CN103468813A - 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
- Publication number
- CN103468813A CN103468813A CN2013104263923A CN201310426392A CN103468813A CN 103468813 A CN103468813 A CN 103468813A CN 2013104263923 A CN2013104263923 A CN 2013104263923A CN 201310426392 A CN201310426392 A CN 201310426392A CN 103468813 A CN103468813 A CN 103468813A
- Authority
- CN
- China
- Prior art keywords
- alk
- eml4
- fusion gene
- hypotype
- seq
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Images
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 highest malignant tumour of M & M in the world.Over nearly more than 50 years, countries in the world are industrially developed country particularly, and the sickness rate of lung cancer and case fatality rate all rise rapidly.Nineteen ninety-five, the whole world had 600,000 people to die from lung cancer, and annual number all rising, and the mortality ratio that the World Health Organization (WHO) in 2003 announces is 1,100,000/year, 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), 80% male lung cancer and 75% female lung cancer are caused by smoking, in addition with the contacting of the industrial cancerogens such as asbestos, radon, arsenic, ionizing rays, halogen alkene class, polycyclic aromatic compound, nickel, race, family members' history are all influential to the morbidity of lung cancer.
Lung cancer is divided 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 lung cancer cases, the serious threat human health.The treatment of NSCLC comprises the several different 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 20%-30% case 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 characteristics efficiently in theory with it, more and more becomes the focus of Treatment for Non-small Cell Lung.The treatment that targeted therapy is lung cancer has increased a new field, also for the treatment of lung cancer, has brought new hope.
Molecular targeted therapy is the treatment means that a kind of cell signaling for the tumorigenesis process and other biological are learned approach, be characterized in optionally acting on tumour cell, the propagation of directed blocking-up cancer cells, transfer, signal conduction, 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 a plurality of important steps of growth of tumour cell, at present research is comparatively ripe and be applied to clinical two point of application that mainly contain, one is vascular endothelial growth factor receptor (VEGFR), and another is EGF-R ELISA (EGFR).In addition, become in addition lymphoma kinases (echinoderm microtubule associated protein like4, EML4-ALK) fusion gene between the echinoderms microtubule bindin 4-of latest find.Existing this fusion gene of research prompting may be present in lung cancer at interior multiple noumenal tumour, but the recall rate with NSCLC is the highest, while is because of it and EGFR suddenlys change, the distinct Clinical symptoms that does not coexist phenomenon and contain EML4-ALK gene patient of K-ras sudden change, and pointing out this target spot is the molecular marked compound that the adenocarcinoma of lung specificity is higher.The discovery of EML4-ALK has defined the new subtype of NSCLC, can be rated as in recent years a landmark event on the NSCLC research history.The gene of coding EML4 is positioned at No. 21 locus of No. 2 the short arm of a chromosome, and the gene of coding ALK is positioned at No. 23 locus of No. 2 karyomit(e), both on karyomit(e), be separated by approximately 12,700,000 base pairs and transcriptional orientation opposites.During gene fusion, EML4 gene fracture on karyomit(e), form the exon splicing fragment of different lengths, turn direction, insert between relatively conservative ALK gene 19,20 exons in people position, thereby form the various EML4-ALK fusion variants that length does not wait, at present, at least have been found that 9 kinds of variation hypotypes, be respectively hypotype 1(E13; A20) (this nomenclature refers to 13 exons of EML4 and 20 exons of ALK merge), hypotype 2(E20; A20), hypotype 3(E6a/b; A20), hypotype 4(E14; A20), hypotype 5(E2a/b; A20), hypotype 6(E13; A20) (annotate: hypotype 6 is connected with the 20th exon of ALK for the 13rd exon of EML4 adds the small segment of a 49bp 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 EML4-ALK fusion gene NSCLC patient's incidence.All these fusion genes all have biological function, and expression product is a kind of mosaic type Tyrosylprotein kinase.EML4-ALK merges the molecular isoform that variation has been accredited as the uniqueness of NSCLC, particularly adenocarcinoma of lung a few days ago, and EML4-ALK fusion gene positive patient has its unique clinical pathologic characteristic.For the detection of this target spot, will contribute to screen the treatment crowd that EGFR-TKI is suitable the frontier of opening the NSCLC targeted therapy.
At present detecting in the world the common technological method of EML4-ALK fusion gene 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 there is a kind of fast diagnosis method of ALK fusion gene in NSCLC, and in theory, the advantage of this technology is that it detects the hypersensitivity of sudden change transcript, as found that amplified production means the ALK fusion gene.But, in clinical practice, this technology faces lot of challenges: (1) primer diversification, and research needs a plurality of primers of design to detect 9 varients and 2 non-EMIA transpositions; (2) the fixing paraffin-embedded tissue DNA height fragmentation of formalin, be unfavorable for detecting; (3) false positive often appears in the PCR result, is difficult to as the routine clinical detection method.
FISH is hybridized with specific fluorescent label probe and target DNA, by the immunocytochemistry process, connects upper fluorescein-labelled thing, the technology in fluorescence microscopy Microscopic observation probe mark or site.FISH detects the more special method of ALK fusion gene, but it is advantageous that business development series probe, is applied to adenocarcinoma of lung and detects the 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 interpretation as a result are at present still without unified standard.
IHC refer to specific antibody with the developer mark in the histocyte original position by antigen antibody reaction and histochemical color reaction, corresponding antigens is carried out to a new technology of qualitative, location, quantitative assay.The sharpest edges of IHC are to detect the expression of tumour specific antigen and do not lose cell and the characteristics of organizational structure that can differentiate healthy tissues and pathological tissue.Dye to detect the ALK egg from expressing for biopsy section using-system, the possibility of result is faint and local, and always there is certain subjectivity in the IHC method simultaneously, to the judgement of weak positive findings, need to further by technology such as FISH, verify.IHC can not identify that ALK merges the patient and specifically belongs to that variant hypotype equally in addition.
The imperial teach problem group of the Wu of Guangdong Province lung cancer institute one adopts RACE-coupling PCR sequencing technologies to detect the fusion variation of analyzing the ALK gene, the distinctive feature of the method is to adopt cDNA end rapid amplifying (RACE) technology to carry out the fusion variant of Enrichment Amplification ALK gene in conjunction with the two-wheeled round pcr, 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 further the means by order-checking can clearly to merge be specifically any from many kinds of variants of EML4-ALK.But this method relates to two-wheeled PCR and order-checking, complex steps and consuming time, the cost costliness, be 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, detect the PCR product by means of fluorescent signal, having solved the normal PCR technology can not be quantitatively and the problem polluted of amplified production, avoided 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 characteristics such as high, the reproducible and quantitative scope of susceptibility and specificity is wide.Detection by quantitative requires that detection method is responsive, special, accurate, accurate, reproducible, linearity range is wide, 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) in 1996, by U.S. Applied Biosystems company, released, it is a kind ofly to add a specific fluorescent probe when adding pair of primers when pcr amplification, this probe is an oligonucleotide, and two ends are a report fluorophor of mark and a cancellation fluorophor respectively.When probe is complete, the fluorescent signal of reporter group emission is quenched group and absorbs; While just starting, probe is combined on the arbitrary strand of DNA; During pcr amplification, 5 ' end-3 ' end 5 prime excision enzyme activity of Taq enzyme is cut degraded by the probe enzyme, make to report that fluorophor separates with the cancellation fluorophor, thereby the fluorescence monitoring system can receive fluorescent signal, it is DNA chain of every amplification, just there is a fluorescence molecule to form, realized that the accumulation of fluorescent signal and PCR product form Complete Synchronization.This technology not only realized to DNA profiling quantitatively, and there is highly sensitive, specificity and the characteristics such as reliability is stronger, can realize multiple reaction, level of automation is high, nonstaining property, tool real-time and accuracy, be widely used at present the fields such as molecular biology research and medical research.
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 energy is quick, accurate, high sensitive ground detects 9 kinds of modal EML4-ALK fusion gene variants, 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, thereby set up the real-time fluorescence quantitative PCR system that detects 9 kinds of EML4-ALK fusion gene variants the most common.
The present invention adopts following technical measures: a kind of fluorescent quantificationally PCR detecting kit that fast, accurately detects the 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 of fluorescent probe ' end flag F AM fluorescence report group, 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 of fluorescent probe ' end flag F AM fluorescence report group, 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.
The 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 of fluorescent probe ' end flag F AM fluorescence report group, 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 of fluorescent probe ' end flag F AM fluorescence report group, 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 of fluorescent probe ' end flag F AM fluorescence report group, 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 of fluorescent probe ' end flag F AM fluorescence report group, 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 of fluorescent probe ' end flag F AM fluorescence report group, 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 of fluorescent probe ' end flag F AM fluorescence report group, 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 of fluorescent probe ' end flag F AM fluorescence report group, 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 the EML4-ALK fusion gene.A pair of for the 9 kinds of special design of conserved sequence TaqMan probe and primers that merge variant of EML4-ALK fusion gene respectively, when running into the corresponding fusion variant of EML4-ALK fusion gene specific gene sequence, the combination fully with it of TaqMan probe, during pcr amplification, the fluorescence report group sends fluorescence because enzymolysis separates, can detect in real time the accumulation of corresponding fluorescent signal, thereby realize detecting the purpose of EML4-ALK fusion gene content.
Although 9 kinds of complete genome sequences that merge variant provided by the invention are the published sequences of GENBANK, but the definite of conserved sequence is to need screening comparison and experimental verification, after selected good conserved sequence, designed primer and probe sequence by Primer3 software again, and pass through again a large amount of experimental verifications after the upper blast of NCBI verifies its specificity, and optimize the 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 mutually close, 5 ' end fluorescence of sending of reporter group is because FRET (fluorescence resonance energy transfer) (FRET) and by the group cancellation of going out of 3 ' end quenching, so there is no the variation of fluorescent signal in system.Once and the combination of it and template specificity, its binding site is between two primers, extension along with primer, the Taq archaeal dna polymerase runs into the probe combined with template in the chain extension process, its 5 '-3 ' 5 prime excision enzyme activity will cut off probe, the fluorescence report group, away from the fluorescent quenching group, has so just destroyed the FRET between two fluorophors, and the photofluorometer that the fluorescence that reporter group discharges just can be built in instrument detects.PCR is every through a circulation, and fluorescent signal is also the same with the purpose fragment, the process that has sync index to increase, the power of fluorescent signal just represented template DNA copy number the 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 detection method, compare, present technique 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 has designed Auele Specific Primer and fluorescent probe for 9 kinds of varients of EML4-ALK fusion gene respectively, correlated response liquid is in charge of and is detected, can get rid of the interference between each primer, more can greatly improve detection specificity and sensitivity than the situation that will adopt mixed reaction solution, and false positive is low
2. linear relationship is good, but detection by quantitative because the logarithm of the power of fluorescent signal and template amplification product is linear, by the detection of fluorescent signal, sample original template concentration is carried out quantitatively, error is little;
3. simple to operate, level of automation is high, and the FQ-PCR technology is to the amplification of PCR product and detect that next step completes in the situation of stopped pipe, does not need to uncap, and crossed contamination and contaminate environment chance are few, have therefore also just reduced the probability of result error;
4. interpretation as a result is clear and definite, directly perceived, can carry out quantitative analysis to result.The interpretation of fluorescence quantitative PCR method result: more than the thresholding line, have the sample of amplification curve to be positive sample, interpretation as a result is very simple, directly perceived;
5. the sample size detected is large, once can detect 384 examples at most;
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 hybridization, electrophoresis, take pictures.
8. 9 kinds of varients of the specific detection test kit disposable examination EML4-ALK fusion gene of energy provided by the invention, fast and convenient.
The accompanying drawing explanation
Fig. 1 is EML4-ALK hypotype 1(E13; A20) FQ-PCR of fusion gene figure.
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) FQ-PCR of fusion gene figure.
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) FQ-PCR of fusion gene figure.
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) FQ-PCR of fusion gene figure.
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) FQ-PCR of fusion gene figure.
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) FQ-PCR of fusion gene figure.
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) FQ-PCR of fusion gene figure.
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) FQ-PCR of fusion gene figure.
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) FQ-PCR of fusion gene figure.
Figure 18 is EML4-ALK hypotype 7(E14; A20) the sequencing result figure of fusion gene.
The FQ-PCR figure that Figure 19 is EML4-ALK fusion gene specific detection.
Embodiment
The sample explanation: in following embodiment, positive sample used is collected the wax stone tissue from the EML4-ALK gene fusion of Pathology Deparment of Tumor Hospital Attached to Zhongshan Univ. Clinicopathologic Diagnosis.Extract genomic dna 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 are a pair of:
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 is carried 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), 2*Taqman universal PCR.
Master Mix(is purchased from U.S. applying biological company) 7.5 μ l, ddH
2o4.3 μ l.
PCR reaction conditions: 95 ℃ of denaturation 30sec; And by 95 ℃ 5 seconds, 58 ℃ 33 seconds, 65 circulations of amplified reaction.In the fluorescent quantitation test, when needing to detect sample, also need to arrange the sample reference material, with the validity of confirmed test, as shown in Figure 1, be EML4-ALK hypotype 1(E13 simultaneously; A20) the fluorescent quantitative PCR curve of fusion gene positive sample; In addition, the sample in above-described embodiment 1 is carried out to sequence verification, find that detecting test by above-mentioned fluorescent quantitation in the present embodiment detects EML4-ALK hypotype 1(E13; A20) fusion gene, as shown in Figure 2, can find out really, in this site, corresponding gene fusion occur by sequencing result, and it is in full accord that itself and fluorescent quantitation detect the result of testing.With this, find to adopt EML4-ALK fusion gene fluorescent quantificationally PCR detecting kit of the present invention can determine exactly EML4-ALK hypotype 1(E13; A20) fusion gene sudden change situation.
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 are a pair of:
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 is carried 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(is purchased from U.S. applying biological company) 7.5 μ l, ddH
2o4.3 μ l.
PCR reaction conditions: 95 ℃ of denaturation 30sec; And by 95 ℃ 5 seconds, 58 ℃ 33 seconds, 65 circulations of amplified reaction.In the fluorescent quantitation test, when needing to detect sample, also need to arrange the sample reference material, with the validity of confirmed test, as shown in Figure 3, be EML4-ALK hypotype 2(E20 simultaneously; A20) the fluorescent quantitative PCR curve of fusion gene positive sample; In addition, the sample in above-described embodiment 2 is carried out to sequence verification, find that detecting test by above-mentioned fluorescent quantitation in the present embodiment detects EML4-ALK hypotype 2(E20; A20) fusion gene, as shown in Figure 4, can find out really, in this site, corresponding gene fusion occur by sequencing result, and it is in full accord that itself and fluorescent quantitation detect the result of testing.With this, find to adopt EML4-ALK fusion gene fluorescent quantificationally PCR detecting kit of the present invention can determine exactly EML4-ALK hypotype 2(E20; A20) fusion gene sudden change situation.
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 are a pair of:
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 is carried 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), 2*Taqman universal PCR
Master Mix(is purchased from U.S. applying biological company) 7.5 μ l, ddH
2o4.3 μ l.
PCR reaction conditions: 95 ℃ of denaturation 30sec; And by 95 ℃ 5 seconds, 58 ℃ 33 seconds, 65 circulations of amplified reaction.In the fluorescent quantitation test, when needing to detect sample, also need to arrange the sample reference material, with the validity of confirmed test, as shown in Figure 5, be EML4-ALK hypotype 3a(E6a simultaneously; A20) the fluorescent quantitative PCR curve of fusion gene positive sample; In addition, the sample in above-described embodiment 3 is carried out to sequence verification, find that detecting test by above-mentioned fluorescent quantitation in the present embodiment detects EML4-ALK hypotype 3a(E6a; A20) fusion gene, as shown in Figure 6, can find out really, in this site, corresponding gene fusion occur by sequencing result, and it is in full accord that itself and fluorescent quantitation detect the result of testing.With this, find to adopt EML4-ALK fusion gene fluorescent quantificationally PCR detecting kit of the present invention can determine exactly EML4-ALK hypotype 3a(E6a; A20) fusion gene sudden change situation.
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 are a pair of:
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 is carried 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(is purchased from U.S. applying biological company) 7.5 μ l, ddH
2o4.3 μ l.
PCR reaction conditions: 95 ℃ of denaturation 30sec; And by 95 ℃ 5 seconds, 58 ℃ 33 seconds, 65 circulations of amplified reaction.In the fluorescent quantitation test, when needing to detect sample, also need to arrange the sample reference material, with the validity of confirmed test, as shown in Figure 7, be EML4-ALK hypotype 3b(E6b simultaneously; A20) the fluorescent quantitative PCR curve of fusion gene positive sample; In addition, the sample in above-described embodiment 4 is carried out to sequence verification, find that detecting test by above-mentioned fluorescent quantitation in the present embodiment detects EML4-ALK hypotype 3b(E6b; A20) fusion gene, as shown in Figure 8, can find out really, in this site, corresponding gene fusion occur by sequencing result, and it is in full accord that itself and fluorescent quantitation detect the result of testing.With this, find to adopt EML4-ALK fusion gene fluorescent quantificationally PCR detecting kit of the present invention can determine exactly EML4-ALK hypotype 3b(E6b; A20) fusion gene sudden change situation.
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 are a pair of:
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 is carried 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(is purchased from U.S. applying biological company) 7.5 μ l, ddH
2o4.3 μ l.
PCR reaction conditions: 95 ℃ of denaturation 30sec; And by 95 ℃ 5 seconds, 58 ℃ 33 seconds, 65 circulations of amplified reaction.In the fluorescent quantitation test, when needing to detect sample, also need to arrange the sample reference material, with the validity of confirmed test, as shown in Figure 9, be EML4-ALK hypotype 4(E14 simultaneously; A20) the fluorescent quantitative PCR curve of fusion gene positive sample; In addition, the sample in above-described embodiment 5 is carried out to sequence verification, find that detecting test by above-mentioned fluorescent quantitation in the present embodiment detects EML4-ALK hypotype 4(E14; A20) fusion gene, as shown in figure 10, can find out really, in this site, corresponding gene fusion occur by sequencing result, and it is in full accord that itself and fluorescent quantitation detect the result of testing.With this, find to adopt EML4-ALK fusion gene fluorescent quantificationally PCR detecting kit of the present invention can determine exactly EML4-ALK hypotype 4(E14; A20) fusion gene sudden change situation.
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 are a pair of:
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 is carried 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(is purchased from U.S. applying biological company) 7.5 μ l, ddH
2o4.3 μ l.
PCR reaction conditions: 95 ℃ of denaturation 30sec; And by 95 ℃ 5 seconds, 58 ℃ 33 seconds, 65 circulations of amplified reaction.In the fluorescent quantitation test, when needing to detect sample, also need to arrange the sample reference material, with the validity of confirmed test, as shown in figure 11, be EML4-ALK hypotype 5a(E2a simultaneously; A20) the fluorescent quantitative PCR curve of fusion gene positive sample; In addition, the sample in above-described embodiment 6 is carried out to sequence verification, find that detecting test by above-mentioned fluorescent quantitation in the present embodiment detects EML4-ALK hypotype 5a(E2a; A20) fusion gene, as shown in figure 12, can find out really, in this site, corresponding gene fusion occur by sequencing result, and it is in full accord that itself and fluorescent quantitation detect the result of testing.With this, find to adopt EML4-ALK fusion gene fluorescent quantificationally PCR detecting kit of the present invention can determine exactly EML4-ALK hypotype 5a(E2a; A20) fusion gene sudden change situation.
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 are a pair of:
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 is carried 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(is purchased from U.S. applying biological company) 7.5 μ l, ddH
2o4.3 μ l.
PCR reaction conditions: 95 ℃ of denaturation 30sec; And by 95 ℃ 5 seconds, 58 ℃ 33 seconds, 65 circulations of amplified reaction.In the fluorescent quantitation test, when needing to detect sample, also need to arrange the sample reference material, with the validity of confirmed test, as shown in figure 13, be EML4-ALK hypotype 5b(E2b simultaneously; A20) the fluorescent quantitative PCR curve of fusion gene positive sample; In addition, the sample in above-described embodiment 7 is carried out to sequence verification, find that detecting test by above-mentioned fluorescent quantitation in the present embodiment detects EML4-ALK hypotype 5b(E2b; A20) fusion gene, as shown in figure 14, can find out really, in this site, corresponding gene fusion occur by sequencing result, and it is in full accord that itself and fluorescent quantitation detect the result of testing.With this, find to adopt EML4-ALK fusion gene fluorescent quantificationally PCR detecting kit of the present invention can determine exactly EML4-ALK hypotype 5b(E2b; A20) fusion gene sudden change situation.
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 are a pair of:
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 is carried 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(is purchased from U.S. applying biological company) 7.5 μ l, ddH
2o4.3 μ l.
PCR reaction conditions: 95 ℃ of denaturation 30sec; And by 95 ℃ 5 seconds, 58 ℃ 33 seconds, 65 circulations of amplified reaction.In the fluorescent quantitation test, when needing to detect sample, also need to arrange the sample reference material, with the validity of confirmed test, as shown in figure 15, be EML4-ALK hypotype 6(E18 simultaneously; A20) the fluorescent quantitative PCR curve of fusion gene positive sample; In addition, the sample in above-described embodiment 8 is carried out to sequence verification, find that detecting test by above-mentioned fluorescent quantitation in the present embodiment detects EML4-ALK hypotype 6(E18; A20) fusion gene, as shown in figure 16, can find out really, in this site, corresponding gene fusion occur by sequencing result, and it is in full accord that itself and fluorescent quantitation detect the result of testing.With this, find to adopt EML4-ALK fusion gene fluorescent quantificationally PCR detecting kit of the present invention can determine exactly EML4-ALK hypotype 6(E18; A20) fusion gene sudden change situation.
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 are a pair of:
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 is carried 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(is purchased from U.S. applying biological company) 7.5 μ l, ddH
2o4.3 μ l.
PCR reaction conditions: 95 ℃ of denaturation 30sec; And by 95 ℃ 5 seconds, 58 ℃ 33 seconds, 65 circulations of amplified reaction.In the fluorescent quantitation test, when needing to detect sample, also need to arrange the sample reference material, with the validity of confirmed test, as shown in figure 17, be EML4-ALK hypotype 7(E14 simultaneously; A20) the fluorescent quantitative PCR curve of fusion gene positive sample; In addition, the sample in above-described embodiment 9 is carried out to sequence verification, find that detecting test by above-mentioned fluorescent quantitation in the present embodiment detects EML4-ALK hypotype 7(E14; A20) fusion gene, as shown in figure 18, can find out really, in this site, corresponding gene fusion occur by sequencing result, and it is in full accord that itself and fluorescent quantitation detect the result of testing.With this, find to adopt EML4-ALK fusion gene fluorescent quantificationally PCR detecting kit of the present invention can determine exactly EML4-ALK hypotype 7(E14; A20) fusion gene sudden change situation.
Embodiment 10: the specific detection experiment
Adopt this test kit to detect respectively following several fusion gene: RET-KIF5B fusion gene, ROS1 fusion gene, EML4-ALK fusion gene.
Detected result is: there is specificity fluorescent quantitative pcr amplification curve in the corresponding hole of detecting of EML4-ALK fusion gene, and fused type is hypotype 1, and detected result is positive; All the other two all without the specific amplification curve, detected result negative (seeing Figure 19).
As from the foregoing: the 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, comprise 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) probe that the reverse primer that the forward primer that the sequence of fusion gene is SEQ ID NO.1, sequence are SEQ ID NO.2 and sequence are SEQ ID NO.3;
Described fluorescent quantitation reaction solution B comprises: detect EML4-ALK hypotype 2(E20; A20) probe that the reverse primer that the forward primer that the sequence of fusion gene is SEQ ID NO.4, sequence are SEQ ID NO.5 and sequence are SEQ ID NO.6;
Described fluorescent quantitation reaction liquid C comprises: detect EML4-ALK hypotype 3a(E6a; A20) probe that the reverse primer that the forward primer that the sequence of fusion gene is SEQ ID NO.7, sequence are SEQ ID NO.8 and sequence are SEQ ID NO.9;
Described fluorescent quantitation reaction solution D comprises: detect EML4-ALK hypotype 3b(E6b; A20) probe that the reverse primer that the forward primer that the sequence of fusion gene is SEQ ID NO.10, sequence are SEQ ID NO.11 and sequence are SEQ ID NO.12;
Described fluorescent quantitation reaction solution E comprises: detect EML4-ALK hypotype 4(E14; A20) probe that the reverse primer that the forward primer that the sequence of fusion gene is SEQ ID NO.13, sequence are SEQ ID NO.14 and sequence are SEQ ID NO.15;
Described fluorescent quantitation reaction solution F comprises: detect EML4-ALK hypotype 5a(E2a; A20) probe that the reverse primer that the forward primer that the sequence of fusion gene is SEQ ID NO.16, sequence are SEQ ID NO.17 and sequence are SEQ ID NO.18;
Described fluorescent quantitation reaction solution G comprises: detect EML4-ALK hypotype 5b(E2b; A20) probe that the reverse primer that the forward primer that the sequence of fusion gene is SEQ ID NO.19, sequence are SEQ ID NO.20 and sequence are SEQ ID NO.21;
Described fluorescent quantitation reaction solution H comprises: detect EML4-ALK hypotype 6(E18; A20) probe that the reverse primer that the forward primer that the sequence of fusion gene is SEQ ID NO.22, sequence are SEQ ID NO.23 and sequence are SEQ ID NO.24;
Described fluorescent quantitation reaction solution I comprises: detect EML4-ALK hypotype 7(E14; A20) probe that the reverse primer that the forward primer that the sequence of fusion gene is SEQ ID NO.25, sequence are SEQ ID NO.26 and sequence are 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, it is characterized in that: described probe is fluorescent probe, 5 ' end flag F AM fluorescence report group, 3 ' end mark TAMRA fluorescent quenching group.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310426392.3A CN103468813B (en) | 2013-09-17 | 2013-09-17 | EML4-ALK (Echinoderm microtubule associated protein like4-anaplastic lymphoma kinase) fusion gene fluorescent quantitative PCR (polymerase chain reaction) assay kit |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310426392.3A CN103468813B (en) | 2013-09-17 | 2013-09-17 | EML4-ALK (Echinoderm microtubule associated protein like4-anaplastic lymphoma kinase) fusion gene fluorescent quantitative PCR (polymerase chain reaction) assay kit |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN103468813A true CN103468813A (en) | 2013-12-25 |
| CN103468813B CN103468813B (en) | 2015-06-03 |
Family
ID=49793853
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201310426392.3A Active CN103468813B (en) | 2013-09-17 | 2013-09-17 | EML4-ALK (Echinoderm microtubule associated protein like4-anaplastic lymphoma kinase) fusion gene fluorescent quantitative PCR (polymerase chain reaction) assay kit |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN103468813B (en) |
Cited By (21)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105039580A (en) * | 2015-09-06 | 2015-11-11 | 武汉海吉力生物科技有限公司 | Human ALK fusion gene detection primer set and detection kit |
| CN106148323A (en) * | 2015-04-22 | 2016-11-23 | 北京贝瑞和康生物技术股份有限公司 | A kind of method and test kit merging abrupt climatic change library for building ALK gene |
| CN106414768A (en) * | 2014-03-27 | 2017-02-15 | 生命技术公司 | Gene fusions and gene variants associated with cancer |
| WO2017095486A1 (en) | 2015-12-01 | 2017-06-08 | Seracare Life Sciences, Inc. | Multiplex cellular reference materials |
| CN106978497A (en) * | 2017-04-26 | 2017-07-25 | 武汉友芝友医疗科技股份有限公司 | Detection primer, probe and the detection kit of EML4 ALK fusion gene mutations |
| CN107058305A (en) * | 2017-03-15 | 2017-08-18 | 中国人民解放军总医院 | One group of nucleotide sequence and the application in EML4 ALK fusion gene quick detections |
| CN107475387A (en) * | 2017-08-22 | 2017-12-15 | 上海格诺生物科技有限公司 | Quality-control product for detecting fragmentation DNA mutation and preparation method thereof |
| CN107653320A (en) * | 2017-11-15 | 2018-02-02 | 深圳华大生命科学研究院 | EML4 ALK fusion gene noninvasive detection kits |
| CN108148912A (en) * | 2018-03-08 | 2018-06-12 | 四川大学 | Biomarker, application and the lesion detection kit of tumour |
| CN108703967A (en) * | 2018-05-29 | 2018-10-26 | 中国人民解放军第二军医大学第二附属医院 | Method rice replaces application of the Buddhist nun in preparing EML4-ALK kinases inhibitors |
| CN109022581A (en) * | 2018-08-13 | 2018-12-18 | 上海佰臻生物科技有限公司 | The kit of EML4-ALK fusion fluorescence quantitative PCR detection |
| CN109136343A (en) * | 2018-03-14 | 2019-01-04 | 江苏苏博生物医学股份有限公司 | One kind detecting EML4-ALK fusion hypotype fluorescence quantifying PCR method from human peripheral CTC |
| CN109563547A (en) * | 2016-04-15 | 2019-04-02 | 外来体诊断公司 | The detection based on blood plasma of anaplastic lymphoma kinase (ALK) nucleic acid and ALK fusion transcript and its purposes in cancer diagnosis and treatment |
| CN109706232A (en) * | 2019-03-04 | 2019-05-03 | 合肥欧创基因生物科技有限公司 | For detecting primer, probe and the kit and its detection method of the fusion mutation of mankind's ALK gene |
| CN109837275A (en) * | 2019-01-08 | 2019-06-04 | 大连医科大学附属第二医院 | A kind of preparation method of fusion positive control standard items |
| CN113136419A (en) * | 2021-06-09 | 2021-07-20 | 广州华银健康医疗集团股份有限公司 | Fluorescent quantitative PCR detection method for fusion gene mutation |
| CN113373233A (en) * | 2021-07-07 | 2021-09-10 | 远辰生物科技(苏州)有限公司 | Reaction system for detecting EML4-ALK fusion gene based on digital PCR and application thereof |
| CN115927564A (en) * | 2022-09-29 | 2023-04-07 | 杭州联川基因诊断技术有限公司 | Primer combination, kit and method for detecting gene fusion in biological sample |
| US11746379B2 (en) | 2013-04-17 | 2023-09-05 | Life Technologies Corporation | Gene fusions and gene variants associated with cancer |
| CN117025766A (en) * | 2023-07-07 | 2023-11-10 | 银丰基因科技有限公司 | DNA standard for human ALK-E13 and A20 fusion gene detection, and preparation method and application thereof |
| CN117025766B (en) * | 2023-07-07 | 2024-04-30 | 银丰基因科技有限公司 | DNA standard for human ALK-E13 and A20 fusion gene detection, and preparation method and application thereof |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP1914240A1 (en) * | 2006-10-11 | 2008-04-23 | Astellas Pharma Inc. | EML4-ALK fusion gene |
| CN102719525A (en) * | 2012-04-12 | 2012-10-10 | 厦门艾德生物医药科技有限公司 | Primer, probe and detection kit for detection of EML4-ALK fusion gene mutation |
-
2013
- 2013-09-17 CN CN201310426392.3A patent/CN103468813B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP1914240A1 (en) * | 2006-10-11 | 2008-04-23 | Astellas Pharma Inc. | EML4-ALK fusion gene |
| CN102719525A (en) * | 2012-04-12 | 2012-10-10 | 厦门艾德生物医药科技有限公司 | Primer, probe and detection kit for detection of EML4-ALK fusion gene mutation |
Non-Patent Citations (3)
| Title |
|---|
| NIU HAITAO: "Identification of anaplastic lymphoma kinase break points and oncogenic mutation profiles in acral_mucosal melanomas", 《PIGMENT CELL MELANOMA RES》 * |
| YUN-GANG ZHANG: "Evaluation of ALK Rearrangement in Chinese Non-Small Cell Lung Cancer Using FISH, Immunohistochemistry,and Real-Time Quantitative RT- PCR on Paraffin-Embedded Tissues", 《PLOS ONE》 * |
| 杨彦卓: "非小细胞肺癌中EML 4-ALK 融合基因的生物学特性及其治疗", 《肿瘤》 * |
Cited By (27)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US11746379B2 (en) | 2013-04-17 | 2023-09-05 | Life Technologies Corporation | Gene fusions and gene variants associated with cancer |
| CN106414768A (en) * | 2014-03-27 | 2017-02-15 | 生命技术公司 | Gene fusions and gene variants associated with cancer |
| CN106414768B (en) * | 2014-03-27 | 2020-05-29 | 生命技术公司 | Gene fusions and gene variants associated with cancer |
| CN106148323A (en) * | 2015-04-22 | 2016-11-23 | 北京贝瑞和康生物技术股份有限公司 | A kind of method and test kit merging abrupt climatic change library for building ALK gene |
| CN106148323B (en) * | 2015-04-22 | 2021-03-05 | 北京贝瑞和康生物技术有限公司 | Method and kit for constructing ALK gene fusion mutation detection library |
| CN105039580A (en) * | 2015-09-06 | 2015-11-11 | 武汉海吉力生物科技有限公司 | Human ALK fusion gene detection primer set and detection kit |
| WO2017095486A1 (en) | 2015-12-01 | 2017-06-08 | Seracare Life Sciences, Inc. | Multiplex cellular reference materials |
| CN109563547A (en) * | 2016-04-15 | 2019-04-02 | 外来体诊断公司 | The detection based on blood plasma of anaplastic lymphoma kinase (ALK) nucleic acid and ALK fusion transcript and its purposes in cancer diagnosis and treatment |
| CN107058305A (en) * | 2017-03-15 | 2017-08-18 | 中国人民解放军总医院 | One group of nucleotide sequence and the application in EML4 ALK fusion gene quick detections |
| CN106978497A (en) * | 2017-04-26 | 2017-07-25 | 武汉友芝友医疗科技股份有限公司 | Detection primer, probe and the detection kit of EML4 ALK fusion gene mutations |
| CN107475387A (en) * | 2017-08-22 | 2017-12-15 | 上海格诺生物科技有限公司 | Quality-control product for detecting fragmentation DNA mutation and preparation method thereof |
| CN107475387B (en) * | 2017-08-22 | 2020-12-04 | 上海格诺生物科技有限公司 | Quality control product for detecting fragmented DNA mutation and preparation method thereof |
| CN107653320A (en) * | 2017-11-15 | 2018-02-02 | 深圳华大生命科学研究院 | EML4 ALK fusion gene noninvasive detection kits |
| CN108148912A (en) * | 2018-03-08 | 2018-06-12 | 四川大学 | Biomarker, application and the lesion detection kit of tumour |
| CN109136343A (en) * | 2018-03-14 | 2019-01-04 | 江苏苏博生物医学股份有限公司 | One kind detecting EML4-ALK fusion hypotype fluorescence quantifying PCR method from human peripheral CTC |
| CN108703967A (en) * | 2018-05-29 | 2018-10-26 | 中国人民解放军第二军医大学第二附属医院 | Method rice replaces application of the Buddhist nun in preparing EML4-ALK kinases inhibitors |
| CN109022581A (en) * | 2018-08-13 | 2018-12-18 | 上海佰臻生物科技有限公司 | The kit of EML4-ALK fusion fluorescence quantitative PCR detection |
| CN109837275A (en) * | 2019-01-08 | 2019-06-04 | 大连医科大学附属第二医院 | A kind of preparation method of fusion positive control standard items |
| CN109837275B (en) * | 2019-01-08 | 2022-09-06 | 大连医科大学附属第二医院 | Preparation method of fusion gene positive control standard substance |
| CN109706232A (en) * | 2019-03-04 | 2019-05-03 | 合肥欧创基因生物科技有限公司 | For detecting primer, probe and the kit and its detection method of the fusion mutation of mankind's ALK gene |
| CN109706232B (en) * | 2019-03-04 | 2022-03-22 | 合肥欧创基因生物科技有限公司 | Primer, probe and kit for detecting human ALK gene fusion mutation and detection method thereof |
| CN113136419A (en) * | 2021-06-09 | 2021-07-20 | 广州华银健康医疗集团股份有限公司 | Fluorescent quantitative PCR detection method for fusion gene mutation |
| CN113373233A (en) * | 2021-07-07 | 2021-09-10 | 远辰生物科技(苏州)有限公司 | Reaction system for detecting EML4-ALK fusion gene based on digital PCR and application thereof |
| CN115927564A (en) * | 2022-09-29 | 2023-04-07 | 杭州联川基因诊断技术有限公司 | Primer combination, kit and method for detecting gene fusion in biological sample |
| CN115927564B (en) * | 2022-09-29 | 2023-09-12 | 杭州联川基因诊断技术有限公司 | Primer combination, kit and method for detecting gene fusion in biological sample |
| CN117025766A (en) * | 2023-07-07 | 2023-11-10 | 银丰基因科技有限公司 | DNA standard for human ALK-E13 and A20 fusion gene detection, and preparation method and application thereof |
| CN117025766B (en) * | 2023-07-07 | 2024-04-30 | 银丰基因科技有限公司 | DNA standard for human ALK-E13 and A20 fusion gene detection, and preparation method and application thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| CN103468813B (en) | 2015-06-03 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN103468813B (en) | EML4-ALK (Echinoderm microtubule associated protein like4-anaplastic lymphoma kinase) fusion gene fluorescent quantitative PCR (polymerase chain reaction) assay kit | |
| JP2020127416A (en) | Methods and materials for assessing loss of heterozygosity | |
| JP2018019720A (en) | Recurrent gene fusions in prostate cancer | |
| US20230127823A1 (en) | Non-Invasive Gene Mutation Detection in Lung Cancer Patients | |
| Afrin et al. | Targeted next-generation sequencing for detecting MLL gene fusions in leukemia | |
| Kim et al. | Simultaneous diagnostic platform of genotyping EGFR, KRAS, and ALK in 510 Korean patients with non‐small‐cell lung cancer highlights significantly higher ALK rearrangement rate in advanced stage | |
| CN104774962A (en) | Kit for detecting BRAF gene mutation and detecting method thereof | |
| Takashima et al. | Target amplicon exome-sequencing identifies promising diagnosis and prognostic markers involved in RTK-RAS and PI3K-AKT signaling as central oncopathways in primary central nervous system lymphoma | |
| Xie et al. | The diagnostic accuracy of circulating free DNA for the detection of KRAS mutation status in colorectal cancer: A meta‐analysis | |
| Barrow et al. | Confirmation that somatic mutations of beta‐2 microglobulin correlate with a lack of recurrence in a subset of stage II mismatch repair deficient colorectal cancers from the QUASAR trial | |
| Manderson et al. | Comparative analysis of loss of heterozygosity of specific chromosome 3, 13, 17, and X loci and TP53 mutations in human epithelial ovarian cancer | |
| Smolle et al. | Liquid biopsy in non-small cell lung cancer—current status and future outlook—a narrative review | |
| Yang et al. | Detecting ultralow frequency mutation in circulating cell‐free DNA of early‐stage nonsmall cell lung cancer patients with unique molecular identifiers | |
| Nigro et al. | Clinical utility and application of liquid biopsy genotyping in lung cancer: a comprehensive review | |
| Shen et al. | Genomic alterations identification and resistance mechanisms exploration of NSCLC With Central nervous system metastases using liquid biopsy of cerebrospinal fluid: a real-World study | |
| CN113646443A (en) | Composition for diagnosing glioma or predicting prognosis and method for providing information related thereto | |
| Germetaki et al. | Blood-based RAS mutation testing: concordance with tissue-based RAS testing and mutational changes on progression | |
| Pinheiro et al. | KRAS and NRAS mutational analysis in plasma ctDNA from patients with metastatic colorectal cancer by real-time PCR and digital PCR | |
| JP2022551488A (en) | Using Concurrent Marker Detection to Assess Diffuse Glioma and Responsiveness to Therapy | |
| CN102776287A (en) | Kit for quantitatively detecting expression level of specific gene 1 mRNA (messenger Ribonucleic Acid) in human breast cancer | |
| CN105603069A (en) | Bim (Bcl-2 interacting mediator of cell death) gene deletion fluorescent quantitative PCR (polymerase chain reaction) detection primer, probe and detection reagent kit | |
| CN105506140B (en) | ROS1 fusion ARMS fluorescence quantitive PCR typing detection kit | |
| CN103898232B (en) | A kind of method detecting low abundant species and change | |
| CN102747159A (en) | B-raf mutation genotyping fluorescent quantitative PCR (polymerase chain reaction) kit and detection method | |
| CN103436621A (en) | Method and kit for quickly detecting expression quantity of CK19 mRNA (messenger ribonucleic acid) |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| C41 | Transfer of patent application or patent right or utility model | ||
| TR01 | Transfer of patent right |
Effective date of registration: 20160728 Address after: 241000 Anhui city of Wuhu Province Economic and Technological Development Zone Branch Valley Park No. 9 Building Patentee after: ANHUI DAJIAN MEDICAL TECHNOLOGY CO., LTD. Address before: 510006 Guangdong city of Guangzhou province Panyu District Xiaoguwei Street Outer Ring Road No. 280 Building No. 401 room a department of Guangdong Pharmaceutical University Patentee before: Guangzhou Dajian Biotechnology Co., Ltd. |
|
| TR01 | Transfer of patent right | ||
| TR01 | Transfer of patent right |
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. |