CN110373453A - A kind of detection primer, probe and the kit in KRAS gene mutation site - Google Patents
A kind of detection primer, probe and the kit in KRAS gene mutation site Download PDFInfo
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Abstract
The present invention provides detection primer, probe and the kit in a kind of KRAS gene mutation site, the abrupt climatic change site of KRAS gene is 7 hot spot mutation points on 2 exon the 12nd of KRAS gene and the 13rd bit codon at present: G12S, G12R, G12C, G12D, G12A, G12V, G13D, this 7 kinds mutation have accounted for 98.5% or more of all mutation.KRAS gene mutation site primer kit on the market is all made of point 7 hot spot mutation points of 7 pipes detection KRAS, detection process is cumbersome, cost is expensive, and detects sample type mainly for tissue or blood sample, cannot detect many samples of this inhibitor content of excrement.The present invention will test 7 reaction tubes needed when 7 catastrophe points, be reduced to 2 reaction tubes, and operating procedure is simple, and cost is lower, time-consuming less.And the sensitivity and specificity of detection are further improved by cleverly design of primers, the present invention can detect the mutation in the sample DNA of 10ng down to 1%, be suitable for clinical practice application and popularization.
Description
Technical field
The present invention relates to oncogene abrupt climatic change fields, and in particular to a kind of for detecting KRAS gene mutation site
Detection primer, kit and its application.
Background technique
The main reason for colorectal cancer and lung cancer are global cancer related mortalities.Non-small cell lung cancer (non-small
Cell lung cancer, NSCLC) 80%-85% in Zhan Suoyou patients with lung cancer, most of patients with lung cancer located when making a definite diagnosis
In late stage.Colorectal cancer includes colon cancer and the carcinoma of the rectum, also known as colorectal cancer.Normal Colon and rectum can all have number with ten thousand daily
In the epithelial cell shedding to enteron aisle of meter, if having cancer or Precancerous Lesion in our enteron aisle, these lesion groups
A part of abnormal cell knitted can also fall off together with normal cell, and excrete with excrement.Currently, based on oncogene
The development of body quick diagnosis and targeted therapy changes the diagnosing and treating mode of the patient of colorectal cancer and lung cancer, many institute's weeks
Know that KRAS gene is one of most common mutant oncogene in NSCLC and colorectal cancer, KRAS gene was in NSCLC base in 1984
It is described for the first time because in, belongs to RAS gene family, there are three types of RAS gene family genes relevant to human tumor: HRAS,
KRAS and NRAS is respectively positioned on 11,12 and No. 1 chromosomes, and wherein KRAS gene pairs human cancer influences maximum, KRAS base
Because it is normal when can control regulating cell growth path；When KRAS gene mutation, which is permanently activated, and cannot be generated normal
RAS albumen, make Cellular Signaling Transduction Mediated disorder, uncontrolled cellular proliferation and canceration.Therefore, Kras gene mutation state and prediction
The validity of tumor patient application targeted drug and preferably carry out individualized treatment in play an important role, detect tissue or
Kras gene mutation has important for instructing cancer patients' clinical application such as colorectal cancer, non-small cell lung cancer in blood plasma
Reference value.However, KRAS gene mutation is somatic mutation, detection method is detected also different from general genetic mutation.Institute
Mainly there is two o'clock with the requirement to detection method: on the one hand since the mutant proportion in sample form is unknown, content may be mutated
Only 1% is even lower, so needing highly sensitive detection method to detect these rare cells；On the other hand due to sample
It is middle possibility the overwhelming majority be wild type, so accurately to be detected in most wild type backgrounds mutant cell just need it is higher
Specificity.Currently, KRAS gene mutation site primer kit on the market is all made of 7 of point 7 pipes detection KRAS gene
Hot spot mutation point, detection process is cumbersome, and time-consuming, and cost is expensive, and sensitivity and specificity are not high, and detects the main needle of sample type
To tissue or blood sample, many samples of this inhibitor content of excrement cannot be detected.
Summary of the invention
In order to solve above-mentioned problems of the prior art, it is an object of the present invention to provide a kind of high special
Property, high sensitivity is at low cost, and easy to operate, sample application type is wide, can detect 7 kinds of common KRAS gene mutations simultaneously
Primer and probe composition, the KRAS gene mutation include in 2 exons the 12nd and the 13rd following codon mutation
One of point is a variety of: G12S, G12R, G12C, G12D, G12A, G12V, G13D；The primer is to set the catastrophe point
First at the end primer sequence 3' is counted, and in the second at the end 3', third position and/or the one or more bases of the 4th increase
Mispairing, prevent the sequence of wild type from conjunction with the primer sequence of design, and the sequence of saltant type can be expanded efficiently.
Preferably, the corresponding base mutation of the codon mutation be followed successively by 34G > A, 34G > C, 34G > T, 35G > A,
35G>C、35G>T、38G>A。
Preferably, the primer includes one of following upstream and downstream primers or a variety of:
For KRAS gene mutation point G12S detection design upstream primer sequence as shown in SEQ ID NO.1；
For KRAS gene mutation point G12R detection design upstream primer sequence as shown in SEQ ID NO.2；
For KRAS gene mutation point G12C detection design upstream primer sequence as shown in SEQ ID NO.3；
For the downstream primer sequence such as SEQ ID NO.4 institute of KRAS gene mutation point G12S, G12R, G12C detection design
Show；
For KRAS gene mutation point G12D detection design downstream primer sequence as shown in SEQ ID NO.6；
For KRAS gene mutation point G12A detection design downstream primer sequence as shown in SEQ ID NO.7；
For KRAS gene mutation point G12V detection design downstream primer sequence as shown in SEQ ID NO.8；
For KRAS gene mutation point G13D detection design downstream primer sequence as shown in SEQ ID NO.9；
For the upstream primer sequence such as SEQ ID of KRAS gene mutation point G12D, G12A, G12V, G13D detection design
Shown in NO.5.
Preferably, the probe includes following any one or more:
For KRAS gene mutation point G12S, G12R, G12C detection design probe sequence as shown in SEQ ID NO.10；
For the probe sequence such as SEQ ID NO.11 of KRAS gene mutation point G12D, G12A, G12V, G13D detection design
It is shown.
Preferably, the primer and probe composition further includes the upstream and downstream primer and probe of reference gene GAPDH, institute
The upstream primer sequence of GAPDH is stated as shown in SEQ ID NO.12, the downstream primer sequence of the GAPDH such as SEQ ID NO.13
Shown, the probe sequence of the GAPDH is as shown in SEQ ID NO.14.
Another object of the present invention is to provide above-mentioned primer and probe compositions in preparation detection KRAS gene mutation
Application in kit.
Preferably, the KRAS gene mutation is the KRAS gene mutation of tumor sample, and the tumour includes lung cancer, ties directly
Intestinal cancer, the sample include fecal sample, the whole blood sample of the mankind, plasma sample, tissue samples, oral cavity sample, urine specimen,
Saliva sample, FFPE sample.
Another object of the present invention is to provide a kind of kit of KRAS gene mutation detection, the kit includes inspection
Survey probe groups, upstream detection primer sets, downstream detector primer group, reference gene upstream primer, reference gene downstream primer, internal reference
Genetic test probe, 5 ' of the detection probe in the detection probe group are terminal modified the first fluorescent reporter group, the internal reference
The 5 ' of genetic test probe are terminal modified the second fluorescent reporter group；Preferably, detection probe in the detection probe group
3 ' are modified with the first fluorescent quenching group, and the 3 ' of the reference gene detection probe are modified with the second fluorescent quenching group；
The upstream detection primer sets include following any one or more:
For KRAS gene mutation point G12S detection design upstream primer sequence as shown in SEQ ID NO.1；
For KRAS gene mutation point G12R detection design upstream primer sequence as shown in SEQ ID NO.2；
For KRAS gene mutation point G12C detection design upstream primer sequence as shown in SEQ ID NO.3；
For the upstream primer sequence such as SEQ ID of KRAS gene mutation point G12D, G12A, G12V, G13D detection design
Shown in NO.5.
The downstream detector primer group includes following any one or more:
For the downstream primer sequence such as SEQ ID NO.4 institute of KRAS gene mutation point G12S, G12R, G12C detection design
Show；
For KRAS gene mutation point G12D detection design downstream primer sequence as shown in SEQ ID NO.5；
For KRAS gene mutation point G12A detection design downstream primer sequence as shown in SEQ ID NO.7；
For KRAS gene mutation point G12V detection design downstream primer sequence as shown in SEQ ID NO.8；
For KRAS gene mutation point G13D detection design downstream primer sequence as shown in SEQ ID NO.9；
The detection probe group includes following any one or more:
For KRAS gene mutation point G12S, G12R, G12C detection design probe sequence as shown in SEQ ID NO.10；
For the probe sequence such as SEQ ID NO.11 of KRAS gene mutation point G12D, G12A, G12V, G13D detection design
It is shown.
Preferably, the reference gene is GADPH, the reference gene upstream primer sequence such as SEQ ID NO.12 institute
Show, the reference gene downstream primer sequence is as shown in SEQ ID NO.13, the reference gene detection probe sequence such as SEQ
Shown in ID NO.14.
Preferably, the kit is the detection kit of quantitative fluorescent PCR (QPCR) or multiple fluorescence PCR, described glimmering
Light reporter group is FAM, HEX, ROX or VIC, and the quenching group is BHQ1 or TAMRA；First fluorescent reporter group with
Second fluorescent reporter group is different.
Preferably, the kit further includes having additive, and the additive is selected from following one or more: dimethyl is sub-
Sulfone (DMSO) and formamide (formamide), glycerol, dithiothreitol (DTT) (DTT), bovine serum albumin (BSA), gelatin
(gelatin), Tween-20, NP-40, polyethylene glycol (PEG), ammonium sulfate tetramethylamine chloride.
Preferably, the kit further includes having reaction solution, and the reaction solution is selected from following one or more: AmpliTaq
GoldTM360Master Mix、T aKaRa LAWith GC Buffer, section T5_Direct_PCR_Kit is held up
(Blood), Hotstart HiTaq Master Mix (enzyme containing UDG) thermal starting pollution-proof, Hotstart HiTaq DNA
Polymerase, Anstart Master PCR mix (enzyme containing UDG) thermal starting pollution-proof,Universal
Master Mix II with UNG、TAQMAN GT Master Mix。
Preferably, the kit further include have nuclease free water, it is negative referring to product, it is positive referring to product, magnesium chloride, EDTA,
Taq enzyme, Tris, dNTPs；The negative reference product are human genome KRAS mutation feminine gender DNA, and the positive reference product are people's base
Because of a group KRAS mutation positive DNA.
Preferably, the kit includes A pipe and B pipe, and the A pipe is examined containing described for KRAS gene mutation point G12S
Survey the upstream primer of the design, upstream primer for KRAS gene mutation point G12R detection design, described for KRAS base
It is described for KRAS gene mutation point G12S, G12R, G12C detection design because of the upstream primer of catastrophe point G12C detection design
Downstream primer, the probe for KRAS gene mutation point G12S, G12R, G12C detection design；The B pipe contains the needle
It is described to be directed to KRAS gene mutation point to the upstream primer of KRAS gene mutation point G12D, G12A, G12V, G13D detection design
The downstream primer of G12D detection design, described is directed to the downstream primer for KRAS gene mutation point G12A detection design
The downstream primer of KRAS gene mutation point G12V detection design, the downstream for KRAS gene mutation point G13D detection design
Primer, the probe for KRAS gene mutation point G12D, G12A, G12V, G13D detection design, the A pipe and B pipe
It carries out closing pipe detection respectively.
Preferably, the kit, primer and/or the detectable sample type of probe include: fecal sample, the mankind it is complete
Blood sample, plasma sample, tissue samples, oral cavity sample, urine specimen, saliva sample and/or FFPE sample.
Compared with prior art, the invention has the following advantages:
(1) the mutated gene detection site of KRAS gene mutation detection kit provided by the invention is KRAS gene 2
7 hot spot mutation points on exon the 12nd and the 13rd bit codon: G12S, G12R, G12C, G12D, G12A, G12V, G13D,
This 7 kinds mutation have accounted for 98.5% or more of all mutation.Although KRAS gene mutation site primer kit on the market
It may be implemented to detect above-mentioned 7 kinds of mutation, but be all made of point 7 hot spot mutation points of 7 pipes detection KRAS, detection process is cumbersome, at
This is expensive, and detects sample type mainly for tissue or blood sample, cannot detect many samples of this inhibitor content of excrement
This.However, the present invention provides a kind of detection kit in KRAS gene mutation site, by the KRAS mutation point of other commercializations
7 reaction tubes for needing to detect when detection kit detects single 7 catastrophe points of sample, are reduced to 2 reaction tubes, by multiple glimmering
Light PCR closes pipe detection simultaneously, and operating procedure is simpler, and cost is lower, time-consuming less.
(2) the not examined sample type limitation of kit provided by the invention, detectable sample type is extensive, including excrement
Just sample, the whole blood sample of the mankind, plasma sample, tissue samples, oral cavity sample, urine specimen, saliva sample, FFPE sample
Deng.
(3) design of primers provided by the present invention is ingenious, and method is unique, greatly improve testing result sensitivity and
Specificity.In the primer sequence of design detection catastrophe point, using the method for ARMS-PCR, by mutation point design in primer sequence
First of the end 3', and the artificial second at the end 3'/third position/four increases the mispairing of one or more bases, makes
The sequence of wild type cannot be in conjunction with the mutant primer of design, and the sequence of saltant type also can be expanded efficiently, be further increased
The sensitivity and specificity of detection, the present invention can detect the mutation in the sample DNA of 10ng down to 1%, it is seen that the present invention provides
Kit detection sensitivity is high, high specificity, can satisfy the actual demand of clinical application, be suitable for large-scale promotion and make
With.
Detailed description of the invention
Fig. 1 is kit overhaul flow chart of the invention.
Fig. 2 is the KRAS gene mutation detection FAM fluorescence channel result of sample 1-7.
Fig. 3 is the FAM Air conduct measurement knot that the KRAS mutation frequency of 10ng is 10%, 5%, 2%, 1%, 0.5% and 0.1%
Fruit.
Fig. 4 is the FAM Air conduct measurement result of KRAS positive sample A and KRAS negative sample B.
Specific embodiment
Below by way of specific embodiment, invention is further described in detail, so that those skilled in the art can be more preferable
Ground understands the present invention and is practiced, but embodiment is not intended as restriction of the invention.
Experimental method used in following embodiment is conventional method unless otherwise specified.Material used, reagent
Deng being commercially available unless otherwise specified.
Detection primer, probe and the kit of embodiment 1KRAS gene mutation site
1. a kind of KRAS gene mutation detection site
KRAS gene mutation detection site is 7 hot spots on 2 exon the 12nd of KRAS gene and the 13rd bit codon
Catastrophe point: G12S, G12R, G12C, G12D, G12A, G12V, G13D, this 7 kinds be mutated accounted for the 98.5% of all mutation with
On, specific mutation such as the following table 1:
1 KRAS gene mutation detection site of table
2. design of primers principle and method and primer and probe sequence
In the primer sequence of design detection catastrophe point, using the method for ARMS-PCR, by mutation point design in primer sequence
First of the end 3' is arranged, and the artificial second at the end 3'/third position/four increases the mispairing of one or more bases,
Prevent the sequence of wild type from conjunction with the mutant primer of design, and the sequence of saltant type also can be expanded efficiently, further be mentioned
The sensitivity and specificity of high detection.
It is as follows for the particular sequence of the primer and probe of the KRAS gene mutation detection site design in table 1:
For the upstream primer sequence of KRAS gene mutation point G12S detection design are as follows:
TGAATATAAACTTGTGGTAGTTGGAGCGA(SEQ ID NO.1)；
For the upstream primer sequence of KRAS gene mutation point G12R detection design are as follows:
TGAATATAAACTTGTGGTAGTTGGAGATC(SEQ ID NO.2)；
For the upstream primer sequence of KRAS gene mutation point G12C detection design are as follows:
TGAATATAAACTTGTGGTAGTTGGAGCTT(SEQ ID NO.3)；
For the downstream primer sequence of KRAS gene mutation point G12S, G12R, G12C detection design are as follows:
GATTCTGAATTAGCTGTATCGT(SEQ ID NO.4)；
For the probe sequence of KRAS gene mutation point G12S, G12R, G12C detection design are as follows:
CAAGAGTGCCTTGACGATA(SEQ ID NO.10)；
For the upstream primer sequence of KRAS gene mutation point G12D, G12A, G12V, G13D detection design are as follows:
CACATTTTCATTATTTTTATTATAAGGC(SEQ ID NO.5)；
For the downstream primer sequence of KRAS gene mutation point G12D detection design are as follows:
TCAAGGCACTCTTGCCTACGCAAT(SEQ ID NO.6)；；
For the downstream primer sequence of KRAS gene mutation point G12A detection design are as follows:
TCAAGGCACTCTTGCCTACGCGCG(SEQ ID NO.7)；
For the downstream primer sequence of KRAS gene mutation point G12V detection design are as follows:
TCAAGGCACTCTTGCCTACGCTAA(SEQ ID NO.8)；
For the downstream primer sequence of KRAS gene mutation point G13D detection design are as follows: TCAAGGCACTCTTGCCTAGGT
(SEQ ID NO.9)；
For the probe sequence of KRAS gene mutation point G12D, G12A, G12V, G13D detection design are as follows:
TATATTCAGTCATTTTCA(SEQ ID NO.11)；
For the upstream primer sequence of the GAPDH of KRAS gene mutation point detection design are as follows: GAGGTCCTCTTGTGTCC
(SEQ ID NO.12)；
For the downstream primer sequence of the GAPDH of KRAS gene mutation point detection design are as follows: AACTACCCATGACTCAGC
(SEQ ID NO.13)；
For the probe sequence of the GAPDH of KRAS gene mutation point detection design are as follows: TGGCTGTGGCATGGTGCCAAG
(SEQ ID NO.14)。
The composition of 3.KRAS gene mutation site detection kit
A kind of main component of KRAS gene mutation site primer kit of the present invention includes as shown in table 2 below:
The composition of 2 KRAS gene mutation site primer kit of table
It will be detected for the upstream primer of KRAS gene mutation point G12S detection design, for KRAS gene mutation point G12R
The upstream primer of design, for the upstream primer of KRAS gene mutation point G12C detection design, for KRAS gene mutation point
The downstream primer of G12S, G12R, G12C detection design, for the spy of KRAS gene mutation point G12S, G12R, G12C detection design
Needle is combined into pipe detection, manages labeled as A；
It will be directed to the upstream primer of KRAS gene mutation point G12D, G12A, G12V, G13D detection design, for KRAS base
Because the downstream primer of catastrophe point G12D detection design, for KRAS gene mutation point G12A detection design downstream primer, be directed to
The downstream primer of KRAS gene mutation point G12V detection design is drawn for the downstream of KRAS gene mutation point G13D detection design
Object is combined into pipe detection for the probe of KRAS gene mutation point G12D, G12A, G12V, G13D detection design, manages labeled as B；
The concrete composition of primer and probe in kit: 0.1-20 μM of target gene primer pair, 0.1-20 μM of target gene
Probe, 0.1-20 μM of reference gene primer pair, 0.1-20 μM of reference gene probe.
Additive is also added into when doing QPCR detection, the additive is selected from one of following substance or several
Kind: dimethyl sulfoxide (DMSO) and formamide (formamide), glycerol, dithiothreitol (DTT) (DTT), bovine serum albumin (BSA),
Gelatin (gelatin),- 20, NP-40, polyethylene glycol (PEG), ammonium sulfate tetramethylamine chloride etc., reaction system body
Additive is added in system to increase the amplification efficiency of PCR reaction and reduce nonspecific products.
QPCR reaction solution is selected from one of following reaction solution: AmpliTaq GoldTM360Master Mix、T aKaRa
LAWith GC Buffer, section T5_Direct_PCR_Kit (Blood), Hotstart HiTaq Master Mix are held up
(enzyme containing UDG) thermal starting pollution-proof, Hotstart HiTaq DNA polymerase, Anstart Master PCR mix
(enzyme containing UDG) thermal starting pollution-proof,Universal Master Mix II with UNG、TAQMAN GT
Master Mix etc..
Embodiment 2KRAS gene mutation site testing process and result interpretation standard
Using the KRAS gene mutation site primer kit in embodiment 1, the QPCR platform of detection can apply to
It include: Roche, ABI, Bio-Rad, Agilent, Hangzhou BIOER Technology Co., Ltd (BIOER).
The concrete operation step of mentioned reagent box testing process is as shown in Fig. 1, and detectable sample type includes: excrement sample
Sheet, the whole blood sample of the mankind, plasma sample, tissue samples, oral cavity sample, urine specimen, saliva sample, FFPE sample etc..Often
When secondary detection, negative referring to product, the positive requires to detect with sample together synchronous participation referring to product, to guarantee the accuracy of experiment
And validity.
Testing result interpretation (by taking Lightcycler 480II as an example):
Before interpretation, Noiseband (Auto) is changed to Noiseband (Fluoresc) at the interface Noise Band, it will
Number behind Noiseband in box is changed to 2.0000；
If the Cp value of pattern detection KRAS mutation reference gene GAPDH (channel VIC) is respectively less than or is equal to 40, illustrate
The input amount of sample DNA is enough when detection, and experiment effectively, then can carry out data analysis detection KRAS mutation (FAM to the channel FAM
Channel) Cp value.Interpretation is carried out shown according to the form below 3:
3 result interpretation standard of table
The optimization of 3 additive of embodiment
Select multiple additives single type or combined test to the effectiveness of experiment, respectively with KRAS positive DNA and wild
Type DNA is tested, and the mutation in the sample DNA of 10ng down to 1% is examined, and the optimum addn that suitable fecal sample can detect is
PEG and BSA is simultaneously in use, testing result such as table 4:
The optimization of 4 additive of table
4 primer mispairing of embodiment screening
First of the end primer sequence 3', second/third position one or more base mispairing at the end 3', such as table 5.Point
It is not tested with KRAS positive DNA and wild type DNA, is detected according to the application method of kit in embodiment three, examined
In the sample DNA of 10ng down to 1% mutation, such as table 6.
5 primer mispairing of table designs table
6 primer mispairing the selection result of table
It obtains according to testing result, the kit detection most suitable mispairing scheme of fecal sample of the present invention is table 7.
The most suitable mispairing scheme of table 7
The application method of the kit of the present invention of embodiment 5
Kit of the present invention specific steps are as follows:
Step 1: the human gene group DNA inside sample is extracted with the DNA extraction kit of commercialization；
Step 2: column purification step one is purified using the DNA Clean&Concentrator of ZYMO RESEARCH company
The human gene group DNA of middle extraction, with further removal inhibit ingredient (if the DNA purity extracted in step 1 is preferable, can be with
Save this step)；
Step 3: with KRAS gene mutation site primer kit of the invention, the DNA of 10-50ng after purification is taken to carry out
Fluorescence quantitative PCR detection, quantitative fluorescent PCR reaction system such as the following table 8:
8 quantitative fluorescent PCR reaction system of table
Each sample does 2 wells, detects mutation and the KRAS of KRAS gene mutation point G12S, G12R, G12C respectively
The mutation of gene mutation point G12D, G12A, G12V, G13D.
Quantitative fluorescent PCR response procedures are as follows:
Yin and yang attribute quality-control product testing result such as the following table 9:
The detection of 9 yin and yang attribute quality-control product of table
Note: 1) PC-A, NC-A are the Quality Control for detecting KRAS gene mutation point G12S, G12R, G12C mutation；
2) PC-B, NC-B are the Quality Control for detecting KRAS gene mutation point G12D, G12A, G12V, G13D mutation；
Next interpretation can be carried out to single sample, if reference gene GAPDH (VIC when pattern detection KRAS mutation
Channel) Cp value be respectively less than or be equal to 40, then illustrate detection when sample DNA input amount it is enough, single sample can be pressed
Interpretation is carried out shown in table 10.
10 testing result interpretation standard of table
| Reaction tube | KRAS(GAPDH) | KRAS(FAM) | As a result interpretation |
| A/B | Cp≦40 | Cp≦42 | KRAS is positive |
| A&B | Cp≦40 | Cp>42 | KRAS is negative |
The detection of 6 KRAS gene mutation positive sample of embodiment
The following table 11 show 7 samples of 7 point mutation of KRAS screened by sequencing.
The detection of 11 KRAS gene mutation positive sample of table
| Sample ID | Gene | Exon | Base mutation | Amino acid mutation | The frequency of mutation |
| 1 | KRAS | Exon2 | 34G>A | G12S | 64.60% |
| 2 | KRAS | Exon2 | 34G>C | G12R | 37% |
| 3 | KRAS | Exon2 | 34G>T | G12C | 27.70% |
| 4 | KRAS | Exon2 | 35G>A | G12D | 37% |
| 5 | KRAS | Exon2 | 35G>C | G12A | 34.40% |
| 6 | KRAS | Exon2 | 35G>T | G12V | 21.90% |
| 7 | KRAS | Exon2 | 38G>A | G13D | 20% |
KRAS gene mutation is carried out to seven samples (10ng) in table according to the application method of the kit in embodiment 5
Detection, when detection, negative quality-control product, positive quality control product and sample synchronous participation detects together, negative quality-control product, positive quality control
Product meet the interpretation standard in embodiment one, and testing result is as shown in Fig. 2, from the results, it was seen that kit energy of the present invention
The sample of the detection KRAS gene mutation positive of specificity.
Embodiment 7 adds inhibitor detection
With 1 containing mutated gene sample and 1 normal person's sample, after the genomic DNA for extracting people, it is added a certain amount of
Inhibitor enhances background interference with KRAS gene mutation site primer kit of the invention and takes the DNA of 10-50ng after purification
Carry out fluorescence quantitative PCR detection, when detection, negative quality-control product, positive quality control product and sample synchronous participation detects together, detect
As a result such as the following table 12:
Table 12 adds inhibitor sample testing result
To single sample by carrying out interpretation shown in table 10, from the results, it was seen that kit of the present invention can specificity inspection
Difficult inspection, complexity sample out
The performance of 8 kit of embodiment
1. the sensitivity of kit
It is respectively 10%, 5%, 2%, 1% with the KRAS mutation frequency of KRAS positive DNA and wild type DNA blending 10ng,
0.5% and 0.1%, it is detected according to the application method of kit in embodiment two, kit of the present invention can detect 10ng's
In sample DNA down to 1% mutation, testing result is as shown in Figure 3.
2. the repeatability of kit detection
According to the application method of kit in embodiment two, KRAS positive sample A and KRAS negative sample B is carried out 20 times
Detection, three repetitions are done in detection every time, and KRAS gene FAM Air conduct measurement result is as shown in figure 4, the Cp value that KRAS mutation point detects
The coefficient of variation is respectively less than 5%.
The foregoing is merely presently preferred embodiments of the present invention, is not intended to limit the invention, it is all in spirit of the invention and
Within principle, any modification, equivalent replacement, improvement and so on be should all be included in the protection scope of the present invention
Sequence table
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Claims (10)
1. one kind can detect the primer and probe composition of 7 kinds of KRAS gene mutations simultaneously, the KRAS gene mutation includes
It is any one or more of in 2 exons the 12nd and the 13rd following codon mutation point: G12S, G12R, G12C,
G12D,G12A,G12V,G13D；The primer is by the mutation point design at first of the end primer sequence 3', and at the end 3'
Second, third position and/or the 4th increase the mispairing of one or more bases, prevent the sequence of wild type from design
Primer sequence combines, and the sequence of saltant type can be expanded efficiently；Preferably, the corresponding base mutation of the codon mutation
It is followed successively by 34G > A, 34G > C, 34G > T, 35G > A, 35G > C, 35G > T, 38G > A.
2. primer and probe composition according to claim 1, the primer includes following any one or more:
For KRAS gene mutation point G12S detection design upstream primer sequence as shown in SEQ ID NO.1；
For KRAS gene mutation point G12R detection design upstream primer sequence as shown in SEQ ID NO.2；
For KRAS gene mutation point G12C detection design upstream primer sequence as shown in SEQ ID NO.3；
For KRAS gene mutation point G12S, G12R, G12C detection design downstream primer sequence as shown in SEQ ID NO.4；
For the upstream primer sequence such as SEQ ID NO.5 of KRAS gene mutation point G12D, G12A, G12V, G13D detection design
It is shown；
For KRAS gene mutation point G12D detection design downstream primer sequence as shown in SEQ ID NO.6；
For KRAS gene mutation point G12A detection design downstream primer sequence as shown in SEQ ID NO.7；
For KRAS gene mutation point G12V detection design downstream primer sequence as shown in SEQ ID NO.8；
For KRAS gene mutation point G13D detection design downstream primer sequence as shown in SEQ ID NO.9；
The probe includes following any one or more:
For KRAS gene mutation point G12S, G12R, G12C detection design probe sequence as shown in SEQ ID NO.10；
For the probe sequence such as SEQ ID NO.11 institute of KRAS gene mutation point G12D, G12A, G12V, G13D detection design
Show.
3. primer and probe composition according to claim 1 or 2, the primer and probe composition further includes internal reference base
Because of the upstream and downstream primer and probe of GAPDH, the upstream primer sequence of the GAPDH is described as shown in SEQ ID NO.12
The downstream primer sequence of GAPDH is as shown in SEQ ID NO.13, and the probe sequence of the GAPDH is as shown in SEQ ID NO.14.
4. the described in any item primer and probe compositions of claim 1-3 are in the kit of preparation detection KRAS gene mutation
Application, the KRAS gene mutation be tumor sample KRAS gene mutation, the tumour includes lung cancer, colorectal cancer, institute
Stating sample includes fecal sample, the whole blood sample of the mankind, plasma sample, tissue samples, oral cavity sample, urine specimen, saliva sample
Originally, FFPE sample；Preferably, the tumour is colorectal cancer；Preferably, the sample is fecal sample.
5. a kind of kit of KRAS gene mutation detection, the kit include detection probe group, upstream detection primer sets, under
Swim detection primer group, reference gene upstream primer, reference gene downstream primer, reference gene detection probe, the detection probe
5 ' of detection probe in group are terminal modified the first fluorescent reporter group, and the 5 ' of the reference gene detection probe are terminal modified
Two fluorescent reporter groups；Preferably, 3 ' of the detection probe in the detection probe group are modified with the first fluorescent quenching group, institute
It states the 3 ' of reference gene detection probe and is modified with the second fluorescent quenching group；
The upstream detection primer sets include following any one or more:
For KRAS gene mutation point G12S detection design upstream primer sequence as shown in SEQ ID NO.1；
For KRAS gene mutation point G12R detection design upstream primer sequence as shown in SEQ ID NO.2；
For KRAS gene mutation point G12C detection design upstream primer sequence as shown in SEQ ID NO.3；
For the upstream primer sequence such as SEQ ID NO.5 of KRAS gene mutation point G12D, G12A, G12V, G13D detection design
It is shown；
The downstream detector primer group includes following any one or more:
For KRAS gene mutation point G12S, G12R, G12C detection design downstream primer sequence as shown in SEQ ID NO.4；
For KRAS gene mutation point G12D detection design downstream primer sequence as shown in SEQ ID NO.6；
For KRAS gene mutation point G12A detection design downstream primer sequence as shown in SEQ ID NO.7；
For KRAS gene mutation point G12V detection design downstream primer sequence as shown in SEQ ID NO.8；
For KRAS gene mutation point G13D detection design downstream primer sequence as shown in SEQ ID NO.9；
The detection probe group includes following any one or more:
For KRAS gene mutation point G12S, G12R, G12C detection design probe sequence as shown in SEQ ID NO.10；
For the probe sequence such as SEQ ID NO.11 institute of KRAS gene mutation point G12D, G12A, G12V, G13D detection design
Show.
6. kit according to claim 5, the reference gene is GADPH, the reference gene upstream primer sequence
As shown in SEQ ID NO.12, the reference gene downstream primer sequence is as shown in SEQ ID NO.13, the reference gene inspection
Probe sequence is surveyed as shown in SEQ ID NO.14.
7. kit according to claim 5, the kit is quantitative fluorescent PCR (QPCR) or multiple fluorescence PCR
Detection kit, the fluorescent reporter group are FAM, HEX, ROX or VIC, and the quenching group is BHQ1 or TAMRA；It is described
First fluorescent reporter group is different from the second fluorescent reporter group.
8. according to claim 5-7 described in any item kits, the kit further includes having additive, the additive choosing
From following one or more: dimethyl sulfoxide (DMSO) and formamide (formamide), glycerol, dithiothreitol (DTT) (DTT), ox
Haemocyanin (BSA), gelatin (gelatin), Tween-20, NP-40, polyethylene glycol (PEG), ammonium sulfate tetramethylamine chloride；
Preferably, the kit further includes having reaction solution, and the reaction solution is selected from following one or more: AmpliTaq
GoldTM360 Master Mix、T aKaRa LAWith GC Buffer, section T5_Direct_PCR_Kit is held up
(Blood), Hotstart HiTaq Master Mix (enzyme containing UDG) thermal starting pollution-proof, Hotstart HiTaq DNA
Polymerase, Anstart Master PCR mix (enzyme containing UDG) thermal starting pollution-proof,Universal
Master Mix II with UNG,TAQMAN GT Master Mix；Preferably, the kit further include have it is seedless
Enzyme water, negative reference product, positive reference product, magnesium chloride, EDTA, Taq enzyme, Tris, dNTPs；The negative reference product are people's base
Because of a group KRAS mutation feminine gender DNA, the positive reference product are human genome KRAS mutation positive DNA.
9. the kit includes A pipe and B pipe, and the A pipe is containing according to claim 5-7 described in any item kits
It states the upstream primer for KRAS gene mutation point G12S detection design, is described for KRAS gene mutation point G12R detection design
Upstream primer, the upstream primer for KRAS gene mutation point G12C detection design, it is described be directed to KRAS gene mutation
The downstream primer of point G12S, G12R, G12C detection design, it is described to be set for KRAS gene mutation point G12S, G12R, G12C detection
The probe of meter；The B pipe draws containing the upstream for KRAS gene mutation point G12D, G12A, G12V, G13D detection design
Object, it is described to be examined for the downstream primer of KRAS gene mutation point G12D detection design, the KRAS gene mutation point G12A that is directed to
Downstream primer, the downstream primer for KRAS gene mutation point G12V detection design of design are surveyed, it is described to be directed to KRAS base
It is described to be detected for KRAS gene mutation point G12D, G12A, G12V, G13D because of the downstream primer of catastrophe point G13D detection design
The probe of design, the A pipe and the B pipe carry out closing pipe detection respectively.
10. the KRAS gene mutation is the KRAS gene of tumor sample according to claim 5-7 described in any item kits
Mutation, the tumour include lung cancer, colorectal cancer, the sample include fecal sample, the whole blood sample of the mankind, plasma sample,
Tissue samples, oral cavity sample, urine specimen, saliva sample, FFPE sample；Preferably, the tumour is colorectal cancer；Make
For preferably, the sample is fecal sample.
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| CN115927637A (en) * | 2022-12-20 | 2023-04-07 | 苏州普熙生生物医药科技有限公司 | Primer group, kit and detection method for KRAS gene mutation detection |
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