CN105039580A - Human ALK fusion gene detection primer set and detection kit - Google Patents
Human ALK fusion gene detection primer set and detection kit Download PDFInfo
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Abstract
The invention discloses a human ALK fusion gene detection primer set and a detection kit. The primer set comprises one or more detection primers in sixteen ALK fusion gene types A01 to A16 and Taqman probes corresponding to the gene types. As many as sixteen fusion gene types except for EML4 can be detected through the detection kit formed by the primer set and the probes, the requirement for the sample RNA obtaining quality is low, and the ideal detection effect can be achieved through samples in different existence forms; by means of the kit, an operator only needs to directly feed extracted sample RNA once, inverse transcription and PCR amplification are carried out in one closed tube reaction system, and the pollution probability and the result error probability are reduced. One-time detection only takes eighty minutes. The human ALK fusion gene detection primer set and the detection kit have the remarkable advantages that the number of the detection types is large, sensitivity is high, experiment operation is simple, the period is short, the human ALK fusion gene detection primer set and the detection kit are safe and nontoxic, and cost is low.
Description
Technical field
The present invention relates to biotechnology and clinical molecular diagnosis technical field, primer, probe and the test kit of particularly mankind's ALK fusion gene detection.
Background technology
Lung cancer is modal malignant tumour in world wide, and wherein the case of 80% ~ 85% is nonsmall-cell lung cancer (nonsmallcelllungcancer, NSCLC).The sickness rate of lung cancer in China and case fatality rate are all in the trend significantly raised.According to the statistics of the Ministry of Health of China, lung cancer related mortality occupies the first place of malignant tumour general mortality rate.Operation, radiation and chemotherapy are the primary treatments of NSCLC always.In recent years, along with deepening continuously of studying Tumorigenesis and biological behaviour thereof, the molecular targeted therapy field of lung cancer becomes study hotspot.In a kind of hypotype of primary cutaneous type (ALCL), find that ALK (Nucleophosmin-anaplastic lymphoma kinase anaplasticlymphomakinase) resets.Detect ALK fusion gene mutation situation develop the generation of tumour and to tumor individual therapy and prognosis, all there is important directive significance.ALK fusion gene, suddenlys change with EGFR, and do not coexist phenomenon and the distinct Clinical symptoms containing this gene patient of K-ras sudden change, point out this target spot to be the molecular marked compound that adenocarcinoma of lung specificity is higher.Small molecule tyrosine kinase inhibitors gram azoles is evident in efficacy to the patients with lung cancer containing ALK fusion gene mutation for Buddhist nun (Crizotinib).Clinical practice proves the difference existed between each organization type of NSCLC on therapeutic effects of molecular targeted agents.Negative or gene appearance is unknown for EGFR, or EGFR genetic mutation and occur the patient of former or secondary resistance after using EGFR-TKI treatment, suggestion row ALK fusion gene detects.
In the case of nonsmall-cell lung cancer, the sudden change of multiple EMLA-ALK fusion gene is intimate through being found, majority is because the breaking point difference of EML4 gene causes, also just like KIF5B, the non-EML4 gene such as TFG and ALK fused type, therefore Precise Diagnosis ALK positive tumor needs the various amalgamation modes detecting the genes such as EML4, KIF5B, TFG and ALK gene cDNA.
Develop FISH that ALK gene merges in detection of lung cancer technology is DNA level the earliest and immunohistochemical method detects.
FISH remarkable advantage to be recombinated by specific detection ALK, the commercialization of its probe, but there is many deficiencies for current clinical demand, as: the concrete fusion variant that ALK fusion gene can not be specified; Advanced NSCLC patients can only provide the little biopsy of about 2mm usually, is difficult to ensure that each visual field exists the cancer cells of more than 50 for result interpretation; Difficult resolution weave construction form under mirror is organized in for FFPE; For operation and the requirement of interpretation technology very high, doctor's result of determination that operating experience is abundant just has reliability, technology popularization difficulty and the cost intensive of detection.In view of above reason present stage FISH detects extensive examination and diagnosis that the method that there is no is applicable to the positive NSCLC patient of ALK.
The expression status that ALK detects in monoclonal antibody binding immunoassay histochemical method has simple and rapid advantage, histocytology equal samples for lung cancer has screening and is worth, but also need to consider immunohistochemical method susceptibility simultaneously and judge that yin and yang attribute always exists certain subjectivity, making a definite diagnosis further further with FISH or sequence-specific round pcr is needed to the interpretation of weak positive findings.
Summary of the invention
For the problems referred to above, the present invention, for adapting to current clinical rapid detection demand, develops a kind of cheap fast, mankind's ALK fusion gene detection method simple to operate and detection kit.Present method only needs a step fluorescent PCR amplification, detects ALK fusion kind many, not only comprises EML4 gene fusion type and also comprise KIF5B and TFG gene type; Highly sensitive, the sudden change sample of 1-10 copy can effectively detect.
Mankind's ALK fusion gene provided by the invention detects primer sets, comprises the detection primer of 16 kinds of ALK fusion gene types of following A01 ~ A16 one or more groups:
A01:EML4-ALK-V1 hypotype (E13; A20) primer pair is detected: nucleotide sequence shown in SEQIDNO.1 ~ 2,
SEQIDNO.1：5'ATGGAGGACAAAACTACTGTAGA3'，
SEQIDNO.2：5'ACCATCATGACCGACTACAACCC3'；
A02:EML4-ALK-V6 hypotype (E13; Ins69A20) primer pair is detected: nucleotide sequence shown in SEQIDNO.4 ~ 5,
SEQIDNO.4：5'GAAGTGGCCTGTGTAGTGCTTCA3'，
SEQIDNO.5：5'CTGCAGAGCCCTGAGTACAAGCT3'；
A03:EML4-ALK-V2 hypotype (E20; A20) primer pair is detected: nucleotide sequence shown in SEQIDNO.7 ~ 8,
SEQIDNO.7：5'CCAGACAACAAGTATATAATGTCT3'，
SEQIDNO.8：5'CAACTACTGCTTTGCTGGCAAGA3'；
A04:EML4-ALK-V3a hypotype (E6a; A20) primer pair is detected: nucleotide sequence shown in SEQIDNO.10 ~ 11,
SEQIDNO.10：5'GCATAAAGATGTCATCATCA3'，
SEQIDNO.11：5'CTGGCAAGACCTCCTCCATC3'；
A05:EML4-ALK-V3b hypotype (E6b; A20) primer pair is detected: nucleotide sequence shown in SEQIDNO.13 ~ 14,
SEQIDNO.13：5'GATGTCATCATCAACCAAGC3'，
SEQIDNO.14：5'TCCGCACCTCGACCATCATG3'；
A06:EML4-ALK-V4 hypotype (E14; Ins11del49A20) primer pair is detected: nucleotide sequence shown in SEQIDNO.16 ~ 17,
SEQIDNO.16：5'ATTCTGTGGGATCATGATCTGAATC3'；
SEQIDNO.17：5'ACCTCCTCCATCAGTGACCTGAAG3'；
A07:EML4-ALK-V7 hypotype (E14; Del12A20) primer pair is detected: nucleotide sequence shown in SEQIDNO.19 ~ 20,
SEQIDNO.19：5'TGTTATTAACTGGAGGAG3'，
SEQIDNO.20：5'GACCATCATGACCGACTA3'；
A08:EML4-ALK-V5a hypotype (E2; A20) primer pair is detected: nucleotide sequence shown in SEQIDNO.22 ~ 23,
SEQIDNO.22：5'CGGCTTTGGCTGATGTTTTGAG3'，
SEQIDNO.23：5'ACCGACTACAACCCCAACTACT3'；
A09:EML4-ALK-V5b hypotype (E2; Ins117A20) primer pair is detected: nucleotide sequence shown in SEQIDNO.25 ~ 26,
SEQIDNO.25：5'GAAGTGGCCTGTGTAGTGCT3'，
SEQIDNO.26：5'CATCATGACCGACTACAACC3'；
A10:EML4-ALK-V8a hypotype (E17; A20) primer pair is detected: nucleotide sequence shown in SEQIDNO.28 ~ 29,
SEQIDNO.28：5'CCAAGTGGCACAGTGGTGGC3'，
SEQIDNO.29：5'CAAGCTGAGCAAGCTCCGCA3'；
A11:EML4-ALK-V8b hypotype (E17; Ins68A20) primer pair is detected: nucleotide sequence shown in SEQIDNO.31 ~ 32,
SEQIDNO.31：5'CACAGTGGTGGCCATAGGA3'，
SEQIDNO.32：5'ATGGAGCTGCAGAGCCCTG3'；
A12:EML4-ALK-v4alt hypotype (E15del19; Del20A20) primer pair is detected: nucleotide sequence shown in SEQIDNO.34 ~ 35,
SEQIDNO.34：5'GCACATCACGAAACTTTATTT3'，
SEQIDNO.35：5'TGACCGACTACAACCCCAAC3'；
A13:EML4-ALK-v5alt hypotype (E18; A20) primer pair is detected: nucleotide sequence shown in SEQIDNO.37 ~ 38,
SEQIDNO.37：5'GAACAGCTCTCTGTGATGCG3'，
SEQIDNO.38：5'CCCAACTACTGCTTTGCTGG3'；
A14:KIF5B-ALK hypotype (K17; A20) primer pair is detected: nucleotide sequence shown in SEQIDNO.40 ~ 41,
SEQIDNO.40：5'CAGTTGGAGGAATCTGTCGATGCC3'，
SEQIDNO.41：5'TCGACCATCATGACCGACTACAAC3'；
A15:KIF5B-ALK hypotype (K24; A20) primer pair is detected: nucleotide sequence shown in SEQIDNO.43 ~ 44,
SEQIDNO.43：5'GGCCAGAAGAGGGCATTCTGC3'，
SEQIDNO.44：5'CAACCCCAACTACTGCTTTGC3'；
A16:TFG-ALK hypotype (T5; A20) primer pair is detected: nucleotide sequence shown in SEQIDNO.46 ~ 47,
SEQIDNO.46：5'TGTTATGTCAGCGTTTGGC3'，
SEQIDNO.47：5'GCAAGCTCCGCACCTCGA3'。
The detectable gene type of table 1 the present invention
The fluorescence RT-PCR that present invention also offers mankind's ALK fusion gene detects primer sets, comprises the detection primer of 16 kinds of ALK fusion gene types of above-described A01 ~ A16 one or more groups, and the following probe corresponding to this group gene type:
A01:EML4-ALK-V1 hypotype (E13; A20) detection probes: nucleotide sequence as shown in SEQIDNO.3, SEQIDNO.03:TAAAGTGTACCGCCGG;
A02:EML4-ALK-V6 hypotype (E13; Ins69A20) detection probes: nucleotide sequence as shown in SEQIDNO.6, SEQIDNO.06:ACCGCCGGAAGCACC;
A03:EML4-ALK-V2 hypotype (E20; A20) detection probes: nucleotide sequence as shown in SEQIDNO.9, SEQIDNO.09:ATTGTACTTGTACCGCC;
A04:EML4-ALK-V3a hypotype (E6a; A20) detection probes: nucleotide sequence as shown in SEQIDNO.12, SEQIDNO.12:CCAAGTGTACCGCCGG;
A05:EML4-ALK-V3b hypotype (E6b; A20) detection probes: nucleotide sequence as shown in SEQIDNO.15, SEQIDNO.15:CCGCCGGAAGCACCAG;
A06:EML4-ALK-V4 hypotype (E14; Ins11del49A20) detection probes: nucleotide sequence as shown in SEQIDNO.18, SEQIDNO.18:CCCTGAGTACAAGCTGA;
A07:EML4-ALK-V7 hypotype (E14; Del12A20) detection probes: nucleotide sequence as shown in SEQIDNO.21, SEQIDNO.21:AGAAATAGAGCACCAG;
A08:EML4-ALK-V5a hypotype (E2; A20) detection probes: nucleotide sequence as shown in SEQIDNO.24, SEQIDNO.24:CCCTGAGTACAAGCTGA;
A09:EML4-ALK-V5b hypotype (E2; Ins117A20) detection probes: nucleotide sequence as shown in SEQIDNO.27, SEQIDNO.27:CCACCTGCAGTGTACCG;
A10:EML4-ALK-V8a hypotype (E17; A20) detection probes: nucleotide sequence as shown in SEQIDNO.30, SEQIDNO.30:CCTGCAGTGTACCGCC;
A11:EML4-ALK-V8b hypotype (E17; Ins68A20) detection probes: nucleotide sequence as shown in SEQIDNO.33, SEQIDNO.33:CCACCTGCAGTGTACC;
A12:EML4-ALK-v4alt hypotype (E15del19; Del20A20) detection probes: nucleotide sequence as shown in SEQIDNO.36, SEQIDNO.36:CTGCAAGCCATGCAGAT;
A13:EML4-ALK-v5alt hypotype (E18; A20) detection probes: nucleotide sequence as shown in SEQIDNO.39, SEQIDNO.39:CTCAATAGTGTACCGC;
A14:KIF5B-ALK hypotype (K17; A20) detection probes: nucleotide sequence as shown in SEQIDNO.42, SEQIDNO.42:CACAAGTGTACCGCCGG;
A15:KIF5B-ALK hypotype (K24; A20) detection probes: nucleotide sequence as shown in SEQIDNO.45, SEQIDNO.45:CACCAGGAGCTGCAAGCC;
A16:TFG-ALK hypotype (T5; A20) detection probes: nucleotide sequence as shown in SEQIDNO.48, SEQIDNO.48:CAGTGTACCGCCGGAAGC;
The nucleotide sequence 5 ' of detection probes holds mark fluorescent reporter group, 3 ' end mark fluorescent quenching group.
Preferably, described fluorescent reporter group is FAM (6-Fluoresceincarboxylic acid), and fluorescent quenching group is MGB (MinorGrooveBinder).
Present invention also offers the fluorescent RT-PCR detection reagent box of mankind's ALK fusion gene, it fluorescence RT-PCR comprising above-described mankind's ALK fusion gene detects primer sets.
Preferably, this test kit also comprises the internal control gene primer pair of nucleotide sequence as SEQIDNO.49 ~ 50, with the internal control gene probe of nucleotide sequence as shown in SEQIDNO.51, probe nucleotide sequence 5 ' holds mark fluorescent reporter group JOE (2,7-dimethyl-4, the chloro-6-Fluoresceincarboxylic acid of 5-bis-), 3 ' end mark fluorescent quenching group BHQ1 (Black hole quencher 1 of BiosearchTechnologies company).
SEQIDNO.49：5'CTGGCATTGCCGACAGGA3'，
SEQIDNO.50：5'AGGAGCAATGATCTTGATC3'，
SEQIDNO.51：AAGGAGATCACTGCCCTGGCACCCAG。
Preferably; mentioned reagent box also comprises one or more groups the positive reference substance in 16 kinds of ALK fusion gene types of A01 ~ A16, and positive reference substance is one section of specific base sequence of this ALK fusion gene type and the plasmon DNA of house-keeping gene ACTB mono-section of base sequence.
Preferably, in the fluorescent RT-PCR detection reagent box of above-mentioned mankind's ALK fusion gene, comprise in test kit:
The detection reagent of different genes type: the detection primer of each ALK fusion gene type and probe are in a reaction system, also containing internal control gene test primer and probe in the reaction system of this gene type, and PCR damping fluid, form the detection reagent of this gene type of pipe;
One pipe contains the enzyme reagent of ThermoScript II and DNA polymerase;
Have positive reference substance, also have a pipe positive reference substance mixed solution, positive control mixed solution includes the positive reference substance of this kind ALK fusion gene type corresponding to detection primer gene types all in this test kit.
Wherein, PCR damping fluid is containing buffer, dNTP, Mg
2+deng, directly can buy commercial goods, also can prepare voluntarily, be prior art contents known.
Preferably, test kit of the present invention comprises detection primer and the probe of whole 16 kinds of ALK fusion gene types, the detection primer of often kind of gene type and probe, together with internal control primer and probe and the independent packaging of PCR damping fluid in a pipe.
Present invention also offers the using method of the fluorescent RT-PCR detection reagent box of above arbitrary described mankind's ALK fusion gene, that measuring samples is extracted RNA, join in the detection reaction system of various ALK fusion gene type, carry out quantitative fluorescent PCR reaction, collect reaction signal, JOE signal should reach setting threshold value Ct value and be less than 26, otherwise the failure of an experiment; Detecting JOE signal meets after Ct value is less than 26, carries out following judgement:
FAM signal Ct value > 30 or without Ct value in often kind of detection system, shows that this sample feminine gender does not exist ALK fusion gene;
Wherein FAM signal Ct value≤30 in certain detection system, show the ALK fusion gene containing this type in this measuring samples.
Preferably, in above-mentioned using method, quantitative fluorescent PCR response procedures is: 45 DEG C of reaction 5min, 1 circulation; 95 DEG C of reaction 5min, 1 circulation; 95 DEG C of reaction 30s, 58 DEG C of reactions 45s, totally 10 circulations; 95 DEG C of reaction 15s, 58 DEG C of reactions 45s, totally 35 circulations, collect signal 58 DEG C time.
Compared with prior art, the present invention has following beneficial effect:
(1) detection fusion type is many: the fusion gene kind that can detect the other types except EML4, in table 1.
(2) clear and definite fused type: the corresponding reacting hole of a kind of fused type, makes pattern detection fused type out very clear and definite, in table 2.
(3) multiple sample type is applicable to: it is low that this technology obtains specification of quality to sample RNA, and no matter be flesh tissue or paraffin organization, serum, blood plasma can obtain desirable Detection results.
(4) simple to operate, detection time is short: existing market product and technology are all two-step pcrs, and the first step reverse transcription becomes cDNA rna transcription, and second step open pipe joins PCR reaction tubes cDNA.But this test kit only needs an application of sample directly to add the sample rna extracted, reverse transcription and pcr amplification carry out in the same tube reaction system closed, and so not only reduce pollution probability, and reduce the probability that deviation appears in result.One-time detection only needs complete for 80 minutes.
(5) specificity is good: detect A549 clone with this test kit and extract RNA, detect ROS1 fusion gene, detect RET fusion gene, detected result is feminine gender, sees Fig. 1.
(6) highly sensitive: sensitivity checking 1 ~ 10copyDNA genome can detect by analysis, sees Fig. 3.
(7) result interpretation is clearly directly perceived: directly see that amplification curve and Ct value just can judge.
In a word, the mankind's ALK fusion gene detection kit that the present invention relates to is applicable to clinical multiple sample type and selects, and have detection kind many, highly sensitive, experimental implementation is simple, and the cycle is short, safety non-toxic, and cost is low waits remarkable advantage.
Accompanying drawing explanation
Fig. 1 is that test kit of the present invention detects not containing the A549 clone RNA (Fig. 1-1) of ALK fusion gene, ROS1 fusion gene (Fig. 1-2), RET fusion gene (Fig. 1-3) amplification curve diagram.
Fig. 2 is the non-small cell adenocarcinoma of lung FFPE sample rna amplification figure that test kit of the present invention detects containing ALK fusion gene.
Fig. 3 is for detecting test kit sensitive amplification graphic representation of the present invention.
Embodiment
Below in conjunction with specific embodiment, the invention will be further described, and to make those skilled in the art the present invention may be better understood and can be implemented, but illustrated embodiment is not as a limitation of the invention.
The extraction of embodiment 1 clinical sample RNA
The present embodiment extracts RNA from the paraffin-embedded tissue section of non-small cell patients with lung adenocarcinoma, and carry out quantitatively, as detection template to it.Adopt the Cat.NO.73504QIAamp paraffin-embedded tissue of Qiagen company to extract test kit, specifically details are as follows.
1, the preparation before experiment
(1) water-bath is transferred to 56 DEG C.
(2) by the BufferPKD in QIAamp test kit in 56 DEG C of placements, until resolution of precipitate.
2, the process of sample
(1) paraffin unnecessary on paraffin section being cut, by being cut into suitable size, being contained on paraffin slicing machine.
(2) slide calliper rule are transferred to 10 μm, then cut wax stone, until uniform tissue is significantly seen in the section of cutting.
(3) remove initial 5, from the 6th, load in the EP pipe of 1.5mL, often pipe 5, then numbers.
3, RNA extracts
(1) add 1mL dimethylbenzene in the sample, cover lid, mixing is put upside down in soft rotation, the centrifugal 2min of 14000rpm under room temperature.
(2) suck supernatant with pipettor, add 1mL dehydrated alcohol, cover lid, mixing is put upside down in soft rotation, the centrifugal 2min of 14000rpm under room temperature.
(3) exhaust supernatant with pipettor, lid is opened, place 10min in room temperature.
(4) add 240 μ LBufferPKD, cover lid, vortex mixes, and the centrifugal 1min of 10000rpm, adds at the bottom of 10 μ L Proteinase Ks to pipe, inhales up and down play mixing with rifle head.
(5) 56 DEG C hatch 15min after 80 DEG C hatch 15min.
(6) sample of hatching is transferred in clean 2mLEP pipe together with precipitation and supernatant, places the centrifugal 15min of 13500rpm after 3min on ice.
(7) draw supernatant in clean 2mLEP pipe, add 25ulDNaseBoosterBuffer and 10 μ LDNaseI, whirlpool mixes, centrifugal by the bottom of the centrifugal on tube wall to pipe fast, and room temperature places 15min.
(8) add 500 μ LBufferRBC whirlpool mixings, then add the dehydrated alcohol of 1200 μ L, mixing of turning upside down, does not need centrifugal.
(9) adsorption column that collection tube is housed is taken out, by the liquid assimilating 700 μ L in centrifuge tube in adsorption column, cover lid, the centrifugal 15s of 10000rpm, discards effluent, is taken out in adsorption column by the liquid be left in centrifuge tube, the centrifugal 15s of 10000rpm, discards effluent.
(10) 500 μ LBufferRPE are added, cover lid, the centrifugal 2min of 10000rpm.Discard effluent.
(11) add 500 μ LBufferRPE, cover lid, the centrifugal 2min of 10000rpm, discards effluent.
(12) be inserted in by adsorption column in the collection tube of a clean 2mL, uncap, the centrifugal 5min of maximum speed of revolution, discards effluent and collection tube.
(13) be inserted in by adsorption column in a clean 1.5mLEP pipe, uncap, adds 14-30 μ LBufferATE in film central authorities, cover lid, and the centrifugal 1min of maximum speed of revolution, effluent is the RNA of extraction.
4, quantitative
The RNA ultraviolet spectrophotometer of extraction is carried out quantitatively, and dilutes RNA concentration to 1ng/ μ L.
Embodiment 2: real-time fluorescence RT-PCR method amplification clinical sample RNA
Mankind's ALK fusion gene detection kit, containing 16 PCR reaction systems, is used for detecting sample ALK fusion gene situation to be checked.Also have a pipe enzyme reagent (containing reversed transcriptive enzyme and archaeal dna polymerase) in test kit, in test kit, PCR reaction system composition and detection fusion type are in table 2.
Table 2: the primer that test kit provides and probe sequence
Detecting step is as follows:
(1) to detect a sample.First 3 centrifuge tubes (PCR level) are got at pipe lid marker samples name S1, STD, NTC respectively; Secondly correspondence adds 80 μ L (2ng/ μ L) sample to be detected, positive control (STD) and non-template contrast NTC (DEPC water) at the bottom of pipe respectively to 3 centrifuge tubes; Finally add enzyme reagent (the precious biological product in Dalian, EnzyMix) 8 μ L separately to 3 centrifuge tubes again; Slightly shake after covering tightly centrifuge tube lid, centrifugal, be placed on ice, stand-by.
(2) get PCR react bar, determine pipe number after open PCR reaction tubes pipe lid gently.Stand-by liquid step 1 mixed is added in corresponding PCR hole respectively, 5 μ L/ holes, recommends cloth plate mode in table 3.Cover tightly PCR reaction tubes pipe lid, mix centrifugal.
In PCR pipe, reaction system is:
2 × PCR damping fluid: 10 μ L
Various primer: 0.1 ~ 1.0 μm of ol
Various probe: 0.1 ~ 1.0 μm of ol
Enzyme reagent: 1 μ L
RNA template: 5 μ L
Mend DEPC water extremely: 25 μ L.
1. centrifugal good PCR reaction tubes is put in quantitative real time PCR Instrument,
2. instrument parameter is arranged: fluorescent signal channel selecting FAM and JOE;
3. fluorescent PCR instrument response procedures is arranged: 45 DEG C of reaction 5min, 1 circulation; 95 DEG C of reaction 5min, 1 circulation; 95 DEG C of reaction 30s, 58 DEG C of reactions 45s, totally 10 circulations; 95 DEG C of reaction 15s, 58 DEG C of reactions 45s, totally 35 circulations, collect signal 58 DEG C time.
The present invention judges detected result by the fluorescence intensity of FAM and JOE of reaction system; JOE is internal control signal, and whether add sample for detecting to leak and judge sample quality, JOE signal should reach setting threshold value (CT value < 26); FAM is detection signal, for detecting whether there is position of fusion; After internal control signal reaches requirement, using FAM signal reach setting threshold value needed for cycle index CT value as judging criterion, if the Ct value > 30 of the FAM signalling channel of 16 hole PCR pipe or prove that sample feminine gender does not exist fused type without Ct, if there is amplification curve in any hole of the FAM signalling channel of 16 hole PCR pipe and Ct value < 30, prove that the sample positive exists fused type, which hole has amplification curve just to judge time sample is as which kind of ALK fused type.Concrete judgement is in table 3.
Table 3: kit results judges reference value
After positive reference substance checking, detected result entirely accurate, see Fig. 2.
Fig. 1 is the amplification curve using test kit of the present invention to detect other samples, and wherein do not contain the A549 clone RNA detected result of ALK fusion gene as Fig. 1-1, display only has JOE to have curve, and FAM signalling channel is without curve; The detected result of ROS1 fusion gene is shown in Fig. 1-2, and only there is amplification curve in the first hole, and showing its genotype is A01:EML4-ALK-V1 hypotype (E13; A20); The detected result of RET fusion gene is shown in Fig. 1-3, and display does not have 16 kinds of gene types.
The sensitivity test of each detection primer and probe the results are shown in Figure 3, and display 1 ~ 10copyDNA genome can detect.
The above embodiment is only that protection scope of the present invention is not limited thereto in order to absolutely prove the preferred embodiment that the present invention lifts.The equivalent alternative or conversion that those skilled in the art do on basis of the present invention, all within protection scope of the present invention.Protection scope of the present invention is as the criterion with claims.
SEQUENCELISTING
<110> Wuhan Hygiea Bioscience Co., Ltd.
<120> mankind's ALK fusion gene detects primer sets and detection kit
<130>WH150948
<160>51
<170>PatentInversion3.3
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caactactgctttgctggcaaga23
<210>9
<211>17
<212>DNA
<213> artificial sequence
<400>9
attgtacttgtaccgcc17
<210>10
<211>20
<212>DNA
<213> artificial sequence
<400>10
gcataaagatgtcatcatca20
<210>11
<211>20
<212>DNA
<213> artificial sequence
<400>11
ctggcaagacctcctccatc20
<210>12
<211>16
<212>DNA
<213> artificial sequence
<400>12
ccaagtgtaccgccgg16
<210>13
<211>20
<212>DNA
<213> artificial sequence
<400>13
gatgtcatcatcaaccaagc20
<210>14
<211>20
<212>DNA
<213> artificial sequence
<400>14
tccgcacctcgaccatcatg20
<210>15
<211>16
<212>DNA
<213> artificial sequence
<400>15
ccgccggaagcaccag16
<210>16
<211>25
<212>DNA
<213> artificial sequence
<400>16
attctgtgggatcatgatctgaatc25
<210>17
<211>24
<212>DNA
<213> artificial sequence
<400>17
acctcctccatcagtgacctgaag24
<210>18
<211>17
<212>DNA
<213> artificial sequence
<400>18
ccctgagtacaagctga17
<210>19
<211>18
<212>DNA
<213> artificial sequence
<400>19
tgttattaactggaggag18
<210>20
<211>18
<212>DNA
<213> artificial sequence
<400>20
gaccatcatgaccgacta18
<210>21
<211>16
<212>DNA
<213> artificial sequence
<400>21
agaaatagagcaccag16
<210>22
<211>22
<212>DNA
<213> artificial sequence
<400>22
cggctttggctgatgttttgag22
<210>23
<211>22
<212>DNA
<213> artificial sequence
<400>23
accgactacaaccccaactact22
<210>24
<211>17
<212>DNA
<213> artificial sequence
<400>24
ccctgagtacaagctga17
<210>25
<211>20
<212>DNA
<213> artificial sequence
<400>25
gaagtggcctgtgtagtgct20
<210>26
<211>20
<212>DNA
<213> artificial sequence
<400>26
catcatgaccgactacaacc20
<210>27
<211>17
<212>DNA
<213> artificial sequence
<400>27
ccacctgcagtgtaccg17
<210>28
<211>20
<212>DNA
<213> artificial sequence
<400>28
ccaagtggcacagtggtggc20
<210>29
<211>20
<212>DNA
<213> artificial sequence
<400>29
caagctgagcaagctccgca20
<210>30
<211>16
<212>DNA
<213> artificial sequence
<400>30
cctgcagtgtaccgcc16
<210>31
<211>19
<212>DNA
<213> artificial sequence
<400>31
cacagtggtggccatagga19
<210>32
<211>19
<212>DNA
<213> artificial sequence
<400>32
atggagctgcagagccctg19
<210>33
<211>16
<212>DNA
<213> artificial sequence
<400>33
ccacctgcagtgtacc16
<210>34
<211>21
<212>DNA
<213> artificial sequence
<400>34
gcacatcacgaaactttattt21
<210>35
<211>20
<212>DNA
<213> artificial sequence
<400>35
tgaccgactacaaccccaac20
<210>36
<211>17
<212>DNA
<213> artificial sequence
<400>36
ctgcaagccatgcagat17
<210>37
<211>20
<212>DNA
<213> artificial sequence
<400>37
gaacagctctctgtgatgcg20
<210>38
<211>20
<212>DNA
<213> artificial sequence
<400>38
cccaactactgctttgctgg20
<210>39
<211>16
<212>DNA
<213> artificial sequence
<400>39
ctcaatagtgtaccgc16
<210>40
<211>24
<212>DNA
<213> artificial sequence
<400>40
cagttggaggaatctgtcgatgcc24
<210>41
<211>24
<212>DNA
<213> artificial sequence
<400>41
tcgaccatcatgaccgactacaac24
<210>42
<211>17
<212>DNA
<213> artificial sequence
<400>42
cacaagtgtaccgccgg17
<210>43
<211>21
<212>DNA
<213> artificial sequence
<400>43
ggccagaagagggcattctgc21
<210>44
<211>21
<212>DNA
<213> artificial sequence
<400>44
caaccccaactactgctttgc21
<210>45
<211>18
<212>DNA
<213> artificial sequence
<400>45
caccaggagctgcaagcc18
<210>46
<211>19
<212>DNA
<213> artificial sequence
<400>46
tgttatgtcagcgtttggc19
<210>47
<211>18
<212>DNA
<213> artificial sequence
<400>47
gcaagctccgcacctcga18
<210>48
<211>18
<212>DNA
<213> artificial sequence
<400>48
cagtgtaccgccggaagc18
<210>49
<211>18
<212>DNA
<213> artificial sequence
<400>49
ctggcattgccgacagga18
<210>50
<211>19
<212>DNA
<213> artificial sequence
<400>50
aggagcaatgatcttgatc19
<210>51
<211>26
<212>DNA
<213> artificial sequence
<400>51
aaggagatcactgccctggcacccag26
Claims (10)
1. mankind's ALK fusion gene detects primer sets, comprises the detection primer of 16 kinds of ALK fusion gene types of following A01 ~ A16 one or more groups:
A01:EML4-ALK-V1 hypotype (E13; A20) primer pair is detected: nucleotide sequence shown in SEQIDNO.1 ~ 2;
A02:EML4-ALK-V6 hypotype (E13; Ins69A20) primer pair is detected: nucleotide sequence shown in SEQIDNO.4 ~ 5;
A03:EML4-ALK-V2 hypotype (E20; A20) primer pair is detected: nucleotide sequence shown in SEQIDNO.7 ~ 8;
A04:EML4-ALK-V3a hypotype (E6a; A20) primer pair is detected: nucleotide sequence shown in SEQIDNO.10 ~ 11;
A05:EML4-ALK-V3b hypotype (E6b; A20) primer pair is detected: nucleotide sequence shown in SEQIDNO.13 ~ 14;
A06:EML4-ALK-V4 hypotype (E14; Ins11del49A20) primer pair is detected: nucleotide sequence shown in SEQIDNO.16 ~ 17;
A07:EML4-ALK-V7 hypotype (E14; Del12A20) primer pair is detected: nucleotide sequence shown in SEQIDNO.19 ~ 20;
A08:EML4-ALK-V5a hypotype (E2; A20) primer pair is detected: nucleotide sequence shown in SEQIDNO.22 ~ 23;
A09:EML4-ALK-V5b hypotype (E2; Ins117A20) primer pair is detected: nucleotide sequence shown in SEQIDNO.25 ~ 26;
A10:EML4-ALK-V8a hypotype (E17; A20) primer pair is detected: nucleotide sequence shown in SEQIDNO.28 ~ 29;
A11:EML4-ALK-V8b hypotype (E17; Ins68A20) primer pair is detected: nucleotide sequence shown in SEQIDNO.31 ~ 32;
A12:EML4-ALK-v4alt hypotype (E15del19; Del20A20) primer pair is detected: nucleotide sequence shown in SEQIDNO.34 ~ 35;
A13:EML4-ALK-v5alt hypotype (E18; A20) primer pair is detected: nucleotide sequence shown in SEQIDNO.37 ~ 38;
A14:KIF5B-ALK hypotype (K17; A20) primer pair is detected: nucleotide sequence shown in SEQIDNO.40 ~ 41;
A15:KIF5B-ALK hypotype (K24; A20) primer pair is detected: nucleotide sequence shown in SEQIDNO.43 ~ 44;
A16:TFG-ALK hypotype (T5; A20) primer pair is detected: nucleotide sequence shown in SEQIDNO.46 ~ 47.
2. the fluorescence RT-PCR of mankind's ALK fusion gene detects primer sets, it is characterized in that, comprises one or more groups of the detection primer of 16 kinds of ALK fusion gene types of A01 ~ A16 according to claim 1, and the following probe corresponding to this group gene type:
A01:EML4-ALK-V1 hypotype (E13; A20) detection probes: nucleotide sequence is as shown in SEQIDNO.3;
A02:EML4-ALK-V6 hypotype (E13; Ins69A20) detection probes: nucleotide sequence is as shown in SEQIDNO.6;
A03:EML4-ALK-V2 hypotype (E20; A20) detection probes: nucleotide sequence is as shown in SEQIDNO.9;
A04:EML4-ALK-V3a hypotype (E6a; A20) detection probes: nucleotide sequence is as shown in SEQIDNO.12;
A05:EML4-ALK-V3b hypotype (E6b; A20) detection probes: nucleotide sequence is as shown in SEQIDNO.15;
A06:EML4-ALK-V4 hypotype (E14; Ins11del49A20) detection probes: nucleotide sequence is as shown in SEQIDNO.18;
A07:EML4-ALK-V7 hypotype (E14; Del12A20) detection probes: nucleotide sequence is as shown in SEQIDNO.21;
A08:EML4-ALK-V5a hypotype (E2; A20) detection probes: nucleotide sequence is as shown in SEQIDNO.24;
A09:EML4-ALK-V5b hypotype (E2; Ins117A20) detection probes: nucleotide sequence is as shown in SEQIDNO.27;
A10:EML4-ALK-V8a hypotype (E17; A20) detection probes: nucleotide sequence is as shown in SEQIDNO.30;
A11:EML4-ALK-V8b hypotype (E17; Ins68A20) detection probes: nucleotide sequence is as shown in SEQIDNO.33;
A12:EML4-ALK-v4alt hypotype (E15del19; Del20A20) detection probes: nucleotide sequence is as shown in SEQIDNO.36;
A13:EML4-ALK-v5alt hypotype (E18; A20) detection probes: nucleotide sequence is as shown in SEQIDNO.39;
A14:KIF5B-ALK hypotype (K17; A20) detection probes: nucleotide sequence is as shown in SEQIDNO.42;
A15:KIF5B-ALK hypotype (K24; A20) detection probes: nucleotide sequence is as shown in SEQIDNO.45;
A16:TFG-ALK hypotype (T5; A20) detection probes: nucleotide sequence is as shown in SEQIDNO.48;
The nucleotide sequence 5 ' of detection probes holds mark fluorescent reporter group, 3 ' end mark fluorescent quenching group.
3. the fluorescence RT-PCR of mankind's ALK fusion gene according to claim 2 detects primer sets, and it is characterized in that, described fluorescent reporter group is FAM, and fluorescent quenching group is MGB.
4. a fluorescent RT-PCR detection reagent box for mankind's ALK fusion gene, is characterized in that, the fluorescence RT-PCR comprising the mankind's ALK fusion gene described in Claims 2 or 3 detects primer sets.
5. the fluorescent RT-PCR detection reagent box of mankind's ALK fusion gene according to claim 4, it is characterized in that, also comprise the internal control gene primer pair of nucleotide sequence as SEQIDNO.49 ~ 50, with the internal control gene probe of nucleotide sequence as shown in SEQIDNO.51, probe nucleotide sequence 5 ' holds mark fluorescent reporter group JOE, 3 ' end mark fluorescent quenching group BHQ1.
6. the fluorescent RT-PCR detection reagent box of mankind's ALK fusion gene according to claim 5; it is characterized in that; also comprise one or more groups the positive reference substance in 16 kinds of ALK fusion gene types of A01 ~ A16, positive reference substance is one section of specific base sequence of this ALK fusion gene type and the plasmon DNA of house-keeping gene ACTB mono-section of base sequence.
7. the fluorescent RT-PCR detection reagent box of the mankind's ALK fusion gene according to claim 5 or 6, is characterized in that, comprise in test kit:
The detection reagent of different genes type: the detection primer of each ALK fusion gene type and probe are in a reaction system, also containing internal control gene test primer and probe in the reaction system of this gene type, and PCR damping fluid, form the detection reagent of this gene type of pipe;
One pipe contains the enzyme reagent of ThermoScript II and DNA polymerase;
Have positive reference substance, also have a pipe positive reference substance mixed solution, positive control mixed solution includes the positive reference substance of this kind ALK fusion gene type corresponding to detection primer gene types all in this test kit.
8. the fluorescent RT-PCR detection reagent box of mankind's ALK fusion gene according to claim 7, it is characterized in that, detection primer and the probe of whole 16 kinds of ALK fusion gene types is comprised in test kit, the detection primer of often kind of gene type and probe, together with internal control primer and probe and the independent packaging of PCR damping fluid in a pipe.
9. the using method of the fluorescent RT-PCR detection reagent box of the arbitrary described mankind's ALK fusion gene of claim 4 ~ 8, it is characterized in that, measuring samples is extracted RNA, or after extracting DNA, join respectively in the detection reaction system of various ALK fusion gene type, carry out quantitative fluorescent PCR reaction, collect reaction signal, JOE signal should reach setting threshold value Ct value and be less than 26, otherwise the failure of an experiment; Detecting JOE signal meets after Ct value is less than 26, carries out following judgement:
FAM signal Ct value > 30 or without Ct value in often kind of detection system, shows that this sample feminine gender does not exist ALK fusion gene;
Wherein FAM signal Ct value≤30 in certain detection system, show the ALK fusion gene containing this type in this measuring samples.
10. the using method of the fluorescent RT-PCR detection reagent box of mankind's ALK fusion gene according to claim 9, is characterized in that, quantitative fluorescent PCR response procedures is: 45 DEG C of reaction 5min, 1 circulation; 95 DEG C of reaction 5min, 1 circulation; 95 DEG C of reaction 30s, 58 DEG C of reactions 45s, totally 10 circulations; 95 DEG C of reaction 15s, 58 DEG C of reactions 45s, totally 35 circulations, collect signal 58 DEG C time.
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|---|---|---|---|---|
| CN105671176A (en) * | 2016-03-16 | 2016-06-15 | 北京迈基诺基因科技有限责任公司 | Probe set and kit for detecting ALK fusion breaking point |
| CN106636376A (en) * | 2016-12-05 | 2017-05-10 | 武汉海吉力生物科技有限公司 | Nucleic acid and kit for detecting human ROS1 fusion genes and application method of kit |
| 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 |
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| CN108728538A (en) * | 2018-05-31 | 2018-11-02 | 广州迈景基因医学科技有限公司 | ALK gene fusion detection primer, method and kit |
| 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 |
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| CN110982903A (en) * | 2019-12-31 | 2020-04-10 | 中山大学达安基因股份有限公司 | Kit and method for multiplex detection of ALK gene mutation |
| CN111424088A (en) * | 2020-04-14 | 2020-07-17 | 天津普瑞赛斯分子诊断技术有限责任公司 | Kit and detection method for detecting fusion mutation of 12A L K genes |
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103468813A (en) * | 2013-09-17 | 2013-12-25 | 广州达健生物科技有限公司 | EML4-ALK (Echinoderm microtubule associated protein like4-anaplastic lymphoma kinase) fusion gene fluorescent quantitative PCR (polymerase chain reaction) assay kit |
| CN103805684A (en) * | 2012-11-09 | 2014-05-21 | 益善生物技术股份有限公司 | PCR primer, kit and liquid phase chip for EML4-ALK fusion gene detection |
-
2015
- 2015-09-06 CN CN201510560973.5A patent/CN105039580A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103805684A (en) * | 2012-11-09 | 2014-05-21 | 益善生物技术股份有限公司 | PCR primer, kit and liquid phase chip for EML4-ALK fusion gene detection |
| CN103468813A (en) * | 2013-09-17 | 2013-12-25 | 广州达健生物科技有限公司 | EML4-ALK (Echinoderm microtubule associated protein like4-anaplastic lymphoma kinase) fusion gene fluorescent quantitative PCR (polymerase chain reaction) assay kit |
Non-Patent Citations (2)
| Title |
|---|
| MARUJA E. LIRA等: "a single-tube multiplexed assay for detecting ALK, ROS1, and RET fusions in lung cancer", 《THE JOURNAL OF MOLECULAR DIAGNOSTICS》 * |
| SHA FU等: "detection of EML4-ALK fusion gene in chinese non-small cell lung cancer by using a sensitive quantitative real-time reverse transcriptase PCR technique", 《APPL IMMUNOHISTOCHEM MOL MORPHOL》 * |
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| 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 |
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