
One pair of genes.In some cases, allele can correspond to the replacement of the mononucleotide on specific physics locus.At it
In its situation, allele can correspond to (single or multiple) insertions of nucleotide or missing.
" primer " means the starting point that nucleic acid synthesis is potentially acted as when forming duplex with polynucleotide template, and certainly
Its 3 ' end extends along template, to form the natural or synthetic oligonucleotide of the duplex of extension.Add during extending
The nucleotide sequence added is determined by the sequence of template polynucleotide.Primer is usually extended by DNA polymerase.
" probe " is often referred to the oligonucleotide with oligonucleotide or complementary target under study for action.With allow to detect
Mode, such as used in some aspects of fluorescence or other optionally recognizable label label claimed inventions
Probe.For example, SYBRGreen and other DNA binding dyes are all detection probes.Some detection probes can be sequence-specific
, such as Taqman probe.
Be in the present invention, between probe, primer and DNA profiling it is complementary, in particular to: oligonucleotides have in extension
Under conditions of can with include particular sequence target nucleic acid formed double bond state base sequence, but be not required for completely mutually
It mends, several mismatch base-pairs can also be contained.Matching of the present invention refers to: the Bases in Nucleic Acid Sequences of double-stranded state is to shape
As the state of Watson-Crick (Watson-Crick) base-pair, mismatch refers to: not forming Watson-Crick base
Pair state.Watson-Crick base-pair refers to: two polynucleotide molecules of DNA composition adenine (A) and
Thymidine (T), guanine (G) and cytosine (C) group, and the base-pair connected by hydrogen bond.So, probe, primer with
DNA template sequence can exactly match, and can also have the mismatch of several bases, as long as can be with identical detecting target base sequence position
Setting complementation can be realized technical solution of the present invention.
" sensitivity " is to refer to be detected the minimum (copy number) of template.
" specificity " refers to the ability for distinguishing matching template and mispairing template.
The design of probe
The most common active mode of K-ras gene is point mutation, wherein 90% or more occurs the codon 12GGT in exon 2
(rs121913529), codon 13GGC(rs112445441) and codon 61(rs17851045) on, main 10 kinds of mutation
Be respectively as follows: codon 12GGT > GAT, 12GGT > AGT, 12GGT > GTT, 12GGT > GCT, 12GGT > TGT, 12GGT > CGT,
The replacement mutation of 13GGC > GAC, 13GGC > CGC, 61AAG > ATG, 61AAG > ACG.
The sequence of the above mutation, and design primer are found by the snp database of NCBI.
Design principle is: a matched upstream primer is designed for each saltant type in each site,
3 ' end be located at mutational site on, behind second or third base at set a base mismatch；Upstream primer is prominent for detecting
Modification sample；One downstream primer is designed for each site, for expanding with upstream primer to segment.Therefore, on
Trip primer only can be combined and expand with the sequence of saltant type sample, without expanding with wild type sample.
It further include having a fluorescence probe in primer sets, it is glimmering for when upstream primer expands generating, PCR reaction
Light.
Meanwhile needing to design a triggering primer for each site, a part is and fluorescence probe in triggering primer
Sequence complete complementary, another part is complementary with the sequence of mutational site downstream direction.
When carrying out PCR amplification, the upstream primer for detecting mutational site can be expanded with saltant type sample, upstream primer
After the amplification of 5 ' -3 ' directions, the region triggered primer in conjunction with apperance can be moved to and hydrolyzed, the probe of primer is triggered
Complementary region is then released, the probes complementary region released, be based on base pair complementarity principle, triggering primer again can with lead to
Complementation occurs with fluorescence probe, in the presence of quenching group, the fluorescence of fluorophor is quenched, and quick Taq enzyme passes through intermediary
The probes complementary region for connecting primer, it is mobile to 5 ' -3 ' directions, quenching group is hydrolyzed, so that quenching group is far from fluorophor,
To which PCR reaction produces fluorescence.
In order to improve the specificity of detection, it can also be combined, be prevented using the template of retardance primer pair wild type
Swim the amplified reaction of primer and wild type sample form.The wild type that a complete complementary is designed in each site blocks primer,
3 ' ends of primer carry out double deoxidation base modification, and to prevent amplified reaction, 5 ' ends carry out dephosphorylation modification, to prevent
It is degraded by Taq polymerase digestion.
Based on the above design principle, by a large number of experiments, with improve PCR amplification efficiency, detection sensitivity and specificity,
Avoid occurring the problems such as dimer, ring card structure between primer, the primer sets that final design obtains are as follows:
Table 1
Table 2
Table 3
The sequence of the fluorescence probe are as follows:
5 '-FAM-CCTGCAAGAATTCGACAAGCTGAGGTTTTTTTTTTTTTTTT-3 ' (SEQ ID NO .17).
The sequence of the quenching probes is 5 '-BHQ-GGACGTTC-3 ' (SEQ ID NO .18).
Table 4
The preparation of wild type and mutant plasmids
Upstream and downstream in the mutational site K-ras to be detected respectively designs one couple of PCR primers, and the length of amplified fragments can be
It is template with gDNA in the range of 500bp, using conventional PCR method, amplifies this segment, it, will in such a way that AT is cloned
The segment is cloned into the plasmid of sequencing, usually uses the PCR2.1 Topo carrier T kit of Invitrogen, operation is illustratively
Book carries out, it is also possible to the carrier T kit of other producers, or even the carrier T plasmid that can be used oneself to prepare.Positive gram obtained
It is grand, through sequence verification, it was demonstrated that then the correctness of sequence uses the Quickchange kit of Stratagene company, if
Another genotyping primer of meter corresponding site obtains the other positive colony of the saltant type by the method for Quickchange,
It operates by specification to carry out, obtained positive plasmid all must be the use for next step of being correctly allowed for access through sequence verification.
Taqman quantifies plasmid, and is used as template to verify the sensitivity, linear dynamic range, specificity of given measuring method.Plasmid
Extraction using TIANGEN (High Pure Plasmid Kit, DP116) plasmid extraction kit carry out, concrete operations step
Suddenly it is detailed in product description.
Fluorescence PCR condition
Table 5
PCR response procedures are as follows: 95 DEG C 5 minutes；40 circulations: 95 DEG C 15 seconds, 60 DEG C 45 seconds (collect fluorescence).Instrument
For ABI stepone.
Quantitative PCR detecting reagent Realtime PCR Master Mix is purchased from TOYOBO company, Japan.Tissue gene group is taken out
Extraction reagent kit QIAamp DNA FFPE Tissue Kit is purchased from QIAGEN China (shanghai) Co., Ltd., blood plasma gene
Group extraction agent box High Pure PCR Template Preparetion Kit is limited purchased from Roche Diagnistics' product (Shanghai)
Company.Quantitative PCR apparatus is the 7300 realtime PCR system of Applied Biosystems.PCR the primer is purchased from
Sangon Biotech (Shanghai) Co., Ltd., remaining reagent are bought from SigmaAldrich.
Detection of the embodiment 1 to saltant type and wild plasmid
The mutant plasmids and wild plasmid that 10 kinds of mutation are respectively adopted carry out fluorescent PCR amplification, and plasmid copy number is 1.0 ×
105, calculate the difference of the two CT, △ CT=CTWild type–CTSaltant type, difference is bigger, and the differentiation effect for illustrating kit is better.React item
Part is shown in Table 5.
Table 6
The results show that the amplification procedure to saltant type and wild plasmid has biggish △ CT, there is preferable resolving effect.
The effect of the retardance primer of embodiment 2
Using the plasmid and fluorescence PCR method in embodiment 1, but it is added without retardancy primer, the prominent of 10 kinds of mutation is respectively adopted
Modification plasmid and wild plasmid carry out fluorescent PCR amplification, and plasmid copy number is 1.0 × 105, calculate the difference of the two CT, △
CT=CTWild type–CTSaltant type, difference is bigger, and the differentiation effect for illustrating kit is better.Reaction condition is shown in Table 5.
Table 7
The results show that can effectively prevent the amplification of wild-type template by the addition of retardance primer, △ CT is improved, improves inspection
The specificity of test agent box improves resolving effect.
The selection of the retardance primer length of embodiment 3
The retardance primer of 12 codons of different length is separately designed, fluorescent quantitative PCR is carried out, calculates △ CT value, design
Primer it is as shown in table 8:
Table 8
The △ CT obtained after amplification is as shown in table 9:
Table 9
As can be seen from the table, the length for blocking primer is preferably controlled between 16~18nt.
4 kit sensitivity of embodiment
Using the primer sets and fluorescence PCR method in such as embodiment 1,10 kinds of mutant plasmids are proportionally added into wild respectively
In type plasmid, the detection sensitivity of kit is investigated.△ CT is as shown in table 10:
Table 10
As can be seen from the table, kit provided by the invention still has preferable differentiation when mutant proportion 0.001%
Degree.
5 pattern detection of embodiment
Confirmation is obtained to cut containing 10 parts that K-ras is mutated to be originated from colorectal cancer pathology of the hospital pathology department Jing Guo paraffin embedding
Histotomy is put into dye piece cylinder by piece, and dimethylbenzene is added and sufficiently impregnates 2 hours, is rinsed 2 times with 75% alcohol, is added 100%
Ethyl alcohol impregnates 10 minutes, dries.The histotomy dried is laid flat on experimental bench it is fixed, with flat scraper it is careful by tissue
It is removed from slide, tissue sample is put into 1.5mlEP pipe covers lid rapidly.180 μ LATL buffers and 20 μ L albumen are added
Enzyme K, is mixed well with oscillator, and mixture is put into water-bath and is digested 60 minutes for 56 DEG C.Postdigestive mixture is put into
It is digested 60 minutes for 90 DEG C in water-bath.200 μ LAL buffers are added to mix well, adds the mixing of 200 μ L100% ethyl alcohol, will mix
It closes sample to be put into QIAamp Min Elute column, 8000rpm is centrifuged 2min, then respectively with 500 μ l Buffer
AW1 and 500 μ l Buffer AW2 difference 6000 × g (8000rpm) centrifugation 1min elution, last 20000 × g;14000rpm from
Heart 3min drying, is added 100 μ LATE20000 × g;14000rpm collects eluting liquid progress DNA mass after being centrifuged 1min elution
Identification.
K-ras gene mutation is used for quickly detecting using the above method, and is compared with NGS method testing result.
It can be seen that, kit provided by the invention can be used for the quick detection judgement of K-ras gene from upper table, obtain
To result can obtain the verifying of high-flux sequence method.
Sequence table
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<120>a kind of quick detection kit of K-ras gene mutation
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Claims (9)
1. a kind of quick detection kit of K-ras gene mutation characterized by comprising for detecting 10 sensitizing mutations
The ARMS upstream primer in site, downstream primer, triggering primer, fluorescence probe；
The sequence of the upstream primer is as shown in NO.1~10 SEQ ID；
The sequence of the downstream primer is as shown in SEQ ID NO. 11~13；
The sequence of the triggering primer is as shown in SEQ ID NO. 14~16；
The sequence of the fluorescence probe is as shown in SEQ ID NO. 17.
2. the quick detection kit of K-ras gene mutation according to claim 1, which is characterized in that further include being quenched
Probe, sequence is as shown in SEQ ID NO. 18.
3. the quick detection kit of K-ras gene mutation according to claim 1, which is characterized in that fluorescence probe
5 ' ends are modified by FAM.
4. the quick detection kit of K-ras gene mutation according to claim 2, which is characterized in that quenching probes
5 ' ends are modified by BHQ.
5. the quick detection kit of K-ras gene mutation according to claim 1, which is characterized in that further include having resistance
Stagnant primer, sequence is as shown in SEQ ID NO. 19~21.
6. the quick detection kit of K-ras gene mutation according to claim 5, which is characterized in that block primer
3 ' ends carry out double deoxidation base modification, and 5 ' ends carry out dephosphorylation modification.
7. the quick detection kit of K-ras gene mutation according to claim 1, which is characterized in that the reagent
The composition of box is: 2.5 μ l of 10x buffer, Mg2+It is 3.5 μ l, dNTP2.5 μ l, 1.2 μ l of upstream primer, 1.2 μ l of downstream primer, glimmering
0.5 μ l of light probe, 0.2 μ l of Taq enzyme, 3 μ l of sample DNA, ddH2O complements to 25 μ l.
8. a kind of rapid detection method of K-ras gene mutation, which comprises the steps of:
Sample DNA is extracted, Fluorescence PCR is carried out using the described in any item kits of claim 1~7, collects fluorescence；Root
It is compared according to the fluorescence curve of sample and the fluorescence curve of standard items, judgement sample is with the presence or absence of mutation.
9. the rapid detection method of K-ras gene mutation according to claim 8, which is characterized in that PCR response procedures
Are as follows: 95 DEG C 5 minutes；40 circulation: 95 DEG C 15 seconds, 60 DEG C 45 seconds.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN110684849A (en) * | 2019-12-05 | 2020-01-14 | 苏州绘真医学检验有限公司 | Primer, probe, kit and method for detecting KRAS gene mutation of human circulating tumor cell based on ddPCR |
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2018
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN110684849A (en) * | 2019-12-05 | 2020-01-14 | 苏州绘真医学检验有限公司 | Primer, probe, kit and method for detecting KRAS gene mutation of human circulating tumor cell based on ddPCR |
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Application publication date: 20190712 |