CN106755376A - A kind of multiple fluorescence PCR detection reagent box for detecting B RAF gene mutations - Google Patents
A kind of multiple fluorescence PCR detection reagent box for detecting B RAF gene mutations Download PDFInfo
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Abstract
The invention discloses a kind of multiple fluorescence PCR detection reagent box for detecting B RAF gene mutations, including：B primer sets, the primer sets of E 4, B detection probes, the detection probes of E 4 and internal standard detection probe；5 ' ends of K detection probes and the detection probes of E 4 are modified with the first fluorescent reporter group, and 3 ' ends are modified with the first fluorescent quenching group；The sequence of internal standard detection probe is identical with the sequence of the detection probes of E 2, its 5 ' it is terminal modified have second fluorescent reporter groups different from the first fluorescent reporter group, 3 ' ends are modified with the second fluorescent quenching group.With mankind's B RAF gene mutations as detection object, optimum organization and fluorescence probe by special primer so as to realize accurate, simply and rapidly detection B RAF gene mutations simultaneously, and detect that the ability of mutation is high to the present invention.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a multiplex fluorescence PCR detection kit for detecting B-RAF gene mutation.
Background
The B-raf gene, named as murine sarcoma viral virus filterability (V-raf) oncogenic homolog B1, maps to human chromosome 7q34, has a functional coding region consisting of 2150 pairs of bases, encodes a serine threonine protein kinase in the MAPK pathway, which transduces signals from Ras to MEK1/2, and thus is involved in the regulation of various intracellular biological events. The mutation of the B-raf gene can activate ERK signals, induce cell proliferation and prevent cell apoptosis. Somatic B-raf gene missense mutations are present in about 70% of malignant melanoma and 15% of colon cancers.
The B-raf gene is used as an important member in a raf-MEK-ERK signal transduction pathway and plays an important role in the aspects of tumor cell proliferation, differentiation, apoptosis and the like. The normal B-raf protein functions to transmit signals from the cell membrane. B-raf proteins generally remain active only when signaling is required. However, the mutated B-raf remains active at all times and thus interferes with the normal function of the cellular signaling chain, causing cellular abnormalities.
The B-raf gene can be used for clinically diagnosing, prognostically evaluating and treating tumors (including precancerous lesions). The disease condition and prognosis are evaluated by detecting gene mutations to infer the stage and degree of differentiation of the tumor. With the development of the medical industry, a plurality of medicines for blocking a cell signal pathway aiming at tumor lesions are developed, but when the medicines are used, activation mutation of downstream signal molecules of the signal pathway needs to be ensured, which is a problem to be faced by the medical industry at present, and a rapid, simple, safe, high-sensitivity, high-throughput and low-cost B-raf gene mutation detection is urgently needed to assist the clinical application of the medicines.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provides a multiplex fluorescence PCR detection kit for detecting B-RAF gene mutation.
The technical scheme of the invention is as follows:
a multiplex fluorescence PCR detection kit for detecting B-RAF gene mutation, comprising: a primer group B, a primer group E-4, a detection probe B, a detection probe E-4 and an internal standard detection probe; the 5 'ends of the detection probe B and the detection probe E-4 are both modified with a first fluorescence reporter group, and the 3' ends are both modified with a first fluorescence quenching group; the sequence of the internal standard detection probe is the same as that of the E-4 detection probe, a second fluorescence reporter group different from the first fluorescence reporter group is modified at the 5 'end of the internal standard detection probe, and second fluorescence quenching groups are modified at the 3' end of the internal standard detection probe; wherein,
the primer group B consists of a forward primer B-M-F and a reverse primer B-M-R, and sequentially comprises sequences shown as SEQ ID NO 01 and SEQ ID NO 02;
the E-4 primer group consists of a forward primer E-4-F and a reverse primer E-4-R, and sequentially comprises sequences shown as SEQ ID NO 03 and 04;
the composition of each reaction system of the multiple fluorescence PCR detection kit is as follows:
b primer group
B detection probe and/or E-4 detection probe
E-4 primer set
Internal standard detection probe
1 XPCR buffer
MgCl2
dNTPs
Taq enzyme
H2O
A DNA template;
the reaction conditions of each reaction system of the multiple fluorescence PCR detection kit are as follows: pre-denaturation at 95 ℃ for 5min for 1 cycle; denaturation at 95 ℃ for 25s, annealing at 64 ℃ for 20s, extension at 72 ℃ for 15s, 15 cycles; denaturation at 95 ℃ for 25s, annealing at 60 ℃ for 35s, extension at 72 ℃ for 10s, and 30 cycles; the fluorescence signal was detected during the last 30 cycles of annealing.
In a preferred embodiment of the invention, the B detection probe is B-P-C comprising the sequence shown in SEQ ID NO 05.
In a preferred embodiment of the invention, the E-4 detection probe is E-4-P and the internal standard detection probe is E-4-I, both comprising the sequence shown in SEQ ID NO 06.
The specific sequences of the above substances and probes are shown in the following table:
further preferably, the first fluorescence reporter group is FAM, the first fluorescence quenching group is BHQ, the second fluorescence reporter group is HEX, ROX or VIC, and the second fluorescence quenching group is BHQ.
The invention has the beneficial effects that:
1. the kit of the invention is suitable for use by a patient prior to entering a personalized targeted therapy session. Can provide a scientific basis for the individual medication of the patients, and reduce the treatment risk and the burden of the patients.
2. The invention takes human B-RAF gene mutation as a detection object, realizes accurate, simple and rapid simultaneous detection of the B-RAF gene mutation by the optimized combination of specific primers and a fluorescent probe, and has high mutation detection capability.
Drawings
FIG. 1 is a diagram of a multiplex fluorescent PCR reaction procedure in example 2 of the present invention.
FIG. 2 is a graph showing the detection curve of the negative control sample for detecting the V600E mutation in the B-RAF gene by multiplex fluorescent PCR in example 2 of the present invention.
FIG. 3 is a sample detection graph for detecting the mutation of the B-RAF gene V600E by multiplex fluorescent PCR in example 2 of the present invention.
Detailed Description
The technical solution of the present invention will be further illustrated and described below with reference to the accompanying drawings by means of specific embodiments.
A multiplex fluorescence PCR detection kit for detecting B-RAF gene mutation, comprising: a primer group B, a primer group E-4, a detection probe B, a detection probe E-4 and an internal standard detection probe; the 5 'ends of the detection probe B and the detection probe E-4 are both modified with a first fluorescence reporter group, and the 3' ends are both modified with a first fluorescence quenching group; the sequence of the internal standard detection probe is the same as that of the E-4 detection probe, a second fluorescence reporter group different from the first fluorescence reporter group is modified at the 5 'end of the internal standard detection probe, and second fluorescence quenching groups are modified at the 3' end of the internal standard detection probe; preferably, the first fluorescence reporter group is FAM, the first fluorescence quenching group is BHQ, the second fluorescence reporter group is HEX, ROX or VIC, and the second fluorescence quenching group is BHQ, wherein,
the primer group B consists of a forward primer B-M-F and a reverse primer B-M-R, and sequentially comprises sequences shown as SEQ ID NO 01 and SEQ ID NO 02;
the E-4 primer group consists of a forward primer E-4-F and a reverse primer E-4-R, and sequentially comprises sequences shown as SEQ ID NO 03 and 04;
the composition of each reaction system of the multiple fluorescence PCR detection kit is as follows:
b primer group
B detection probe and/or E-4 detection probe
E-4 primer set
Internal standard detection probe
1 XPCR buffer
MgCl2
dNTPs
Taq enzyme
H2O
A DNA template;
the reaction conditions of each reaction system of the multiple fluorescence PCR detection kit are as follows: pre-denaturation at 95 ℃ for 5min for 1 cycle; denaturation at 95 ℃ for 25s, annealing at 64 ℃ for 20s, extension at 72 ℃ for 15s, 15 cycles; denaturation at 95 ℃ for 25s, annealing at 60 ℃ for 35s, extension at 72 ℃ for 10s, and 30 cycles; the fluorescence signal was detected during the last 30 cycles of annealing.
The B detection probe is B-P-C and comprises a sequence shown as SEQ ID NO 05.
The E-4 detection probe is E-4-P, and the internal standard detection probe is E-4-I, which both comprise a sequence shown in SEQ ID NO 06.
The specific sequences of the above substances and probes are shown in the following table:
| name (R) | Sequence of |
| B-M-F | GTGATTTTGGTCTAGCTACAGA |
| B-M-R | CTCAGCAGCATCTCAGG |
| E-4-F | GACTCTGAAGATGTACCTATGGTCCTA |
| E-4-R | CTTCTTGCTAAGTCCTGAGCCTGT |
| B-P-C | CCCATCAGTTTGAACAGTTGTCTGG |
| E-4-P | TGTGATTTGCCTTCTAGAACAGTAGA |
| E-4-I | TGTGATTTGCCTTCTAGAACAGTAGA |
Example 1
In the embodiment, the V600E mutation of the human B-RAF gene V600 is used as a detection object, and the V600E mutation can be detected accurately, simply and quickly through the optimized combination of specific primers and a fluorescent probe, and the mutation detection capacity is up to 1%.
The genome DNA of a wild-type cell line H460 is used as a wild-type template, a V600E mutant plasmid is used as a positive control template, the multiplex PCR detection is carried out by using the multiplex fluorescence PCR detection kit for detecting B-RAF gene mutation, and finally, a cycle number Ct value required when a set threshold value is reached is used as a standard for result judgment (in the embodiment, a first fluorescence reporter group is FAM, a second fluorescence reporter group is VIC, ROX or HEX, and both the first fluorescence quencher group and the second fluorescence quencher group are BHQ).
The amplification reaction system of the multiplex fluorescence PCR detection is as follows:
| serial number | Material(s) | Concentration of material | Dosage (mu L) |
| 1 | 1 × PCR buffer | 1× | 10 |
| 2 | MgCl2 | 25mM | 8 |
| 3 | dNTPs | 10mM | 5 |
| 4 | B-M-F | 50mM | 2 |
| 5 | B-M-R | 50mM | 0.1 |
| 6 | B-P-C-FAM | 50mM | 0.1 |
| 7 | E-4-F | 50mM | 0.1 |
| 8 | E-4-R | 50mM | 0.1 |
| 9 | Internal standard detection probe | 50mM | 0.1 |
| 10 | Taq enzyme | 5U | 0.5 |
| 11 | H2O | Purified water | 18.9 |
| 12 | DNA template | 2ng/ul | 5 |
| 13 | Total volume | 50 |
The reagent components are 1 × PCR buffer solution and MgCl2Taq enzyme, dNTP were purchased from Dalibao Bio, China.
The reaction conditions of the multiplex fluorescence PCR detection are pre-denaturation at 95 ℃ for 5min for 1 cycle; denaturation at 95 ℃ for 25s, annealing at 60 ℃ for 20s, extension at 72 ℃ for 10s, 15 cycles; denaturation at 95 ℃ for 25s, annealing at 60 ℃ for 35s, extension at 72 ℃ for 10s, 30 cycles, and detection of the fluorescent signals of the first and second fluorescent reporter groups during annealing for the last 30 cycles.
The Ct value for detecting the fluorescence signals of the first fluorescence reporter group and the second fluorescence reporter group is obtained by adopting an Mx3000P fluorescence PCR amplification instrument, an Mx3005P fluorescence PCR amplification instrument or an ABI 7500 fluorescence PCR amplification instrument. 96 samples (including negative and positive controls) can be detected at a time. And (3) judging the result according to the Ct value displayed by the fluorescent PCR amplification instrument: detecting the fluorescence intensity of a first fluorescence reporter group and a second fluorescence reporter group of the reaction system, and when the signal of the second fluorescence reporter group reaches a set threshold value (Ct is more than 18), indicating that the amount of the sample DNA is in an allowable range, and the signal result of the first fluorescence reporter group is credible; taking a cycle number Ct value required when the signal of the first fluorescent reporter group reaches a set threshold value as a negative and positive judgment standard, wherein the Ct value is 0 or 30: negative; ct is less than 30: and (4) positive.
Example 2
In the embodiment, the V600E mutation on the B-RAF gene hot spot mutation V600 is taken as an example to analyze the method for detecting the V600E mutation of the B-RAF gene V600 by multiplex fluorescence PCR. The experimental cell line 3 strains and 1 plasmid are respectively H460(B-RAF gene wild type), 293T (B-RAF gene wild type), SW480(B-RAF gene wild type) and V600E mutant plasmids; 45 healthy blood samples from the blood donator for non-compensation, 45 clinical lung cancer samples (including fresh tissue, paraffin sections, pleural effusion, whole blood).
The method for detecting the V600E mutation on the B-RAF gene V600 by using the fluorescent PCR comprises the following steps:
(1) sample treatment and template extraction quality control:
the sample application range comprises specimens such as fresh pathological tissues excised by operation, formaldehyde-fixed paraffin-embedded case tissues, paraffin sections, whole blood, plasma, serum, pleural effusion and the like. Fresh pathological tissues are taken, the size of mung beans is about 1g, genomic DNA is extracted by using a tissue DNA extraction kit of Qiagen company, and the specific steps are specified according to the kit operation. Cutting the wax block sample into 5-8 μm slices, taking 5 slices, or taking 5 slices of the prepared 5-8 μm slices, dewaxing by xylene, extracting genomic DNA by using a Qiagen paraffin-embedded DNA extraction kit, and specifically carrying out the steps according to the kit operation instructions. Whole blood, plasma, serum and pleural effusion samples, genomic DNA was extracted using Qiagen tissue DNA extraction kit, the specific steps were as per kit instructions. Each time, the whole blood is extracted by 200 μ l, and the blood plasma, blood serum and pleural effusion are not less than 800 μ l. The extracted DNA was dissolved in Tris-HCl (10mmol/L, pH 8.0), the mass of extraction was measured by UV spectrophotometer and the concentration was determined, and the concentration of DNA was adjusted to 100 ng/. mu.l or 2 ng/. mu.l with Tris-HCl solution (10mmol/L, pH 8.0) as a template for PCR amplification.
(2) Performing fluorescence PCR amplification detection on the template obtained in the step (1) by using the multiple fluorescence PCR kit (in the embodiment, the first fluorescence reporter group is FAM, the second fluorescence reporter group is VIC, ROX or HEX, and the first fluorescence quenching group and the second fluorescence quenching group are BHQ), and preparing a reaction system:
the reaction conditions of the fluorescence PCR are shown in FIG. 1: pre-denaturation at 95 ℃ for 5min for 1 cycle; denaturation at 95 ℃ for 25s, annealing at 60 ℃ for 20s, extension at 72 ℃ for 10s, 15 cycles; denaturation at 95 ℃ for 25s, annealing at 60 ℃ for 35s, and extension at 72 ℃ for 10s, for 30 cycles.
(3) And (3) detection: 92 samples (including negative and positive controls) were tested at a time using a Mx3000P real-time PCR amplification apparatus (StrataGene) followed by 30 cycles of annealing to detect the fluorescent signals of the first and second fluorescent reporters. And (5) judging a result: and (3) judging the result according to the Ct value displayed by the fluorescent PCR amplification instrument: detecting the fluorescence intensity of a first fluorescence reporter group and a second fluorescence reporter group of the reaction system, and when the signal of the second fluorescence reporter group reaches a set threshold value (Ct is more than 18), indicating that the amount of the sample DNA is in an allowable range, and the signal result of the first fluorescence reporter group is credible; taking a cycle number Ct value required when the signal of the first fluorescent reporter group reaches a set threshold value as a negative and positive judgment standard, wherein the Ct value is 0 or 30: negative; ct is less than 30: the results of the positive test are shown in FIGS. 2 and 3.
The 45 whole blood samples of healthy blood donors and cell lines H460(B-RAF gene wild type), 293T (B-RAF gene wild type), SW480(B-RAF gene wild type) and V600E mutant plasmids are detected by the system of the invention, only the V600E mutant plasmid has a fluorescence signal, and other samples have no fluorescence signal, so that the specificity of fluorescence PCR is further proved.
And (3) sensitivity analysis: the mutant V600E plasmid was serially diluted 10-fold gradient from the power of 4 of 10 to the power of 3 of 10, the power of 2 of 10, the power of 1 of 10, and the power of 0 of 10. mu.L of DNA was added for each reaction. The result shows that the fluorescence PCR method of the invention has high sensitivity, and 5 copies of DNA genome can be detected.
Analysis of the Selectivity Capacity: the total DNA amount per PCR reaction, 100 ng/reaction and 10 ng/reaction, was fixed. The V600E mutant plasmid DNA and wild type cell line (H460) DNA concentrations were initially adjusted to 20 ng/. mu.L and 2 ng/. mu.L. Thus, the addition of 5. mu.L template per reaction was 100 ng/reaction and 10 ng/reaction. The mock DNA template was prepared as follows.
A: 50% is the 10 rd power V600E mutant cell DNA.
B: 60 mu L of 30% solution A is mixed with 40 mu L of 10 3-power V600E mutant cell DNA, and the mixture is shaken and mixed evenly.
C: after 40. mu.L of 20% solution A was taken, 60. mu.L of 10 rd power V600E mutant cell DNA was mixed, and the mixture was shaken and mixed well.
D: 50 μ L of 15% B solution was taken, 50 μ L of 10 3 rd power V600E mutant cell DNA was mixed, and the mixture was shaken and mixed.
E: 50 μ L of 10% C solution was taken, 50 μ L of 10 3 rd power V600E mutant cell DNA was mixed, and the mixture was shaken and mixed well.
F: 50 μ L of the 5% E solution was taken, 50 μ L of 10 3 rd power V600E mutant cell DNA was mixed, and the mixture was shaken and mixed well.
G: 20 μ L of 1% F solution was mixed with 80 μ L of 10 3 rd power V600E mutant cell DNA, and the mixture was shaken and mixed.
H: 50 μ L of 0.5% G solution was taken, 50 μ L of 10 3 rd power V600E mutant cell DNA was mixed, and the mixture was shaken and mixed well.
I: 20 μ L of 0.1% H solution was taken, and 80 μ L of 10 3 rd power V600E mutant cell DNA was mixed and mixed well by shaking.
The results show that the fluorescence PCR method of the invention has the selective detection capability of detecting 5 copies of mutant DNA in 10ng of total DNA, and the detection capability is 1%.
And (3) repeatability test: the mutant V600E plasmid DNA10ng, 1ng and 100pg were added to each reaction, and the fluorescence PCR amplification was repeated 10 times, 10 times with Ct values less than 0.1 cycle apart.
A total of 45 specimens of surgically excised lung cancer, whole blood and plasma specimens, and pleural effusion were collected, 30 tissue samples, 5 plasma samples, 5 pleural effusions, and 5 whole blood samples. 31 male and 14 female. The age is 36-71 years, and the average age is 55 years.
For the V600E mutation of V600, the detection result of the invention is completely consistent with the DNA sequencing result: of the 45 samples, 12 of them underwent the V600E mutation, and 33 were all wild-type.
The multiplex fluorescence PCR reaction can be used for simultaneously detecting the mutation of the B-RAF exon V600V600E, and the detection time is only 90min, so that the invention is accurate, simple and rapid and can meet the rapid diagnosis of the mutation. Moreover, the coincidence rate of the results of the multiple fluorescence PCR method and the traditional sequencing method is 100%, the fluorescence PCR sensitivity and the selective detection capability are higher than those of the traditional sequencing method, and 1% of mutant DNA in 10ng of sample DNA can be detected.
The above description is only a preferred embodiment of the present invention, and therefore should not be taken as limiting the scope of the invention, which is defined by the appended claims.
<110> Xiamen Biotech Co., Ltd
<120> a multiplex fluorescence PCR detection kit for detecting B-RAF gene mutation
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<213> Artificial sequence
<400>6
tgtgatttgc cttctagaac agtaga 26
Claims (4)
1. A multiple fluorescence PCR detection kit for detecting B-RAF gene mutation is characterized in that: the method comprises the following steps: a primer group B, a primer group E-4, a detection probe B, a detection probe E-4 and an internal standard detection probe; the 5 'ends of the detection probe B and the detection probe E-4 are both modified with a first fluorescence reporter group, and the 3' ends are both modified with a first fluorescence quenching group; the sequence of the internal standard detection probe is the same as that of the E-4 detection probe, a second fluorescence reporter group different from the first fluorescence reporter group is modified at the 5 'end of the internal standard detection probe, and second fluorescence quenching groups are modified at the 3' end of the internal standard detection probe; wherein,
the primer group B consists of a forward primer B-M-F and a reverse primer B-M-R, and sequentially comprises sequences shown as SEQ ID NO 01 and SEQ ID NO 02;
the E-4 primer group consists of a forward primer E-4-F and a reverse primer E-4-R, and sequentially comprises sequences shown as SEQ ID NO 03 and 04;
the composition of each reaction system of the multiple fluorescence PCR detection kit is as follows:
b primer group
B detection probe and/or E-4 detection probe
E-4 primer set
Internal standard detection probe
1 XPCR buffer
MgCl2
dNTPs
Taq enzyme
H2O
A DNA template;
the reaction conditions of each reaction system of the multiple fluorescence PCR detection kit are as follows: pre-denaturation at 95 ℃ for 5min for 1 cycle; denaturation at 95 ℃ for 25s, annealing at 64 ℃ for 20s, extension at 72 ℃ for 15s, 15 cycles; denaturation at 95 ℃ for 25s, annealing at 60 ℃ for 35s, extension at 72 ℃ for 10s, and 30 cycles; the fluorescence signal was detected during the last 30 cycles of annealing.
2. The multiplex fluorescence PCR detection kit according to claim 1, wherein: the B detection probe is B-P-C and comprises a sequence shown as SEQ ID NO 05.
3. The multiplex fluorescence PCR detection kit according to claim 1, wherein: the E-4 detection probe is E-4-P, and the internal standard detection probe is E-4-I, which both comprise a sequence shown in SEQ ID NO 06.
4. The multiplex fluorescence PCR detection kit according to any one of claims 1 to 3, wherein: the first fluorescence reporter group is FAM, the first fluorescence quenching group is BHQ, the second fluorescence reporter group is HEX, ROX or VIC, and the second fluorescence quenching group is BHQ.
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Cited By (1)
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| CN110229910A (en) * | 2019-07-08 | 2019-09-13 | 福建省肿瘤医院（福建省肿瘤研究所、福建省癌症防治中心） | MYD88 gene L265P mutation detection kit and detection method |
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Application publication date: 20170531 |