CN108660193A - Mankind's LMNA-NTRK1 Gene Fusion abrupt climatic changes primer, probe and detection kit - Google Patents
Mankind's LMNA-NTRK1 Gene Fusion abrupt climatic changes primer, probe and detection kit Download PDFInfo
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Abstract
The present invention relates to biotechnology and medical domains, specifically disclose detection primer, probe and the detection kit of the mutation of mankind's LMNA NTRK1 fusions, the detection for the mutation of LMNA NTRK1 fusions.The detection primer includes SEQ ID NO:1 and SEQ ID NO:Sequence shown in 2, the probe sequence such as SEQ ID NO:Shown in 3,5 ' it is terminal modified have a FAM or VIC, 3 ' terminal modified have NFQ MGB.The kit contains above-mentioned detection primer and probe and quality control system etc..The present invention can specific recognition LMNA NTRK1 fusion mutation, and high sensitivity can detect the mutation of 5 copies, entire reverse transcription PCR and fluorescent PCR detection process only need can be completed for 80 minutes.
Description
Technical field
The present invention relates to biotechnology and medical domains, specifically, being related to the mutation of mankind's LMNA-NTRK1 Gene Fusions
Detection.
Background technology
In recent years, more and more (prominent comprising point with oncotherapy associated gene mutation with the development of molecular diagnostic techniques
Change, insertion/deletion mutation, Gene Fusion etc.) it is found, it plays a significant role in tumour personalization diagnosis and treatment process.Nerve battalion
Support the factor tyrosine kinase receptor (NTRK) gene include NTRK1, NTRK2 and NTRK3, be each responsible for coding tropomyosin by
The synthesis of body kinases (TrK) family albumen TrKA, TrKB and TrKC, in maincenter and peripheral nervous system development and cells survival mistake
It plays a significant role in journey.Neurotrophic factor can induce receptor dimer, phosphorylation and activate after being combined with TrK protein
The signal cascade access of downstream PI3K, RAS/MAPK/ERK and PLC- γ.The change of TRK signal paths, including Gene Fusion, egg
White overexpression or single nucleotide alteration, have been found to be many diseases pathogenesis, especially NTRK1 Gene Fusions,
The constitutive activation or overexpression that can result in kinases, cause the occurrence and development of tumour.
Entrectinib is a kind of novel oral tyrosine kinase inhibitor for having central nervous system activity
(tyrosine kinase inhibitor, TKI), targeting contain NTRK1/2/3, and ROS1 or ALK gene fusion are mutated swollen
Tumor, FDA authorize it and break through sex therapy identification, positive for treating NTRK Gene Fusions, and part late period or metastatic solid tumors are suffered from
Person, these patients disease after receiving existing therapy is still in progress, or does not have standard treatment.Clinical research shows
Entrectinib to LMNA-NTRK1 Gene Fusion patients have good therapeutic effect, specially 11 exons of LMNA with
10 exons of NTRK1 merge.Therefore carry out LMNA-NTRK1 Gene Fusion abrupt climatic changes, help direction of medication usage and
Detection etc. is of great significance in real time for personalized treatment, curative effect of medication.
Currently, can the detection method of clinically detection fusion gene mutation be mainly real time fluorescent PCR method and directly
PCR sequencing PCR.However direct sequencing is complicated for operation, efficiency is low, usually wants 1-2 talent that can go out testing result.It would therefore be desirable to have appropriate
Detection method it is claimed below to meet:1) LMNA-NTRK1 fusion mutation types can be accurately detected;2) detection time is short;3)
Contain anti-pollution system and blank control in System Design, prevents the generation of false positive results, ensure that the accurate of testing result can
It leans on.
Invention content
In order to solve the problems, such as that there is presently no a kind of detection LMNA-NTRK1 Gene Fusion mutation methods, mesh of the invention
Be to provide the mankind's LMNA-NTRK1 Gene Fusions mutation detection primer, probe and detection kit.
In order to realize the object of the invention, technical scheme is as follows:
In a first aspect, the present invention provides the primer combination for detecting the mutation of mankind's LMNA-NTRK1 Gene Fusions, packet
Include SEQ ID NO:1 and SEQ ID NO:Sequence shown in 2.Primer combination can be prominent with specific recognition LMNA-NTRK1 fusions
Become.
Moreover, the present invention also provides the specific probe being used cooperatively, sequence such as SEQ are combined with the primer
ID NO:Shown in 3,5 ' it is terminal modified have a FAM or VIC, 3 ' terminal modified have NFQ-MGB.
Second aspect, the present invention provides the kits for detecting the mutation of mankind's LMNA-NTRK1 Gene Fusions, contain
There are primer combination above-mentioned and specific probe.
In order to improve the accuracy in detection of the kit, the kit also contains quality control system, positive control solution and
Blank control liquid.
The quality control system includes Quality Control primer and Quality Control probe, the sequence such as SEQ ID NO of Quality Control primer:4 and SEQ
ID NO:Shown in 5, the sequence such as SEQ ID NO of the Quality Control probe:Shown in 6, the Quality Control probe 5 ' it is terminal modified have FAM or
VIC, 3 ' terminal modified have NFQ-MGB.
The positive control solution is the mixed liquor containing 2 kinds of Plasmid DNA, and 2 kinds of Plasmid DNA contain the mankind respectively
LMNA-NTRK1 and B2M sequences, the blank control are Tris-HCl buffer solutions.
Further, the kit further includes the neccessary composition of other fluorescent PCR kits of this field, as PCR is buffered
Liquid, HotStart Taq enzymes and UNG enzymes.
The application method of the kit includes the following steps:
(1) RNA in extraction detection sample, detection sample includes fresh pathological tissue, paraffin-embedded tissue or pleural fluid;
(2) it is cDNA by the RNA reverse transcriptions of extraction, real-time fluorescent PCR amplification reaction is carried out by template of cDNA;
(3) the Ct values shown according to fluorescent PCR amplification instrument judge testing result:Detect the FAM and VIC/HEX of reaction system
Fluorescence intensity is reached required cycle-index Ct values when the threshold value of setting using FAM and is more than as yin and yang attribute criterion, Ct values
Equal to 38:It is negative;Ct values are less than 38:It is positive.
Wherein, the reaction system of pcr amplification reaction is as follows:
RNA reverse transcriptions are that the method for cDNA is related to reverse transcription system and following operating procedure:
Reverse transcription system includes following ingredient:
Reverse transcription mixed liquor (containing reverse transcriptase) 2 μ L
RNA 3μL
Add 10 μ L of DNase/RNase-free ddH2O.
Operating procedure is:
A) it by reverse transcription reaction mixed liquor mixing, is placed on ice, takes 2 μ L reverse transcription reaction mixed liquors that sterile no enzyme is added
In centrifuge tube, mixing.
B) 3 μ L, RNA total amounts of sample to be tested RNA are added in 0.1~5 μ g ranges, moisturizing to 10 μ L.
C) 15 minutes are kept the temperature for 37 DEG C.
D) 85 DEG C heat preservation 5 seconds after cooled on ice, obtain cDNA templates.
The present invention relates to raw material or reagent be ordinary commercial products, the operation being related to is unless otherwise specified
This field routine operation.
The beneficial effects of the present invention are:
The present invention merges mutation type using specific primer identification LMNA-NTRK1, is collected based on Taqman sonde methods glimmering
Optical signal, specificity are ensured very well.The kit of the present invention has the following advantages:(1) real-time fluorescence PCR body is established
System, can detect mankind's LMNA-NTRK1 fusion mutation simultaneously, and the mutation of (2) high sensitivity, 5 copies can detect;(3) detection speed
Degree is fast, and reverse transcription PCR only needs 15 minutes in the present invention, and fluorescent PCR only needs 60 minutes, entire reverse transcription PCR and fluorescent PCR detection
Process only needs can be completed for 80 minutes;(4) sample detection range is wide, and sample can be fresh pathological tissue, paraffin embedding group
It knits or pleural fluid;(5) the anti-pollution systems of UNG enzymes-dUTP and blank control system are introduced in system of the present invention, can preferably be prevented
The generation of false positive results, it is ensured that testing result is accurate and reliable.
Description of the drawings
Fig. 1 is the non-fused sudden change sample detects schematic diagram of the present invention.
Fig. 2 is present invention fusion sudden change sample detects schematic diagram.
Specific implementation mode
The preferred embodiment of the present invention is described in detail below in conjunction with embodiment.It will be appreciated that following real
Providing merely to play the purpose of explanation for example is applied, is not used to limit the scope of the present invention.The skill of this field
Art personnel can carry out various modifications and replace to the present invention without departing substantially from spirit of the invention and spirit.
Experimental method used in following embodiments is conventional method unless otherwise specified.
The materials, reagents and the like used in the following examples is commercially available unless otherwise specified.
Embodiment 1
The embodiment of the present invention is achieved in that a kind of kit for the detection of mankind's LMNA-NTRK1 Gene Fusions,
The kit includes PCR buffer solutions, 2 group-specific primers, 1 specific probe and quality control system, HotStart Taq enzymes,
UNG enzymes.
MgCl containing 1.0~5.0mM in the PCR buffer solutions2, the dNTPs of 1.0~5.0mM, i.e. dATP, dUTP,
Each 1.0~5.0mM of dGTP and dCTP.
The forward primer of the specific primer sequence is SEQ ID NO:1, reverse primer sequences are SEQ ID NO:2.
Each primer sequence is listed below:
LMNA-NTRK1-F:5’-TGGGCAACTCCAGCCC-3’(SEQ ID NO:1);
LMNA-NTRK1-R:5’-CACAGCCACCGAGACCT-3’(SEQ ID NO:2);
The specific probe 5 ' is terminal modified a FAM or VIC, and 3 ' terminal modified have non-fluorescence quenching group NFQ (Non-
Fluorescent Quencher), the group itself does not generate fluorescence, therefore can substantially reduce the intensity of background signal, together
When the specific probe on be also associated with MGB modification groups, the Tm values of the probe can be improved 10 DEG C or so, therefore same
Tm values, MGB probes can be more shorter than what general T aqman probes designed, and specificity is stronger.
Preferably, 1 specific probe sequence is SEQ ID NO:3, probe sequence is listed below:
LMNA-NTRK1-P:5’-CCCAGGTGAGTTGTCTGTCCC-3’(SEQ ID NO:3).
In the embodiment of the present invention, quality control system monitor sample quality is used.RNA be easy degradation, to sample preparation with preserve,
RNA is extracted and process of reverse-transcription is more demanding.The gene constructed quality control system stablized using expression quantity, can effective monitor sample
Quality avoids generating false negative result.Therefore above-mentioned hidden danger is eliminated using quality control system in the embodiment of the present invention, ensure that inspection
Survey the accuracy of result.The quality control system includes Quality Control primer and Quality Control probe, and this field skill can be used in the embodiment of the present invention
Other quality control systems known to art personnel.Preferably, the quality control system includes Quality Control primer and Quality Control probe.Quality Control primer sequence
It is classified as SEQ ID NO:4 and SEQ ID NO:5, the Quality Control probe sequence is SEQ ID NO:6, the end of Quality Control probe 5 ' is repaiied
It is decorated with FAM or VIC, 3 ' terminal modified have NFQ-MGB.
Preferably, the quality control system in the embodiment of the present invention is designed for people's B2M genes.Quality Control primer sequence wherein included
Row are as follows:
B2M-F:5’-ATGAGTATGCCTGCCGTGTG-3’(SEQ ID NO:4);
B2M-R:5’-GCTTACATGTCTCGATCCCACT-3’(SEQ ID NO:5).
The end of Quality Control probe 5 ' in the embodiment of the present invention is marked with reporter group, such as FAM, VIC, and 3 ' ends, which are marked with, not to be sent out
Light fluorescent quenching group, such as NFQ-MGB.When probe is complete, the fluorescent energy that reporter group is emitted is quenched base
Group absorbs, and instrument can't detect signal.With the progress of PCR, Taq enzyme encounters during DNA chain extends to be combined with template
Probe, 5 ' → 3 ' exonuclease activities will cut off probe, and reporter group generates fluorescence letter far from fluorescent quenching group
Number.Therefore the signal strength detected just represents the copy number of template DNA.
Preferably, the Quality Control probe is:
B2M-P:5’-CCATGTGACTTTGTCACAGC-3’(SEQ ID NO:6).
Preferably, contain Taq enzyme in the enzyme solutions in the embodiment of the present invention, necessary to being reacted for PCR and the enzyme is molten
Liquid also contains UNG enzymes, and wherein UNG (uracil-N-glycosylase) enzyme is uracil-N-glycosylase, its main feature is that most preferably
Active temperature is 50 DEG C, 95 DEG C of inactivations, and action principle is the urine in selective hydrolysis double-strand of the fracture containing dU or single stranded DNA
Pyrimidine glycosylase key, the DNA chain for having missing base of formation.In PCR reactions, non-specific PCR amplification can be prevented using UNG enzymes
And pollution.
Preferably, the detection kit in the embodiment of the present invention further includes positive control solution and blank control liquid, setting sun
Property control and blank control can monitor real-time quantitative PCR reaction be normally carried out, the positive control solution contains 2 kinds of Plasmid DNA
Mixed liquor, 2 kinds of Plasmid DNA contain mankind's LMNA-NTRK1 and B2M sequence respectively, which can be those skilled in the art
Well known plasmid, this 4 kinds of plasmid concentrations are identical, are 2000copies/ μ L;The blank control liquid is Tris-HCl (10mM)
Buffer solution.
The another object of the embodiment of the present invention is to provide a kind of detection method of mankind LMNA-NTRK1 Gene Fusions, be somebody's turn to do
Detection method includes the following steps:
(1) it designs and screens specific amplification and combined and probe groups with the primer of detection mankind's LMNA-NTRK1 Gene Fusions
It closes;Prepare mankind's LMNA-NTRK1 Gene Fusion detection kits of the present invention.
(2) sample to be tested total serum IgE is obtained;
(3) it is cDNA by RNA reverse transcriptions;
(4) fluorescent quantitative PCR is carried out by template of cDNA
Preferably, it by taking 25 μ l reaction systems as an example, is added into reaction tube each in the mixture obtained after detected sample
Component final concentration and content are as follows:
Above-mentioned system is only illustrative, can proportionally expand or shrink in practical applications the volume of mixture and its
Middle each component content.
Specifically, in above-mentioned 25 μ L reaction systems, the primer includes the specific primer and 1 in above-mentioned steps (1)
To Quality Control primer, the probe includes specific probe and Quality Control probe in above-mentioned steps (1), and the sample is cDNA.
Specifically, the quantitative fluorescent PCR reaction condition in step (4) is:
37 DEG C are handled 10 minutes, 95 DEG C of pre-degenerations 5 minutes,
40 cycles:95 DEG C 15 seconds, 55 DEG C 35 seconds and are collected fluorescence signal.
After above-mentioned PCR reactions, acquired results carry out result judgement according to the following steps:
According to weakly positive control and blank control, to kit availability deciding:
According to quality control system as a result, carrying out sample availability judgement, Quality Control PCR reaction solution:Value≤30 Ct, sample rna matter
Amount is preferable, and dosage is moderate.
Pattern detection result is judged, determines sample with the presence or absence of fusion.
Value >=38 pattern detection Ct or without Ct values, then the sample be non-fused mutation or be to melt outside this kit detection range
Close mutation;
Pattern detection Ct values < 38, then sample is LMNA-NTRK1 fusion mutation.
The detection method of the present invention has the advantages that high sensitivity, high specificity, real result are credible, while the detection side
Method is easy to operate quickly, detection can be completed in 80 minutes, and result interpretation is simply objective, convenient for analysis.
The present invention is expanded on further below by way of specific embodiment.
Embodiment 1
The mankind's LMNA-NTRK1 Gene Fusion detection kits for preparing the present invention, include the following steps:
1, primer and probe synthesis:
1 pair of specific primer is designed and synthesized, wherein forward primer is SEQ ID NO:1, reverse primer is SEQ ID
NO:2, specific probe is SEQ ID NO:3.Primer, probe are prepared into 100 μM of mother liquor storage respectively.
2, quality control system is prepared:
1 pair of Quality Control primer for people's B2M genes is designed and synthesized, which is SEQ ID NO:4 and SEQ
ID NO:5;Quality Control probe is designed and synthesized, which is SEQ ID NO:6.The Quality Control primer and probe is configured to respectively
100 μM of mother liquor storage.
3, other reagents are prepared:
PCR buffer solutions are prepared, wherein each 1.0mM of the MgCl2 containing 1.0mM, dATP, dUTP, dGTP and dCTP;Prepare enzyme
Mixed liquor, wherein containing 0.1 × 103U/ml of Taq enzyme 0.5 × 103U/ml, UNG enzyme.
4, positive control solution and blank control liquid are prepared, in the positive control solution contain 2 kinds of Plasmid DNA mixed liquors, described 2
Kind of Plasmid DNA contains mankind's LMNA-NTRK1 and B2M sequence respectively, the selection of the plasmid and to be designed as those skilled in the art ripe
Know, is 2000copies/ μ L;The blank control liquid is Tris-HCl (10mM) buffer solution.
5, PCR reaction solution is prepared:
Four different system PCR reaction solutions are carried out according to the following table to prepare
Table 1:PCR reaction solution is prepared
6, kit is assembled:
Include that it is each to calculate 20 person-portion specifications according to each ingredient usage amount of PCR reaction systems for 2 pipe PCR reaction solutions in kit
Ingredient usage amount ingredient and assembles in each pipe of reagent preparation box.
Embodiment 2
The mankind's LMNA-NTRK1 Gene Fusion detection kits prepared with embodiment 1 are treated side sample and are detected.
Kit forms:
The FFPE samples of 200 colorectal cancer patients are collected in the present embodiment, from RNA is wherein extracted, reverse transcription is at cDNA
Afterwards, the mankind LMNA- of measuring samples is detected with the mankind's LMNA-NTRK1 Gene Fusion detection kits obtained in embodiment 1
NTRK1 merges catastrophe.
1, FFPE sample rnas extract
Its RNA is extracted using the paraffin-embedded tissue RNA extracts kits of Qiagen.Above-mentioned concrete operation step presses reagent
Box specification operates.
2, RNA reverse transcriptions are at cDNA:
With UV spectrophotometer measuring RNA extraction quality and concentration, determine that its concentration OD260/OD280 is 1.8-2.0.
The template for taking the RNA of 0.1~5 μ g to be synthesized as its cDNA, using the reagent of Wuhan You Zhiyou medical science and technologies limited liability company
Box synthesizes cDNA, and cDNA synthetic systems are as follows:
Reverse transcription mixed liquor (containing reverse transcriptase) 2 μ L
RNA 3μL
Add 10 μ L of DNase/RNase-free ddH2O.
(a) it by reverse transcription reaction mixed liquor mixing, is placed on ice, takes 2 μ L reverse transcription reaction mixed liquors that sterile no enzyme is added
Centrifuge tube in, mixing.
(b) 3 μ L, RNA total amounts of sample to be tested RNA are added in 0.1~5 μ g ranges, moisturizing to 10 μ L.
(c) 15 minutes are kept the temperature for 37 DEG C.
(d) 85 DEG C heat preservation 5 seconds after cooled on ice, obtain cDNA templates.
3, the fluorescence quantitative PCR detection of sample
The cDNA obtained in 2 μ L steps 2 is taken, is added in four reaction systems, it is 25 μ L to make reaction system total volume, and
It is put into fluorescence quantitative PCR instrument, is carried out amplification reaction after PCR response procedures are set as described below:
95 DEG C 5 minutes;
95 DEG C of 15s, 60 DEG C of 35s, 40 cycles;The fluorescence signal of FAM and VIC/HEX is collected after each cycle.
4, the Analysis of test results of sample:
Detect FAM fluorescence signal Ct values, the Ct value judging results shown according to fluorescent PCR amplification instrument:Detect reaction system
FAM fluorescence intensities, required cycle-index Ct values are reached when the threshold value of setting using FAM as yin and yang attribute criterion, Ct
Value is more than or equal to 38:It is negative;Ct values are less than 38:It is positive.
Testing result, which shows to detect, has 2 to have mankind LMNA-NTRK1 fusions in 200 colorectal cancer samples
Mutation, mutation rate 1%, while tradition sequencing detection is carried out to above-mentioned all samples, the coincidence rate of two kinds of detection methods is
100%, the accuracy of system detection of the present invention is further demonstrated, it is as a result as follows:
The foregoing is merely illustrative of the preferred embodiments of the present invention, is not intended to limit the invention, all essences in the present invention
All any modification, equivalent and improvement etc., should all be included in the protection scope of the present invention made by within refreshing and principle.
Although above the present invention is described in detail with a general description of the specific embodiments,
On the basis of the present invention, it can be made some modifications or improvements, this will be apparent to those skilled in the art.Cause
This, these modifications or improvements, belong to the scope of protection of present invention without departing from theon the basis of the spirit of the present invention.
Claims (10)
1. the primer combination for detecting the mutation of mankind's LMNA-NTRK1 Gene Fusions, which is characterized in that the primer combination packet
Include SEQ ID NO:1 and SEQ ID NO:Sequence shown in 2.
2. combining the specific probe being used cooperatively with primer described in claim 1, which is characterized in that the specific probe sequence
Row such as SEQ ID NO:Shown in 3,5 ' it is terminal modified have a FAM or VIC, 3 ' terminal modified have NFQ-MGB.
3. the kit for detecting the mutation of mankind's LMNA-NTRK1 Gene Fusions, which is characterized in that the kit, which contains, has the right
Profit requires the primer described in 1 to combine.
4. kit according to claim 3, which is characterized in that kit is also containing the specificity described in claim 2
Probe.
5. kit according to claim 4, which is characterized in that kit also contains quality control system, the quality control system
Including Quality Control primer and Quality Control probe, the sequence such as SEQ ID NO of Quality Control primer:4 and SEQ ID NO:Shown in 5, the Quality Control
The sequence of probe such as SEQ ID NO:Shown in 6.
6. kit according to claim 5, which is characterized in that the Quality Control probe of kit 5 ' it is terminal modified have FAM or
VIC, 3 ' terminal modified have NFQ-MGB.
7. kit according to claim 5 or 6, which is characterized in that the kit also contains positive control solution and sky
White comparison liquid, the positive control solution are the Plasmid DNA containing LMNA-NTRK1, and the blank control buffers for Tris-HCl
Liquid.
8. according to the kit described in claim 3-6 any one, which is characterized in that the kit further includes PCR bufferings
Liquid, HotStart Taq enzymes and UNG enzymes.
9. a kind of application method of kit according to claim 8, method include the following steps:
(1) RNA in extraction detection sample, detection sample includes fresh pathological tissue, paraffin-embedded tissue or pleural fluid;
(2) it is cDNA by the RNA reverse transcriptions of extraction, real-time fluorescent PCR amplification reaction is carried out by template of cDNA;
(3) the Ct values shown according to fluorescent PCR amplification instrument judge testing result:FAM the and VIC fluorescence for detecting reaction system is strong
Degree, required cycle-index Ct values when the threshold value of setting are reached using FAM and are more than or equal to as yin and yang attribute criterion, Ct values
38:It is negative;Ct values are less than 38:It is positive.
10. kit according to claim 9, which is characterized in that kit further includes the reactant of pcr amplification reaction
System, ingredient are as follows:
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CN110714065A (en) * | 2019-11-14 | 2020-01-21 | 益善生物技术股份有限公司 | Kit and method for detecting NTRK gene fusion |
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Cited By (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN109988836A (en) * | 2019-03-18 | 2019-07-09 | 厦门艾德生物技术研究中心有限公司 | A kind of the FISH probe group and its application of detection NTRK fusion |
CN109988836B (en) * | 2019-03-18 | 2022-06-07 | 厦门艾德生物技术研究中心有限公司 | FISH probe set for detecting NTRK fusion and application thereof |
CN110714065A (en) * | 2019-11-14 | 2020-01-21 | 益善生物技术股份有限公司 | Kit and method for detecting NTRK gene fusion |
CN110714065B (en) * | 2019-11-14 | 2023-05-23 | 益善生物技术股份有限公司 | Kit and method for detecting NTRK gene fusion |
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