CN103468813B - EML4-ALK (Echinoderm microtubule associated protein like4-anaplastic lymphoma kinase) fusion gene fluorescent quantitative PCR (polymerase chain reaction) assay kit - Google Patents
EML4-ALK (Echinoderm microtubule associated protein like4-anaplastic lymphoma kinase) fusion gene fluorescent quantitative PCR (polymerase chain reaction) assay kit Download PDFInfo
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Abstract
The invention relates to an EML4-ALK (echinoderm microtubule associated protein like4-anaplastic lymphoma kinase) fusion gene fluorescent quantitative PCR (polymerase chain reaction) assay kit, which is applicable to assay of EML4-ALK fusion gene mutation in lung adenocarcinoma. The kit comprises probes, primers and positive controls, which are specially designed for conserved sequences of 9 fusion variations of the EML4-ALK fusion gene. The kit can be used for quickly and accurately assaying 9 most common EML4-ALK fusion gene variations with high sensitivity, namely 9 fusion variations of 7 variant subtypes, which are subtype 1 (E13; A20), subtype 2 (E20; A20), subtype 3 (E6a/b; A20), subtype 4 (E14; A20), subtype 5 (E2a/b; A20), subtype 6 (E18; A20) and subtype 7 (E14; A20), so that a real-time fluorescent quantitative PCR system for assaying 9 most common EML4-ALK fusion gene variations can be established.
Description
Technical field
The invention belongs to biology field, relate to a kind of test kit of EML4-ALK fusion gene fluorescence quantitative PCR detection, be applicable to the detection of EML4-ALK fusion gene sudden change in adenocarcinoma of lung.
Background technology
Lung cancer is the malignant tumour that M & M is the highest in the world.Over nearly more than 50 years, countries in the world are industrially developed country particularly, and sickness rate and the case fatality rate of lung cancer rise all rapidly.Nineteen ninety-five, the whole world had 600,000 people to die from lung cancer, and annual number is all in rising, and the mortality ratio that the World Health Organization (WHO) in 2003 announces is 1,100,000/year, and sickness rate is 1,200,000/year.The cause of disease of lung cancer is still not exclusively clear and definite so far, medical statistics shows that the paathogenic factor of lung cancer is mainly smoking (comprising second hand smoking), the male lung cancer of 80% and the female lung cancer of 75% are caused by smoking, in addition with the contact of the industrial cancerogen such as asbestos, radon, arsenic, ionizing rays, halogen alkene class, polycyclic aromatic compound, nickel, race, the morbidity of family members' history on lung cancer all have impact.
Lung cancer divides small cell lung cancer (SCLC) and nonsmall-cell lung cancer (NSCLC), and NSCLC comprises squama cancer, gland cancer, adenosquamous carcinoma, large cell carcinoma, carcinoid etc.NSCLC accounts for 80%-90% of all cases of lung cancer, serious threat human health.The treatment of NSCLC comprises the multiple methods such as operation, chemotherapy, radiotherapy, molecular targeted therapy and biological immune treatment.Operative treatment is NSCLC optimal treatment method, but when NSCLC finds, only the case of 20%-30% has surgical indication, and postoperative recurrence and the rate of transform are still up to more than 50%.Molecular targeted therapy meets physiology, low toxicity and efficient feature in theory with it, more and more becomes the focus of Treatment for Non-small Cell Lung.Targeted therapy is that the treatment of lung cancer adds a new field, also for the treatment of lung cancer brings new hope.
Molecular targeted therapy is the treatment means of a kind of cell signaling for tumorigenesis process and other biological approach, be characterized in optionally to act on tumour cell, the propagation of directed blocking-up cancer cells, transfer, intracellular signaling, stop tumor neovasculature generation, tumoricidal metabolism, therefore specificity is high, untoward reaction is little, has good development prospect.Targeted therapy of lung cancer relates to multiple important steps of growth of tumour cell, current research is comparatively ripe and be applied to and clinical mainly contain two point of application, one is vascular endothelial growth factor receptor (VEGFR), and another is EGF-R ELISA (EGFR).In addition, also have between the echinoderms microtubule bindin 4-of latest find and become lymphom kinase (echinoderm microtubule associated protein like4, EML4-ALK) fusion gene.Existing this fusion gene of research prompting may be present in lung cancer at interior multiple noumenal tumour, but it is the highest with the recall rate of NSCLC, simultaneously because of itself and EGFR suddenly change, K-ras suddenlys change do not coexist phenomenon and the distinct Clinical symptoms containing EML4-ALK gene patient, this target spot is pointed out to be the molecular marked compound that adenocarcinoma of lung specificity is higher.The discovery of EML4-ALK defines a new subtype of NSCLC, can be rated as a landmark event on NSCLC research history in recent years.The gene of coding EML4 is positioned at No. 21 locus of No. 2 the short arm of a chromosome, and the gene of the ALK that encodes is positioned at No. 2 karyomit(e)s No. 23 locus, and both are separated by about 12,700,000 base pairs on chromosome, and transcriptional orientation is contrary.During gene fusion, EML4 gene ruptures on chromosome, form the exon splicing fragment of different lengths, turn direction, between ALK gene 19,20 exon that slotting people position is guarded relatively, thus the various EML4-ALK that formation length does not wait merge variants, at present, at least have been found that 9 kinds of variation hypotypes, be respectively hypotype 1(E13; A20) (20 exons of 13 exons and ALK that this nomenclature refers to EML4 merge), hypotype 2(E20; A20), hypotype 3(E6a/b; A20), hypotype 4(E14; A20), hypotype 5(E2a/b; A20), hypotype 6(E13; A20) (note: hypotype 6 is that the 13rd exon of EML4 adds that the small segment of a 49bp is connected with the 20th exon of ALK again), hypotype 7(E14; A20), hypotype 8(E15; A20), hypotype 9(E18; A20).Modal fusion variant is hypotype 1(E13; A20), be secondly hypotype 3(E6a/b; A20), after testing, these two kinds of variants are respectively 33% and 29% in the incidence of EML4-ALK fusion gene NSCLC patient.All these fusion genes all have biological function, and expression product is a kind of mosaic type Tyrosylprotein kinase.EML4-ALK merges variation and is accredited as NSCLC a few days ago, particularly one of adenocarcinoma of lung unique molecular isoform, and EML4-ALK fusion gene positive patient has the clinical pathologic characteristic of its uniqueness.For the detection of this target spot, screen the suitable treatment crowd of EGFR-TKI by contributing to and the frontier of opening NSCLC targeted therapy.
The technological method detecting EML4-ALK fusion gene common in the world at present has: RT-polymerase chain reaction (RT-PCR), fluorescence in situ hybridization (FISH), immunohistochemistry (IHC), RACE-coupling PCR check order (RACE-coupled PCR sequencing).
RT-PCR is the distortion of a kind of widespread use of PCR, and in RT-PCR, a RNA chain is reversed record becomes complementary DNA, then carries out DNA cloning as template by PCR.RT-PCR confirms that NSCLC exists a kind of fast diagnosis method of ALK fusion gene, and in theory, the advantage of this technology is that it detects the hypersensitivity of sudden change transcript, and as found, amplified production then means ALK fusion gene.But in clinical practice, this technology faces lot of challenges: (1) primer diversification, research needs to design multiple primer and detects 9 varients and 2 non-EMIA transpositions; (2) paraffin-embedded tissue DNA height fragmentation fixed by formalin, is unfavorable for detecting; (3) often there is false positive in PCR result, is difficult to as routine clinical detection method.
FISH is hybridized with specific fluorescent label probe and target DNA, connects upper fluorescein-labelled thing by immunocytochemical procedures, in the technology in fluorescence microscopy Microscopic observation probe mark or site.FISH detects the more special method of ALK fusion gene, and it is advantageous that can business development plurality of probes, is applied to adenocarcinoma of lung and detects ALK fusion gene.Although FISH detects a kind of sensitivity of adenocarcinoma of lung ALK fusion gene and special method, also not safe against all possibilities, and can not differentiate different EML4-ALK fusion gene variants.The therapeutic strategy of different quantities break signal and result interpretation are at present still without unified standard.
IHC refer to band developer mark specific antibody in histocyte original position by antigen antibody reaction and histochemical color reaction, to corresponding antigens carry out qualitative, location, quantitative assay a new technology.The sharpest edges of IHC are the expression of detection tumour specific antigen and do not lose the biological cells and tissues constitutional features differentiating healthy tissues and pathological tissue.Detect ALK egg from expressing for the dyeing of biopsy section using-system, the possibility of result is faint and local, and IHC method always exists certain subjectivity simultaneously, needs to verify by technology such as FISH further to the judgement of weak positive findings.In addition IHC can not identify that ALK merges patient and specifically belongs to that variant hypotype equally.
The imperial teach problem group of lung cancer institute of Guangdong Province Wu one adopts RACE-coupling PCR sequencing technologies to detect the fusion variation analyzing ALK gene, the distinctive feature of the method is to adopt cDNA end rapid amplifying (RACE) combine with technique two-wheeled round pcr to carry out the fusion variant of Enrichment Amplification ALK gene, susceptibility is high, not only can detect the fusion of EML4 and ALK, and the fusion of other any genes and ALK can be detected, also can clearly merge further by the means of order-checking is specifically any from EML4-ALK many kinds of variants.But this method relates to two-wheeled PCR and order-checking, complex steps and consuming time, cost intensive, is difficult to penetration and promotion.
The most emerging real-time fluorescence quantitative PCR combines nucleic acid amplification, hybridization, spectroscopic analysis and real-time detection technique, PCR primer is detected by means of fluorescent signal, solve traditional PCR technique can not quantitatively and amplified production pollute problem, avoid the pollution in common quantitative PCR operating process, make it easy and simple to handle, quick, result is accurate, has become the strong instrument of quantitative gene expression, has the features such as high, the reproducible and quantification range of Sensitivity and Specificity is wide.Detection by quantitative requires that responsive, special, accurate, accurate, reproducible, the linearity range of detection method is extensively, and between each genotype indifference.Full automatic quantitative fluorescent PCR, because of its more excellent accuracy, tolerance range and repeatability, becomes the most promising quantitative measurement technology.
Real-Time Fluorescent Quantitative PCR Technique (rea1-time fluorescent quantitative PCR, FQ-PCR) released in 1996 by Applied Biosystems company of the U.S., it a kind ofly adding pair of primers while, adds a specific fluorescent probe when pcr amplification, this probe is an oligonucleotide, and two ends mark a reporter fluorescence group and a quenching fluorescence group respectively.When probe is complete, the fluorescent signal that reporter group is launched is quenched group absorptions; When just starting, probe is combined on the arbitrary strand of DNA; During pcr amplification, 5 ' end-3 of Taq enzyme ' hold 5 prime excision enzyme activity that probe enzyme is cut degraded, reporter fluorescence group is separated with quenching fluorescence group, thus fluorescence monitoring system can receive fluorescent signal, namely often increase a DNA chain, just have a fluorescence molecule to be formed, the accumulation and the PCR primer that achieve fluorescent signal form Complete Synchronization.It is quantitative that this technology not only achieves DNA profiling, and there is highly sensitive, specificity and the feature such as reliability is stronger, can realize multiple reaction, level of automation is high, nonstaining property, tool real-time and accuracy, be widely used in the field such as molecular biology research and medical research at present.
Summary of the invention
The object of the present invention is to provide a kind of fluorescent quantificationally PCR detecting kit of EML4-ALK fusion gene variant, this test kit can detect 9 kinds of modal EML4-ALK fusion gene variants fast, accurately, high sensitive, comprises hypotype 1(E13; A20), hypotype 2(E20; A20), hypotype 3(E6a/b; A20), hypotype 4(E14; A20), hypotype 5(E2a/b; A20), hypotype 6(E18; A20), hypotype 7(E14; A20) 9 kinds of 7 kinds of variation hypotypes merge variant, thus set up the real-time fluorescence quantitative PCR system of the most common EML4-ALK fusion gene variant of detection 9 kinds.
The present invention adopts following technical measures: a kind of fluorescent quantificationally PCR detecting kit that is quick, accurately detection EML4-ALK fusion gene.This test kit comprises fluorescent quantitation reaction solution premixed liquid (PCR Master Mix), fluorescent quantitation reaction solution A, fluorescent quantitation reaction solution B, fluorescent quantitation reaction liquid C, fluorescent quantitation reaction solution D, fluorescent quantitation reaction solution E, fluorescent quantitation reaction solution F, fluorescent quantitation reaction solution G, fluorescent quantitation reaction solution H, fluorescent quantitation reaction solution I, positive control sample A, positive control sample B, positive control sample C, positive control sample D, positive control sample E, positive control sample F, positive control sample G, positive control sample H, positive control sample I.
Fluorescent quantitation reaction solution A: detection be EML4-ALK hypotype 1(E13; A20) fusion gene.
Reaction system comprises: detect EML4-ALK hypotype 1(E13; A20) primer of fusion gene and probe.
Variant 1-forward primer: 5 '-GAGTCATGCTTATATGGAGCAAAACT-3 ' (SEQ ID NO.1).
Variant 1-reverse primer: 5 '-TCAGCTTGTACTCAGGGCTCTG-3 ' (SEQ ID NO.2).
Variant 1-fluorescent probe: 5 '-FAM-CCTAAAGTGTACCGCCGGAAGCACC-TAMRA-3 ' (SEQ ID NO.3).
5 ' end flag F AM fluorescent reporter group of fluorescent probe, 3 ' end mark TAMRA fluorescent quenching group.
Positive control sample A: comprise detected EML4-ALK hypotype 1(E13; A20) fusion gene genomic fragment DNA plasmid.
Fluorescent quantitation reaction solution B: detection be EML4-ALK hypotype 2(E20; A20) fusion gene.
Reaction system comprises: detect EML4-ALK hypotype 2(E20; A20) primer of fusion gene and probe.
Variant 2-forward primer: 5 '-AAGTATATAATGTCTAACTCGGGAGACTATGA-3 ' (SEQ ID NO.4).
Variant 2-reverse primer: 5 '-AGCAGTAGTTGGGGTTGTAGTCG-3 ' (SEQ ID NO.5).
Variant 2-fluorescent probe: 5 '-FAM-TTGTACTTGTACCGCCGGAAGCACCA-TAMRA-3 ' (SEQ ID NO.6).
5 ' end flag F AM fluorescent reporter group of fluorescent probe, 3 ' end mark TAMRA fluorescent quenching group.
Positive control sample B: comprise detected EML4-ALK hypotype 2(E20; A20) fusion gene genomic fragment DNA plasmid.
Fluorescent quantitation reaction liquid C: detection be EML4-ALK hypotype 3a(E6a; A20) fusion gene.
Reaction system comprises: detect EML4-ALK hypotype 3a(E6a; A20) primer of fusion gene and probe.
Variant 3a-forward primer: 5 '-ACCAAAACTGCAGACAAGCATAAAG-3 ' (SEQ ID NO.7).
Variant 3a-reverse primer: 5 '-AGCTTGCTCAGCTTGTACTCAGG-3 ' (SEQ ID NO.8).
Variant 3a-fluorescent probe: 5 '-FAM-TGTCATCATCAACCAAGTGTACCGCCG-TAMRA-3 ' (SEQ ID NO.9).
5 ' end flag F AM fluorescent reporter group of fluorescent probe, 3 ' end mark TAMRA fluorescent quenching group.
Positive control sample C: comprise detected EML4-ALK hypotype 3a(E6a; A20) fusion gene genomic fragment DNA plasmid.
Fluorescent quantitation reaction solution D: detection be EML4-ALK hypotype 3b(E6b; A20) fusion gene
Reaction system comprises: detect EML4-ALK hypotype 3b(E6b; A20) primer of fusion gene and probe.
Variant 3b-forward primer: 5 '-AACACCCAAATTAATACCAAAAGTTACC-3 ' (SEQ ID NO.10).
Variant 3b-reverse primer: 5 '-CAGCTCCTGGTGCTTCCG-3 ' (SEQ ID NO.11).
Variant 3b-fluorescent probe: 5 '-FAM-AATGTCAACTCGCGAAA-TAMRA-3 ' (SEQ ID NO.12).
5 ' end flag F AM fluorescent reporter group of fluorescent probe, 3 ' end mark TAMRA fluorescent quenching group.
Positive control sample D: comprise detected EML4-ALK hypotype 3b(E6b; A20) fusion gene genomic fragment DNA plasmid.
Fluorescent quantitation reaction solution E: detection be EML4-ALK hypotype 4(E14; A20) fusion gene.
Reaction system comprises: detect EML4-ALK hypotype 4(E14; A20) primer of fusion gene and probe.
Variant 4-forward primer: 5 '-AGTAGTTGGGGTTGTAGTCGGTCA-3 ' (SEQ ID NO.13).
Variant 4-reverse primer: 5 '-TTGTCAGATGAGAAATGGGATGTTA-3 ' (SEQ ID NO.14).
Variant 4-fluorescent probe: 5 '-FAM-AGCTTGTACTCAGGGCTCATCCAGCATATCTCTA-TAMRA-3 ' (SEQ ID NO.15).
5 ' end flag F AM fluorescent reporter group of fluorescent probe, 3 ' end mark TAMRA fluorescent quenching group.
Positive control sample E: comprise detected EML4-ALK hypotype 4(E14; A20) fusion gene genomic fragment DNA plasmid.
Fluorescent quantitation reaction solution F: detection be EML4-ALK hypotype 5a(E2a; A20) fusion gene.
Reaction system comprises: detect EML4-ALK hypotype 5a(E2a; A20) primer of fusion gene and probe.
Variant 5a-forward primer: 5 '-GTTTTGAGGCGTCTTGCAATCT-3 ' (SEQ ID NO.16).
Variant 5a-reverse primer: 5 '-GGTTGTAGTCGGTCATGATGGTC-3 ' (SEQ ID NO.17).
Variant 5a-fluorescent probe: 5 '-FAM-CTCAAGTAAAGTGTACCGCCGGAAGCA-TAMRA-3 ' (SEQ ID NO.18).
5 ' end flag F AM fluorescent reporter group of fluorescent probe, 3 ' end mark TAMRA fluorescent quenching group.
Positive control sample F: comprise detected EML4-ALK hypotype 5a(E2a; A20) fusion gene genomic fragment DNA plasmid.
Fluorescent quantitation reaction solution G: detection be EML4-ALK hypotype 5b(E2b; A20) fusion gene.
Reaction system comprises: detect EML4-ALK hypotype 5b(E2b; A20) primer of fusion gene and probe.
Variant 5b-forward primer: 5 '-GTGCTTCCGGCGGTACACT-3 ' (SEQ ID NO.19).
Variant 5b-reverse primer: 5 '-TTGAGGCGTCTTGCAATCTCT-3 ' (SEQ ID NO.20).
Variant 5b-fluorescent probe: 5 '-FAM-CCTCCCCTGAGCTCTGAACCTTTACTTGA-TAMRA-3 ' (SEQ ID NO.21).
5 ' end flag F AM fluorescent reporter group of fluorescent probe, 3 ' end mark TAMRA fluorescent quenching group.
Positive control sample G: comprise detected EML4-ALK hypotype hypotype 5b(E2b; A20) fusion gene genomic fragment DNA plasmid.
Fluorescent quantitation reaction solution H: detection be EML4-ALK hypotype 6(E18; A20) fusion gene
Reaction system comprises: detect EML4-ALK hypotype 6(E18; A20) primer of fusion gene and probe.
Variant 6-forward primer: 5 '-GGGTGGTCAGCTGCAACAT-3 ' (SEQ ID NO.22).
Variant 6-reverse primer: 5 '-GAGACTCAGGTGGAGTCATGCTT-3 ' (SEQ ID NO.23).
Variant 6-fluorescent probe: 5 '-FAM-CCACTTCCTTTAGGTCCTTTCCCAGGTGT-TAMRA-3 ' (SEQ ID NO.24).5 ' end flag F AM fluorescent reporter group of fluorescent probe, 3 ' end mark TAMRA fluorescent quenching group.
Positive control sample H: comprise detected EML4-ALK hypotype 6(E18; A20) fusion gene genomic fragment DNA plasmid.
Fluorescent quantitation reaction solution I: detection be EML4-ALK hypotype 7(E14; A20) fusion gene.
Reaction system comprises: detect EML4-ALK hypotype 7(E14; A20) primer of fusion gene and probe.
Variant 7-forward primer: 5 '-CAGTAGTTGGGGTTGTAGTCGGTC-3 ' (SEQ ID NO.25).
Variant 7-reverse primer: 5 '-TTGTCAGATGAGAAATGGGATGTTAT-3 ' (SEQ ID NO.26).
Variant 7-fluorescent probe: 5 '-FAM-TGGCTTGCAGCTCCTGGTGCTCTATT-TAMRA-3 ' (SEQ ID NO.27).
5 ' end flag F AM fluorescent reporter group of fluorescent probe, 3 ' end mark TAMRA fluorescent quenching group.
Positive control sample I: comprise detected EML4-ALK hypotype 7(E14; A20) fusion gene genomic fragment DNA plasmid.
The principle of EML4-ALK fusion gene fluorescent quantificationally PCR detecting kit of the present invention is: adopt fluorescence quantifying PCR method to carry out detection by quantitative to EML4-ALK fusion gene.The special design TaqMan probe of conserved sequence of variant and primer a pair is merged respectively for EML4-ALK fusion gene 9 kinds, when running into EML4-ALK fusion gene corresponding fusion variant specific gene sequence, TaqMan probe can combine with it completely, during pcr amplification, fluorescent reporter group sends fluorescence because enzymolysis is separated, the accumulation of corresponding fluorescent signal can be detected in real time, thus realize the object detecting EML4-ALK fusion gene content.
Although 9 kinds of complete genome sequences merging variant provided by the invention are the published sequences of GENBANK, but the determination of conserved sequence needs screening comparison and experimental verification, after selected good conserved sequence, design primer and probe sequence is carried out again by Primer3 software, and blast passes through a large amount of experimental verifications after verifying its specificity again on NCBI, and optimize reaction correlated condition.
Two ends provided by the invention all indicate the specificity fluorescent probe of fluorescence radiation group, when probe is complete, two groups distance on space structure is close to each other, the fluorescence that 5 ' end reporter group sends is held quenching group cancellation because of FRET (fluorescence resonance energy transfer) (FRET) by 3 ', so do not have the change of fluorescent signal in system.And once the combination of it and template specificity, its binding site is between two primers, along with the extension of primer, Taq archaeal dna polymerase runs into the probe combined with template in chain extension process, probe will cut off by its 5 '-3 ' 5 prime excision enzyme activity, fluorescent reporter group, away from fluorescent quenching group, destroys the FRET between two fluorophors, and the fluorescence that reporter group discharges the just photofluorometer that can be built in instrument detects.PCR is often through a circulation, and fluorescent signal is also the same with object fragment, has the process that sync index increases, the power of fluorescent signal just represent the copy number of template DNA number.Therefore the present invention not only can be used for simple qualitative detection, also can be used as the detection by quantitative of the concrete content of sample.
With existing Comparison between detecting methods, this technology adopts fluorescent quantitative PCR technique detection EML4-ALK fusion gene to have following advantage:
1. detection sensitivity is high, specificity is good: the present invention devises Auele Specific Primer and fluorescent probe for EML4-ALK fusion gene 9 kinds of varients respectively, correlated response liquid is in charge of and is detected, the interference between each primer can be got rid of, than will the situation of mixed reaction solution be adopted more greatly to improve detection specificity and sensitivity, and false positive be low
2. linear relationship is good, can detection by quantitative, due to the power of fluorescent signal and the logarithm of template amplification product linear, carried out quantitatively sample initial template concentration by the detection of fluorescent signal, error is little;
3. simple to operate, level of automation is high, FQ-PCR technology to the amplification of PCR primer and detect stopped pipe situation next step complete, do not need to uncap, crossed contamination and contaminate environment chance few, therefore also just reduce the probability of result error;
4. result interpretation is clear and definite, directly perceived, can carry out quantitative analysis to result.The interpretation of fluorescence quantitative PCR method result: have the sample of amplification curve to be positive sample more than thresholding line, result interpretation is very simple, directly perceived;
5. the sample size detected is large, once can detect at most 384 examples;
6. safety: do not comprise hazardous and noxious substances in whole system, to operator and environment all without harm;
7. there is no aftertreatment, need not hybridize, electrophoresis, to take pictures.
8. the specific detection agents box disposable examination EML4-ALK fusion gene of energy provided by the invention 9 kinds of varients, fast and convenient.
Accompanying drawing explanation
Fig. 1 is EML4-ALK hypotype 1(E13; A20) the FQ-PCR figure of fusion gene.
Fig. 2 is EML4-ALK hypotype 1(E13; A20) the sequencing result figure of fusion gene.
Fig. 3 is EML4-ALK hypotype 2(E20; A20) the FQ-PCR figure of fusion gene.
Fig. 4 is EML4-ALK hypotype 2(E20; A20) the sequencing result figure of fusion gene.
Fig. 5 is EML4-ALK hypotype 3a(E6a; A20) the FQ-PCR figure of fusion gene.
Fig. 6 is EML4-ALK hypotype 3a(E6a; A20) the sequencing result figure of fusion gene.
Fig. 7 is EML4-ALK hypotype 3b(E6b; A20) the FQ-PCR figure of fusion gene.
Fig. 8 is EML4-ALK hypotype 3b(E6b; A20) the sequencing result figure of fusion gene.
Fig. 9 is EML4-ALK hypotype 4(E14; A20) the FQ-PCR figure of fusion gene.
Figure 10 is EML4-ALK hypotype 4(E14; A20) the sequencing result figure of fusion gene.
Figure 11 is EML4-ALK hypotype 5a(E2a; A20) the FQ-PCR figure of fusion gene.
Figure 12 is EML4-ALK hypotype 5a(E2a; A20) the sequencing result figure of fusion gene.
Figure 13 is EML4-ALK hypotype 5b(E2b; A20) the FQ-PCR figure of fusion gene.
Figure 14 is EML4-ALK hypotype 5b(E2b; A20) the sequencing result figure of fusion gene.
Figure 15 is EML4-ALK hypotype 6(E18; A20) the FQ-PCR figure of fusion gene.
Figure 16 is EML4-ALK hypotype 6(E18; A20) the sequencing result figure of fusion gene.
Figure 17 is EML4-ALK hypotype 7(E14; A20) the FQ-PCR figure of fusion gene.
Figure 18 is EML4-ALK hypotype 7(E14; A20) the sequencing result figure of fusion gene.
Figure 19 is the FQ-PCR figure of EML4-ALK fusion gene specific detection.
Embodiment
Sample illustrates: in embodiment, positive sample used collects the wax stone tissue from the EML4-ALK gene fusion of Pathology Deparment of Tumor Hospital Attached to Zhongshan Univ. Clinicopathologic Diagnosis below.Genomic dna is extracted for following experimental applications from wax stone.
Embodiment 1:EML4-ALK hypotype 1(E13; A20) the detection design energy specific detection EML4-ALK hypotype 1(E13 of fusion gene; A20) probe of fusion gene and primer a pair:
Variant 1-forward primer: 5 '-GAGTCATGCTTATATGGAGCAAAACT-3 ' (SEQ ID NO.1);
Variant 1-reverse primer: 5 '-TCAGCTTGTACTCAGGGCTCTG-3 ' (SEQ ID NO.2);
Variant 1-Taqman-probe: 5 '-FAM-CCTAAAGTGTACCGCCGGAAGCACC-TAMRA-3 ' (SEQ ID NO.3);
Then optimizing reaction system carries out the detection of FQ-PCR: reaction system is 15 μ l, the each 0.15 μ l(20 μ Μ of positive and negative primer), the each 0.2 μ l(20 μ Μ of probe), sample template DNA2.5 μ l(100-300ng/ μ l), 2*Taqman universal PCR.
Master Mix(purchased from American applying biological company) 7.5 μ l, ddH
2o4.3 μ l.
PCR reaction conditions: 95 DEG C of denaturation 30sec; And by 95 DEG C 5 seconds, 58 DEG C 33 seconds, amplified reaction 65 circulation.Simultaneously in fluorescent quantitation test, while needing to detect sample, also needing to arrange samples Reference product, to determine the validity tested, as shown in Figure 1, is EML4-ALK hypotype 1(E13; A20) the fluorescent quantitative PCR curve of fusion gene positive sample; In addition, the sample in above-described embodiment 1 is carried out sequence verification, find to detect EML4-ALK hypotype 1(E13 by above-mentioned fluorescent quantitation detection experiment in the present embodiment; A20) fusion gene, as shown in Figure 2, can be found out by sequencing result and really in this site, corresponding gene fusion occur, and the result of itself and fluorescent quantitation detection experiment is completely the same.Find to adopt EML4-ALK fusion gene fluorescent quantificationally PCR detecting kit of the present invention can determine EML4-ALK hypotype 1(E13 exactly with this; A20) fusion gene catastrophe.
Embodiment 2:EML4-ALK hypotype 2(E20; A20) detection of fusion gene
Design energy specific detection EML4-ALK hypotype 2(E20; A20) probe of fusion gene and primer a pair:
Variant 2-forward primer: 5 '-AAGTATATAATGTCTAACTCGGGAGACTATGA-3 ' (SEQ ID NO.4);
Variant 2-reverse primer: 5 '-AGCAGTAGTTGGGGTTGTAGTCG-3 ' (SEQ ID NO.5);
Variant 2-Taqman-probe: 5 '-FAM-TTGTACTTGTACCGCCGGAAGCACCA-TAMRA-3 ' (SEQ ID NO.6);
Then optimizing reaction system carries out the detection of FQ-PCR: reaction system is 15 μ l, each 0.15 μ l(20 μ Μ of positive and negative primer), each 0.2 μ l(20 μ Μ of probe), sample template DNA2.5 μ l(100-300ng/ μ l) and, 2*Taqman universalPCR
Master Mix(purchased from American applying biological company) 7.5 μ l, ddH
2o4.3 μ l.
PCR reaction conditions: 95 DEG C of denaturation 30sec; And by 95 DEG C 5 seconds, 58 DEG C 33 seconds, amplified reaction 65 circulation.Simultaneously in fluorescent quantitation test, while needing to detect sample, also needing to arrange samples Reference product, to determine the validity tested, as shown in Figure 3, is EML4-ALK hypotype 2(E20; A20) the fluorescent quantitative PCR curve of fusion gene positive sample; In addition, the sample in above-described embodiment 2 is carried out sequence verification, find to detect EML4-ALK hypotype 2(E20 by above-mentioned fluorescent quantitation detection experiment in the present embodiment; A20) fusion gene, as shown in Figure 4, can be found out by sequencing result and really in this site, corresponding gene fusion occur, and the result of itself and fluorescent quantitation detection experiment is completely the same.Find to adopt EML4-ALK fusion gene fluorescent quantificationally PCR detecting kit of the present invention can determine EML4-ALK hypotype 2(E20 exactly with this; A20) fusion gene catastrophe.
Embodiment 3:EML4-ALK hypotype 3a(E6a; A20) detection of fusion gene
Design energy specific detection EML4-ALK hypotype 3a(E6a; A20) probe of fusion gene and primer a pair:
Variant 3a-forward primer: 5 '-ACCAAAACTGCAGACAAGCATAAAG-3 ' (SEQ ID NO.7);
Variant 3a-reverse primer: 5 '-AGCTTGCTCAGCTTGTACTCAGG-3 ' (SEQ ID NO.8);
Variant 3a-Taqman-probe: 5 '-FAM-TGTCATCATCAACCAAGTGTACCGCCG-TAMRA-3 ' (SEQ ID NO.9);
Then optimizing reaction system carries out the detection of FQ-PCR: reaction system is 15 μ l, the each 0.15 μ l(20 μ Μ of positive and negative primer), the each 0.2 μ l(20 μ Μ of probe), sample template DNA2.5 μ l(100-300ng/ μ l), 2*Taqman universal PCR
Master Mix(purchased from American applying biological company) 7.5 μ l, ddH
2o4.3 μ l.
PCR reaction conditions: 95 DEG C of denaturation 30sec; And by 95 DEG C 5 seconds, 58 DEG C 33 seconds, amplified reaction 65 circulation.Simultaneously in fluorescent quantitation test, while needing to detect sample, also needing to arrange samples Reference product, to determine the validity tested, as shown in Figure 5, is EML4-ALK hypotype 3a(E6a; A20) the fluorescent quantitative PCR curve of fusion gene positive sample; In addition, the sample in above-described embodiment 3 is carried out sequence verification, find to detect EML4-ALK hypotype 3a(E6a by above-mentioned fluorescent quantitation detection experiment in the present embodiment; A20) fusion gene, as shown in Figure 6, can be found out by sequencing result and really in this site, corresponding gene fusion occur, and the result of itself and fluorescent quantitation detection experiment is completely the same.Find to adopt EML4-ALK fusion gene fluorescent quantificationally PCR detecting kit of the present invention can determine EML4-ALK hypotype 3a(E6a exactly with this; A20) fusion gene catastrophe.
Embodiment 4:EML4-ALK hypotype 3b(E6b; A20) detection of fusion gene
Design energy specific detection EML4-ALK hypotype 3b(E6b; A20) probe of fusion gene and primer a pair:
Variant 3b-forward primer: 5 '-AACACCCAAATTAATACCAAAAGTTACC-3 ' (SEQ ID NO.10);
Variant 3b-reverse primer: 5 '-CAGCTCCTGGTGCTTCCG-3 ' (SEQ ID NO.11);
Variant 3b-Taqman-probe: 5 '-FAM-AATGTCAACTCGCGAAA-TAMRA-3 ' (SEQ ID NO.12);
Then optimizing reaction system carries out the detection of FQ-PCR: reaction system is 15 μ l, each 0.15 μ l(20 μ Μ of positive and negative primer), each 0.2 μ l(20 μ Μ of probe), sample template DNA2.5 μ l(100-300ng/ μ l) and, 2*Taqman universalPCR
Master Mix(purchased from American applying biological company) 7.5 μ l, ddH
2o4.3 μ l.
PCR reaction conditions: 95 DEG C of denaturation 30sec; And by 95 DEG C 5 seconds, 58 DEG C 33 seconds, amplified reaction 65 circulation.Simultaneously in fluorescent quantitation test, while needing to detect sample, also needing to arrange samples Reference product, to determine the validity tested, as shown in Figure 7, is EML4-ALK hypotype 3b(E6b; A20) the fluorescent quantitative PCR curve of fusion gene positive sample; In addition, the sample in above-described embodiment 4 is carried out sequence verification, find to detect EML4-ALK hypotype 3b(E6b by above-mentioned fluorescent quantitation detection experiment in the present embodiment; A20) fusion gene, as shown in Figure 8, can be found out by sequencing result and really in this site, corresponding gene fusion occur, and the result of itself and fluorescent quantitation detection experiment is completely the same.Find to adopt EML4-ALK fusion gene fluorescent quantificationally PCR detecting kit of the present invention can determine EML4-ALK hypotype 3b(E6b exactly with this; A20) fusion gene catastrophe.
Embodiment 5:EML4-ALK hypotype 4(E14; A20) detection of fusion gene
Design energy specific detection EML4-ALK hypotype 4(E14; A20) probe of fusion gene and primer a pair:
Variant 4-forward primer: 5 '-AGTAGTTGGGGTTGTAGTCGGTCA-3 ' (SEQ ID NO.13);
Variant 4-reverse primer: 5 '-TTGTCAGATGAGAAATGGGATGTTA-3 ' (SEQ ID NO.14);
Variant 4-Taqman-probe: 5 '-FAM-AGCTTGTACTCAGGGCTCATCCAGCATATCTCTA-TAMRA-3 ' (SEQ ID NO.15);
Then optimizing reaction system carries out the detection of FQ-PCR: reaction system is 15 μ l, each 0.15 μ l(20 μ Μ of positive and negative primer), each 0.2 μ l(20 μ Μ of probe), sample template DNA2.5 μ l(100-300ng/ μ l) and, 2*Taqman universalPCR
Master Mix(purchased from American applying biological company) 7.5 μ l, ddH
2o4.3 μ l.
PCR reaction conditions: 95 DEG C of denaturation 30sec; And by 95 DEG C 5 seconds, 58 DEG C 33 seconds, amplified reaction 65 circulation.Simultaneously in fluorescent quantitation test, while needing to detect sample, also needing to arrange samples Reference product, to determine the validity tested, as shown in Figure 9, is EML4-ALK hypotype 4(E14; A20) the fluorescent quantitative PCR curve of fusion gene positive sample; In addition, the sample in above-described embodiment 5 is carried out sequence verification, find to detect EML4-ALK hypotype 4(E14 by above-mentioned fluorescent quantitation detection experiment in the present embodiment; A20) fusion gene, as shown in Figure 10, can be found out by sequencing result and really in this site, corresponding gene fusion occur, and the result of itself and fluorescent quantitation detection experiment is completely the same.Find to adopt EML4-ALK fusion gene fluorescent quantificationally PCR detecting kit of the present invention can determine EML4-ALK hypotype 4(E14 exactly with this; A20) fusion gene catastrophe.
Embodiment 6:EML4-ALK hypotype 5a(E2a; A20) detection of fusion gene
Design energy specific detection EML4-ALK hypotype 5a(E2a; A20) probe of fusion gene and primer a pair:
Variant 5a-forward primer: 5 '-GTTTTGAGGCGTCTTGCAATCT-3 ' (SEQ ID NO.16);
Variant 5a-reverse primer: 5 '-GGTTGTAGTCGGTCATGATGGTC-3 ' (SEQ ID NO.17);
Variant 5a-Taqman-probe: 5 '-FAM-CTCAAGTAAAGTGTACCGCCGGAAGCA-TAMRA-3 ' (SEQ ID NO.18);
Then optimizing reaction system carries out the detection of FQ-PCR: reaction system is 15 μ l, each 0.15 μ l(20 μ Μ of positive and negative primer), each 0.2 μ l(20 μ Μ of probe), sample template DNA2.5 μ l(100-300ng/ μ l) and, 2*Taqman universalPCR
Master Mix(purchased from American applying biological company) 7.5 μ l, ddH
2o4.3 μ l.
PCR reaction conditions: 95 DEG C of denaturation 30sec; And by 95 DEG C 5 seconds, 58 DEG C 33 seconds, amplified reaction 65 circulation.Simultaneously in fluorescent quantitation test, while needing to detect sample, also needing to arrange samples Reference product, to determine the validity tested, as shown in figure 11, is EML4-ALK hypotype 5a(E2a; A20) the fluorescent quantitative PCR curve of fusion gene positive sample; In addition, the sample in above-described embodiment 6 is carried out sequence verification, find to detect EML4-ALK hypotype 5a(E2a by above-mentioned fluorescent quantitation detection experiment in the present embodiment; A20) fusion gene, as shown in figure 12, can be found out by sequencing result and really in this site, corresponding gene fusion occur, and the result of itself and fluorescent quantitation detection experiment is completely the same.Find to adopt EML4-ALK fusion gene fluorescent quantificationally PCR detecting kit of the present invention can determine EML4-ALK hypotype 5a(E2a exactly with this; A20) fusion gene catastrophe.
Embodiment 7:EML4-ALK hypotype 5b(E2b; A20) detection of fusion gene
Design energy specific detection EML4-ALK hypotype 5b(E2b; A20) probe of fusion gene and primer a pair:
Variant 5b-forward primer: 5 '-GTGCTTCCGGCGGTACACT-3 ' (SEQ ID NO.19);
Variant 5b-reverse primer: 5 '-TTGAGGCGTCTTGCAATCTCT-3 ' (SEQ ID NO.20);
Variant 5b-Taqman-probe: 5 '-FAM-CCTCCCCTGAGCTCTGAACCTTTACTTGA-TAMRA-3 ' (SEQ ID NO.21);
Then optimizing reaction system carries out the detection of FQ-PCR: reaction system is 15 μ l, each 0.15 μ l(20 μ Μ of positive and negative primer), each 0.2 μ l(20 μ Μ of probe), sample template DNA2.5 μ l(100-300ng/ μ l) and, 2*Taqman universalPCR
Master Mix(purchased from American applying biological company) 7.5 μ l, ddH
2o4.3 μ l.
PCR reaction conditions: 95 DEG C of denaturation 30sec; And by 95 DEG C 5 seconds, 58 DEG C 33 seconds, amplified reaction 65 circulation.Simultaneously in fluorescent quantitation test, while needing to detect sample, also needing to arrange samples Reference product, to determine the validity tested, as shown in figure 13, is EML4-ALK hypotype 5b(E2b; A20) the fluorescent quantitative PCR curve of fusion gene positive sample; In addition, the sample in above-described embodiment 7 is carried out sequence verification, find to detect EML4-ALK hypotype 5b(E2b by above-mentioned fluorescent quantitation detection experiment in the present embodiment; A20) fusion gene, as shown in figure 14, can be found out by sequencing result and really in this site, corresponding gene fusion occur, and the result of itself and fluorescent quantitation detection experiment is completely the same.Find to adopt EML4-ALK fusion gene fluorescent quantificationally PCR detecting kit of the present invention can determine EML4-ALK hypotype 5b(E2b exactly with this; A20) fusion gene catastrophe.
Embodiment 8:EML4-ALK hypotype 6(E18; A20) detection of fusion gene
Design energy specific detection EML4-ALK hypotype 6(E18; A20) probe of fusion gene and primer a pair:
Variant 6-forward primer: 5 '-GGGTGGTCAGCTGCAACAT-3 ' (SEQ ID NO.22);
Variant 6-reverse primer: 5 '-GAGACTCAGGTGGAGTCATGCTT-3 ' (SEQ ID NO.23);
Variant 6-Taqman-probe: 5 '-FAM-CCACTTCCTTTAGGTCCTTTCCCAGGTGT-TAMRA-3 ' (SEQ ID NO.24);
Then optimizing reaction system carries out the detection of FQ-PCR: reaction system is 15 μ l, each 0.15 μ l(20 μ Μ of positive and negative primer), each 0.2 μ l(20 μ Μ of probe), sample template DNA2.5 μ l(100-300ng/ μ l) and, 2*Taqman universalPCR
Master Mix(purchased from American applying biological company) 7.5 μ l, ddH
2o4.3 μ l.
PCR reaction conditions: 95 DEG C of denaturation 30sec; And by 95 DEG C 5 seconds, 58 DEG C 33 seconds, amplified reaction 65 circulation.Simultaneously in fluorescent quantitation test, while needing to detect sample, also needing to arrange samples Reference product, to determine the validity tested, as shown in figure 15, is EML4-ALK hypotype 6(E18; A20) the fluorescent quantitative PCR curve of fusion gene positive sample; In addition, the sample in above-described embodiment 8 is carried out sequence verification, find to detect EML4-ALK hypotype 6(E18 by above-mentioned fluorescent quantitation detection experiment in the present embodiment; A20) fusion gene, as shown in figure 16, can be found out by sequencing result and really in this site, corresponding gene fusion occur, and the result of itself and fluorescent quantitation detection experiment is completely the same.Find to adopt EML4-ALK fusion gene fluorescent quantificationally PCR detecting kit of the present invention can determine EML4-ALK hypotype 6(E18 exactly with this; A20) fusion gene catastrophe.
Embodiment 9:EML4-ALK hypotype 7(E14; A20) detection of fusion gene
Design energy specific detection EML4-ALK hypotype 7(E14; A20) probe of fusion gene and primer a pair:
Variant 7-forward primer: 5 '-CAGTAGTTGGGGTTGTAGTCGGTC-3 ' (SEQ ID NO.25);
Variant7-reverse primer: 5 '-TTGTCAGATGAGAAATGGGATGTTAT-3 ' (SEQ ID NO.26);
Variant 7-Taqman-probe: 5 '-FAM-TGGCTTGCAGCTCCTGGTGCTCTATT-TAMRA-3 ' (SEQ ID NO.27);
Then optimizing reaction system carries out the detection of FQ-PCR: reaction system is 15 μ l, each 0.15 μ l(20 μ Μ of positive and negative primer), each 0.2 μ l(20 μ Μ of probe), sample template DNA2.5 μ l(100-300ng/ μ l) and, 2*Taqman universalPCR
Master Mix(purchased from American applying biological company) 7.5 μ l, ddH
2o4.3 μ l.
PCR reaction conditions: 95 DEG C of denaturation 30sec; And by 95 DEG C 5 seconds, 58 DEG C 33 seconds, amplified reaction 65 circulation.Simultaneously in fluorescent quantitation test, while needing to detect sample, also needing to arrange samples Reference product, to determine the validity tested, as shown in figure 17, is EML4-ALK hypotype 7(E14; A20) the fluorescent quantitative PCR curve of fusion gene positive sample; In addition, the sample in above-described embodiment 9 is carried out sequence verification, find to detect EML4-ALK hypotype 7(E14 by above-mentioned fluorescent quantitation detection experiment in the present embodiment; A20) fusion gene, as shown in figure 18, can be found out by sequencing result and really in this site, corresponding gene fusion occur, and the result of itself and fluorescent quantitation detection experiment is completely the same.Find to adopt EML4-ALK fusion gene fluorescent quantificationally PCR detecting kit of the present invention can determine EML4-ALK hypotype 7(E14 exactly with this; A20) fusion gene catastrophe.
Embodiment 10: specific detection is tested
This test kit is adopted to detect following several fusion gene respectively: RET-KIF5B fusion gene, ROS1 fusion gene, EML4-ALK fusion gene.
Detected result is: the corresponding detect aperture of EML4-ALK fusion gene has specificity fluorescent quantitative pcr amplification curve, and fused type is hypotype 1, and detected result is positive; All the other two all without specific amplification curve, detected result is negative (see Figure 19).
As from the foregoing: FQ-PCR method is not only quick, efficient, sensitive detection method, and the interpretation of its result is very clear and definite, directly perceived, and result is also reliable, special.
Claims (2)
1. an EML4-ALK fusion gene fluorescent quantificationally PCR detecting kit, comprises fluorescent quantitation reaction solution premixed liquid, fluorescent quantitation reaction solution, positive control sample, it is characterized in that:
Described fluorescent quantitation reaction solution comprises: fluorescent quantitation reaction solution A, fluorescent quantitation reaction solution B, fluorescent quantitation reaction liquid C, fluorescent quantitation reaction solution D, fluorescent quantitation reaction solution E, fluorescent quantitation reaction solution F, fluorescent quantitation reaction solution G, fluorescent quantitation reaction solution H, fluorescent quantitation reaction solution I;
Wherein, described fluorescent quantitation reaction solution A comprises: detect EML4-ALK hypotype 1(E13; A20) sequence of fusion gene be the forward primer of SEQ ID NO.1, the probe of sequence to be the reverse primer of SEQ ID NO.2 and sequence be SEQ ID NO.3;
Described fluorescent quantitation reaction solution B comprises: detect EML4-ALK hypotype 2(E20; A20) sequence of fusion gene be the forward primer of SEQ ID NO.4, the probe of sequence to be the reverse primer of SEQ ID NO.5 and sequence be SEQ ID NO.6;
Described fluorescent quantitation reaction liquid C comprises: detect EML4-ALK hypotype 3a(E6a; A20) sequence of fusion gene be the forward primer of SEQ ID NO.7, the probe of sequence to be the reverse primer of SEQ ID NO.8 and sequence be SEQ ID NO.9;
Described fluorescent quantitation reaction solution D comprises: detect EML4-ALK hypotype 3b(E6b; A20) sequence of fusion gene be the forward primer of SEQ ID NO.10, the probe of sequence to be the reverse primer of SEQ ID NO.11 and sequence be SEQ ID NO.12;
Described fluorescent quantitation reaction solution E comprises: detect EML4-ALK hypotype 4(E14; A20) sequence of fusion gene be the forward primer of SEQ ID NO.13, the probe of sequence to be the reverse primer of SEQ ID NO.14 and sequence be SEQ ID NO.15;
Described fluorescent quantitation reaction solution F comprises: detect EML4-ALK hypotype 5a(E2a; A20) sequence of fusion gene be the forward primer of SEQ ID NO.16, the probe of sequence to be the reverse primer of SEQ ID NO.17 and sequence be SEQ ID NO.18;
Described fluorescent quantitation reaction solution G comprises: detect EML4-ALK hypotype 5b(E2b; A20) sequence of fusion gene be the forward primer of SEQ ID NO.19, the probe of sequence to be the reverse primer of SEQ ID NO.20 and sequence be SEQ ID NO.21;
Described fluorescent quantitation reaction solution H comprises: detect EML4-ALK hypotype 6(E18; A20) sequence of fusion gene be the forward primer of SEQ ID NO.22, the probe of sequence to be the reverse primer of SEQ ID NO.23 and sequence be SEQ ID NO.24;
Described fluorescent quantitation reaction solution I comprises: detect EML4-ALK hypotype 7(E14; A20) sequence of fusion gene be the forward primer of SEQ ID NO.25, the probe of sequence to be the reverse primer of SEQ ID NO.26 and sequence be SEQ ID NO.27;
Described positive control sample comprises: positive control sample A, positive control sample B, positive control sample C, positive control sample D, positive control sample E, positive control sample F, positive control sample G, positive control sample H, positive control sample I;
Wherein, described positive control sample A comprises detected EML4-ALK hypotype 1(E13; A20) fusion gene genomic fragment DNA plasmid;
Described positive control sample B comprises detected EML4-ALK hypotype 2(E20; A20) fusion gene genomic fragment DNA plasmid;
Described positive control sample C comprises detected EML4-ALK hypotype 3a(E6a; A20) fusion gene genomic fragment DNA plasmid;
Described positive control sample D comprises detected EML4-ALK hypotype 3b(E6b; A20) fusion gene genomic fragment DNA plasmid;
Described positive control sample E comprises detected EML4-ALK hypotype 4(E14; A20) fusion gene genomic fragment DNA plasmid;
Described positive control sample F comprises detected EML4-ALK hypotype 5a(E2a; A20) fusion gene genomic fragment DNA plasmid;
Described positive control sample G comprises detected EML4-ALK hypotype hypotype 5b(E2b; A20) fusion gene genomic fragment DNA plasmid;
Described positive control sample H comprises detected EML4-ALK hypotype 6(E18; A20) fusion gene genomic fragment DNA plasmid;
Described positive control sample I comprises detected EML4-ALK hypotype 7(E14; A20) fusion gene genomic fragment DNA plasmid.
2. fluorescent quantificationally PCR detecting kit according to claim 1, is characterized in that: described probe is fluorescent probe, 5 ' end flag F AM fluorescent reporter group, 3 ' end mark TAMRA fluorescent quenching group.
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Effective date of registration: 20200304 Address after: 510000 unit 02, 03, 04, 05, 25th floor, tower a, Guanzhou life science innovation center, No. 51, helix Avenue, international bio Island, Huangpu District, Guangzhou, Guangdong Province Patentee after: Guangzhou Dajian Biotechnology Co., Ltd. Address before: 241000 Anhui city of Wuhu Province Economic and Technological Development Zone Branch Valley Park No. 9 Building Patentee before: ANHUI DAJIAN MEDICAL TECHNOLOGY CO., LTD. |