CN107447028A - Detect the kit of RB1 gene I680T site mutations - Google Patents
Detect the kit of RB1 gene I680T site mutations Download PDFInfo
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- CN107447028A CN107447028A CN201710823406.3A CN201710823406A CN107447028A CN 107447028 A CN107447028 A CN 107447028A CN 201710823406 A CN201710823406 A CN 201710823406A CN 107447028 A CN107447028 A CN 107447028A
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Abstract
The present invention relates to the kit of detection RB1 gene I680T site mutations, including：The specific primer of RB1 gene I680T site mutations is detected, the sense primer nucleotide sequence of the special primer is as shown in SEQ ID NO.1, and the anti-sense primer nucleotide sequence of the special primer is as shown in SEQ ID NO.2.The invention firstly discloses the relation between RB1 gene I680T site mutations and the Patients with Non-small-cell Lung prognosis for receiving adjuvant chemotherapy, and detection kit is designed according to the discovery, provide individuation guidance for the selection of Patients with Non-small-cell Lung adjuvant chemotherapy scheme.
Description
Technical field
The present invention relates to a kind of kit of detection RB1 gene I680T site mutations, belong to biotechnology and medical technology
Field.
Background technology
RB1 is a kind of tumor suppressor gene, because the generation of the gene and retinoblastoma is closely related, therefore is named as
Retinoblastoma gene.Mankind RB1 full length gene 178143bp, No. 13 chromosome long arms (13q14.2) are positioned at, encoded
928 amino acid.RB1 is by cell cycle regulation, cell differentiation, Apoptosis and growth inhibition, to female including retina
Malignant tumour including cytoma, osteosarcoma, cancer of pancreas, breast cancer and small cell carcinoma etc. plays important inhibitory action.Grind
Study carefully and show, the expression by suppressing RB1 in non-small cell lung cancer can promote the invasion and attack and transfer of tumour cell.Late lung
In squamous cell carcinoma patients, the effect of Paclitaxel Chemotherapy scheme is combined in RB1 mutation with platinum class, is closely related, and RB1's is prominent
Change is also determined as independent prognostic indicator.
Taqman probes are a kind of detection technique of fluorescences come out in Real-time round pcrs platform development, are in PCR
A specific fluorescence probe is added during amplification, the probe only combines with template specificity, and its binding site draws at two
Between thing.Fluorescent reporter group such as FAM, VIC etc. are marked with 5 ' ends of probe, base is quenched in 3 ' end mark fluorescents of probe
Group.When probe is complete, quenching group can absorb the fluorescence signal of reporter group transmitting, when entering performing PCR amplification, with template knot
The probe of conjunction can run into Taq archaeal dna polymerases, and probe will be by the 5 prime excision enzyme activity enzyme at 5 ' ends of Taq archaeal dna polymerases to 3 ' ends
Degraded is cut, causes reporter group and quenching group to separate, so as to send fluorescence signal, reaches PCR primer formation and fluorescence signal
Accumulation Complete Synchronization.
Urgent problem is that research one kind being capable of quick detection Patients with Non-small-cell Lung RB1 genes I680T at present
(c.2039T>C) the method for site mutation, so as to provide foundation for the adjuvant chemotherapy of Patients with Non-small-cell Lung individuation.
The content of the invention
In view of the deficiencies of the prior art, the present invention provides a kind of quick, accurate, cheap detection RB1 genes I680T
(c.2039T>C) the kit of site mutation.
Summary of the invention
(c.2039T inventor detects RB1 genes I680T using two generation high throughput sequencing technologies>C) the mutation in site,
And find the site mutation and receive the relation between the prognosis of the patients with lung cancer of adjuvant chemotherapy.When the position nucleotide is T
When, IIB or IIIA phase Patients with Non-small-cell Lung is postoperative to receive carboplatin joint docetaxel scheme or carboplatin joint Changchun
Auspicious shore scheme, the DFS phase (DFS) and Overall survival (OS) no difference of science of statistics of patient；When the position nucleotide is C
When, the postoperative IIB or IIIA phase Patients with Non-small-cell Lung for receiving carboplatin joint docetaxel chemotherapy regimen is compared to receiving
The DFS and OS of the patient of carboplatin joint vinorelbine scheme significantly extends.According to the discovery, inventor uses real time fluorescent quantitative
PCR detection method, design a kind of detection RB1 genes I680T (c.2039T>C) the kit in mutational site, kit tool
There is the characteristics of detection is quick, accurate, cost is relatively inexpensive and easy to operate.
Detailed description of the invention
Detect RB1 genes I680T (c.2039T>C) the kit of site mutation, including：
Detect RB1 genes I680T (c.2039T>C) the specific primer of site mutation, the upstream of the specific primer
Nucleotide sequence is as shown in SEQ ID NO.1, and the downstream nucleotide sequence of the special primer is as shown in SEQ ID NO.2.
According to currently preferred, mentioned reagent box, in addition to：
Specific recognition RB1 genes I680T is (c.2039T>C) the Taqman probes in mutational site, the Taqman probes
Wild-type nucleotide sequences as described in SEQ ID NO.3, the mutant nucleotide sequence such as SEQ ID NO.4 of Taqman probes
It is shown；
According to currently preferred, mentioned reagent box, in addition to：
Buffer solution, dNTPs, Taq DNA polymerase, MgCl2And DNA extraction kit.
According to currently preferred, mentioned reagent box, react and carried out in 20 μ L systems, each composition reaction density is as follows：
0.2 μM of sense primer, 0.2 μM of anti-sense primer, 0.1 μM of wild type Taqman probes, saltant type Taqman probes 0.1
μM, dNTPs 0.25mM, Taq DNA polymerase 0.025U/ μ L, MgCl2The template that 1.5mM, DNA extraction kit are extracted
DNA<250ng。
According to currently preferred, 5 ' end connection VIC fluorescence labelings of the Taqman probes wild type；Taqman probes
5 ' end connection FAM fluorescence labelings of saltant type.
Beneficial effect
1st, the invention firstly discloses RB1 genes I680T (c.2039T>C) site mutation is with receiving the non-of adjuvant chemotherapy
Relation between the prognosis of Patients With Small Cell Carcinoma of The Lung, and the reagent for detecting the RB1 genes site mutation is designed according to the discovery
Box, so as to provide guidance for the adjuvant chemotherapy of Patients with Non-small-cell Lung individuation；
2nd, kit of the present invention using Real-Time Fluorescent Quantitative PCR Technique to RB1 genes I680T (c.2039T>C)
Mutational site is detected, and the kit usage range is wide, can detect and be carried from paraffin section, flesh tissue and peripheral blood
The DNA taken, more existing two generations high throughput sequencing technologies are simple to operate, the time is short, cheap.
Brief description of the drawings
Fig. 1 is to detect RB1 genes I680T (c.2039T with kit of the present invention>C) the detection knot of site wild type
Fruit；
In figure：Dotted line holds upper VIC groups to fluoresce by wild-type probe 5 '；
Fig. 2 is to detect RB1 genes I680T (c.2039T with kit of the present invention>C) the detection knot of site mutation type
Fruit；
In figure：Solid line holds upper FAM groups to fluoresce by saltant type probe 5 '.
Embodiment
Technical scheme is further elaborated with reference to embodiment, but institute's protection domain of the present invention is not limited to
This.
DNA extraction kit includes in embodiment：GeneReadTMDNA FFPE Kit are purchased from QIAGEN companies；
TIANamp Genomic DNA Kit are purchased from TIANGEN companies.
Other reagents such as Taq DNA polymerase are purchased from Dalian treasured biotech firm.
Embodiment 1
(c.2039T inventor detects RB1 genes I680T using two generation high throughput sequencing technologies>C) the mutation in site,
And find the site mutation and receive the relation between the prognosis of the patients with lung cancer of adjuvant chemotherapy.When the position nucleotide is T
When, IIB or IIIA phase Patients with Non-small-cell Lung is postoperative to receive carboplatin joint docetaxel scheme or carboplatin joint Changchun
Auspicious shore scheme, the DFS phase disease-free survival (DFS) and Overall survival overall of patient
Survival (OS) no difference of science of statistics；It is postoperative to receive carboplatin joint docetaxel chemotherapy regimen when the position nucleotide is C
IIB or IIIA phases Patients with Non-small-cell Lung compared to receive carboplatin joint vinorelbine scheme patient DFS and OS
Significantly extend.
Specific research process is as follows：
Enter a group case：Inventor investigated retrospectively from 2 months in July, 2012 in 2010 the Shan great Second Academys go to a doctor it is non-
Patients With Small Cell Carcinoma of The Lung, according to the standard of rejecting, finally determine that a collection of IIB or IIIA phases non-small cell lung cancer enters group this research,
Enter that group patients with lung cancer is postoperative to receive adjuvant chemotherapy, scheme is that carboplatin combines docetaxel or vinorelbine, enters a group lung
Cancer patient has clear and definite clinical pathologic characteristic and complete Follow-up After information.
Research method：Gene sequencing is carried out to above-mentioned tumor tissues using two generation high throughput sequencing technologies, step includes：
1. use GeneReadTMDNA FFPE Kit extract the DNA in paraffin section sample and quantified；
②Ion AmpliSeqTMLibrary prepares；
③Ion PGMTM Hi-QTMSequencing and data processing；
Result of study：RB1 genes I680T is detected in 47.2% patient (c.2039T>C) site mutation, invention
People sends out currently all to detect in the lung cancer patient of the RB1 genes site mutation by Kaplan-Meier analytic approach, carboplatin connection
The DFS phase (DFS) of patient can significantly be extended compared to carboplatin joint vinorelbine chemotherapy regimen by closing docetaxel chemotherapy regimen
With Overall survival (OS)；According to Cox regression models, in the patients with lung cancer of the detection collocation RB1 genes site mutation, carboplatin connection
It is the individual index for influenceing prognosis to close docetaxel chemotherapy regimen.But the patients with lung cancer in the RB1 genes site for wild type
In, carboplatin joint docetaxel chemotherapy regimen combines influence of the vinorelbine chemotherapy regimen to patient's prognosis with carboplatin without statistics
Difference.
Research conclusion：In RB1 genes I680T (c.2039T>C) in the patients with lung cancer of site mutation, the more west of carboplatin joint he
Match chemotherapy regimen is the individual index for influenceing patients with lung cancer prognosis.By the detection to the RB1 genes site, contribute to clinically
The NACT scheme of Patients with Non-small-cell Lung selection more effectively more individuation.
According to above-mentioned discovery, specific primer design is carried out, design of primers principle is as follows：
1. designing primer in nucleic acid series conserved region, it is completely conservative at least ensureing that there are 4 bases at 3 ' ends of primer
's；2. primer sequence length is generally between 18~25bp, and Tm values are between 58~60 DEG C；3. G+C contents in primer sequence
Between 40%~60%；4. the distribution of base will ensure randomness in primer；5. can not exist in primer continuous 4 it is complementary
Base；6. the end of primer 5 ' can be modified, but the end of primer 3 ' can not be modified；7. 3 ' ends of primer are not preferably G or/and C.
Taqman probe design principles are as follows：
1. the specificity of probe designed by probe and guarantee is designed in nucleotide sequence conserved region；2. designed probe
In, it is ensured that the randomness of base distribution；3. probe length typically between 10~30 bases, shortens as far as possible；4. probe sequence
Middle bases G+C content control is between 40%~60%；5. probe itself can not contain 4 continuous complementary bases；6. probe
Quantity of the quantity of bases G not above base C in sequence；7. probe 5' can not be started with bases G；8. fluorescence radiation group adds
Held in probe 5 ', fluorescent quenching group is added in the end of probe 3 '.
Design of primers is carried out according to mentioned above principle：
1st, found in COSMIC databases comprising RB1 genes I680T (c.2039T>C) the 20-30bp pieces of site mutation
Section, identified base sequence is cDNA sequence；
2nd, its corresponding exon sequence on DNA is determined by BLAST according to the cDNA sequence；
3rd, determine that the global DNA sequence corresponding to the exon sequence (including extron and includes in GenomeBrower
Son), it is as follows：T is mutational site
4th, with primer express 3.0.1 software Design primers and probe, it is determined that final upstream primer sequence such as SEQ
Shown in ID NO.1, downstream primer sequence is as shown in SEQ ID NO.2；The wild-type sequence of Taqman probes such as SEQ ID NO.3
Shown, the mutant sequences of Taqman probes are as shown in SEQ ID NO.4.
Embodiment 2
Detect RB1 genes I680T (c.2039T>C) the kit of site mutation, including：
Detect RB1 genes I680T (c.2039T>C) the special primer of site mutation, the sense primer of the special primer
Nucleotide sequence is as shown in SEQ ID NO.1, the anti-sense primer nucleotide sequence such as SEQ ID NO.2 institutes of the special primer
Show；
Specific recognition RB1 genes I680T is (c.2039T>C) the Taqman probes of site mutation, the Taqman probes
Wild-type nucleotide sequences as described in SEQ ID NO.3,5 ' end connection VIC fluorescence labelings；The saltant type core of Taqman probes
Nucleotide sequence is as shown in SEQ ID NO.4,5 ' end connection FAM fluorescence labelings；
Buffer solution, dNTPs, Taq DNA polymerase, MgCl2And DNA extraction kit；
Each composition reaction density is as follows：
0.2 μM of sense primer, 0.2 μM of anti-sense primer, 0.1 μM of wild type Taqman probes, saltant type Taqman probes 0.1
μM, dNTPs 0.25mM, Taq DNA polymerase 0.025U/ μ L, MgCl2The template that 1.5mM, DNA extraction kit are extracted
DNA10~50ng.
Embodiment 3
Kit 10 non-small cell lung cancer paraffin of detection that RB1 gene mutation sites are detected described in Application Example 2 are cut
RB1 genes are c.2039T in piece sample>The mutation in C sites.
Using GeneReadTMDNA FFPE Kit (QIAGEN) extract template DNA；
Enter performing PCR reaction in 20 μ L reaction systems, PCR reaction condition is：
Reaction is carried out on Roche LC96 real-time fluorescence PCR instrument, 94 DEG C of pre-degeneration 180sec, then according to 94 DEG C of denaturation
5sec, 60 DEG C of annealing and extension 30sec, carry out 50 circulations.
After testing, have 3 testing results as shown in figure 1, RB1 genes I680T (c.2039T>C) site is wild type；Have 7
Example testing result as shown in Fig. 2 RB1 genes I680T (c.2039T>C) site is saltant type.
Genetic test, gained 10 are carried out to above-mentioned 10 paraffin section samples respectively using two generation high throughput sequencing technologies
The testing result in the sample RB1 genes site and application kit testing result of the present invention are completely the same.
Embodiment 4
The kit that RB1 gene mutation sites are detected described in Application Example 2 detects 10 fresh cancers of non-small cell lung cancer
RB1 genes are c.2039T in tissue>The mutation in C sites.
Using TIANamp Genomic DNA Kit (TIANGEN) DNA extraction kit respectively from 10 fresh cancerous tissues
Middle extraction template DNA；
Final concentration and result judgement are the same as embodiment 1 when PCR reaction conditions, each component reaction.
6 testing results as shown in figure 1, sample RB1 genes I680T (c.2039T>C) site is wild type；4 inspections
Survey result as shown in Fig. 2 sample RB1 genes I680T (c.2039T>C) site is saltant type.
Genetic test, 10 samples of gained are carried out to 10 fresh cancerous tissue samples respectively using two generation high throughput sequencing technologies
The testing result in this RB1 genes site and application kit testing result of the present invention are completely the same.
Embodiment 5
The kit that RB1 gene mutation sites are detected described in Application Example 2 is detected outside 10 Patients with Non-small-cell Lung
RB1 genes I680T is (c.2039T in all blood>C) the mutation in site.
Using TIANamp Genomic DNA Kit (TIANGEN) DNA extraction kit respectively from 10 blood preparations
Extract template DNA；
Final concentration and result judgement are the same as embodiment 1 when PCR reaction conditions, each component reaction.
5 testing results as shown in figure 1, sample RB1 genes I680T (c.2039T>C) site is wild type；5 inspections
Survey result as shown in Fig. 2 sample RB1 genes I680T (c.2039T>C) site is saltant type.
Genetic test, 10 sample RB1 of gained are carried out to 10 blood preparations respectively using two generation high throughput sequencing technologies
The testing result in the gene site and application kit testing result of the present invention are completely the same.
Embodiment 6
In January, 2011 be have chosen to the paraffin section sample 30 of the Patients with Non-small-cell Lung between in January, 2012, institute
Select case all to receive postoperative adjuvant chemotherapy (carboplatin combines docetaxel or vinorelbine), and have complete Follow-up After
Information.
Kit 30 non-small cell lung cancer paraffin of detection that RB1 gene mutation sites are detected described in Application Example 2 are cut
RB1 genes I680T is (c.2039T in piece sample>C) the catastrophe in site.
Respectively template DNA is extracted from 30 paraffin section samples；
Final concentration and result judgement are the same as embodiment 1 when PCR reaction conditions, each component reaction.
As a result find (c.2039T wherein 19 have RB1 genes I680T>C) site mutation.Inventor further passes through
Kaplan-Meier analytic approach send out currently all 19 detect in RB1 genes I680T (c.2039T>C) the lung cancer in mutational site
In patient, patient can significantly be extended compared to carboplatin joint vinorelbine chemotherapy regimen by receiving carboplatin joint docetaxel chemotherapy regimen
DFS phase (DFS) and Overall survival (OS).So as to demonstrate detection RB1 genes I680T (c.2039T>C) site mutation
There is the kit of situation the adjuvant chemotherapy for Patients with Non-small-cell Lung individuation to provide the application value instructed.
Embodiment described above is only currently preferred specific embodiment, is not intended to limit the present invention, it is every
Within spirit and principle of the invention, any modification, equivalent substitution and improvement for being made etc., the protection of the present invention should be included in
Within the scope of.
SEQUENCE LISTING
<110>Zhao is small firm
<120>Detect the kit of RB1 gene I680T site mutations
<160> 4
<170> PatentIn version 3.5
<210> 1
<211> 24
<212> DNA
<213>It is artificial synthesized
<400> 1
tctgtctgag cacccagaat taga 24
<210> 2
<211> 19
<212> DNA
<213>It is artificial synthesized
<400> 2
tctgcagggt gtgctggaa 19
<210> 3
<211> 17
<212> DNA
<213>It is artificial synthesized
<400> 3
catatcatct ggaccct 17
<210> 4
<211> 16
<212> DNA
<213>It is artificial synthesized
<400> 4
catatcacct ggaccc 16
Claims (5)
1. detect RB1 genes I680T (c.2039T>C) the kit of site mutation, it is characterised in that including：
Detect RB1 genes I680T (c.2039T>C) the specific primer of site mutation, the sense primer of the specific primer
Nucleotide sequence is as shown in SEQ ID NO.1, the anti-sense primer nucleotide sequence such as SEQ ID NO.2 institutes of the special primer
Show.
2. kit as claimed in claim 1, it is characterised in that also include：
Specific recognition RB1 genes I680T is (c.2039T>C) the Taqman probes of site mutation, the open country of the Taqman probes
Raw type nucleotide sequence is as described in SEQ ID NO.3, the mutant nucleotide sequence such as SEQ ID NO.4 institutes of Taqman probes
Show.
3. kit as claimed in claim 1, it is characterised in that also include：
Buffer solution, dNTPs, Taq DNA polymerase, MgCl2And DNA extraction kit.
4. kit as claimed in claim 1, it is characterised in that each composition reaction density is as follows：
0.2 μM of sense primer, 0.2 μM of anti-sense primer, 0.1 μM of wild type Taqman probes, 0.1 μM of saltant type Taqman probes,
DNTPs 0.25mM, Taq DNA polymerase 0.025U/ μ L, MgCl2The template DNA that 1.5mM, DNA extraction kit are extracted<
250ng。
5. kit as claimed in claim 1, it is characterised in that 5 ' end connection VIC of the wild type of the Taqman probes
Fluorescence labeling；5 ' end connection FAM fluorescence labelings of the saltant type of Taqman probes.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN110484623A (en) * | 2019-08-27 | 2019-11-22 | 深圳市宝安区妇幼保健院 | A kind of RB1 mutated gene, primer, detection method, kit and application |
| CN110592212A (en) * | 2019-08-15 | 2019-12-20 | 吴一龙 | Combined marker for lung cancer detection, detection kit and application thereof |
| CN113913521A (en) * | 2021-11-05 | 2022-01-11 | 复旦大学 | Genotyping detection kit and application thereof |
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| CN110592212A (en) * | 2019-08-15 | 2019-12-20 | 吴一龙 | Combined marker for lung cancer detection, detection kit and application thereof |
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| CN113913521A (en) * | 2021-11-05 | 2022-01-11 | 复旦大学 | Genotyping detection kit and application thereof |
| CN113913521B (en) * | 2021-11-05 | 2024-05-14 | 复旦大学 | Genotyping detection kit and application thereof |
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