CN102925559B - Kit for quantitatively detecting W515 site mutation of MPL genes - Google Patents
Kit for quantitatively detecting W515 site mutation of MPL genes Download PDFInfo
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Abstract
The invention relates to a kit for quantitatively detecting the W515 site mutation of MPL genes, belonging to the field of biotechnology. The kit comprises at least one system of the W515L site mutation specific system of the MPL genes and the W515K site mutation specific system of the MPL genes, and an inner reference gene ABL system, wherein each system comprises an upstream primer, a downstream primer and a Taqman fluorescent probe. Research shows that the gain-of-function mutation rates of the W515L site mutation of the MPL genes and the W515K site mutation of the MPL genes are 10% and 3% in PMF and ET respectively, and are not found in PV. Therefore, the W515L site mutation of the MPL genes and the W515K site mutation of the MPL genes are mainly used for diagnosing the PMF and the ET. With the adoption of the fluorescent quantitative PCR (polymerase chain reaction) which is high in sensitivity and specificity for detecting the W515L site mutation of the MPL genes and the W515K site mutation of the MPL genes, the specificity and the sensitivity of the detection result are remarkably improved. Via the kit disclosed by the invention, a novel rapid, simple and convenient gene diagnosis technology is provided for prediction for myeloproliferative diseases.
Description
Technical field
The present invention relates to the fluorescent quantitative PCR technique of biological technical field, be specifically related to a kind of test kit of detection by quantitative MPL gene W515 site mutation.
Background technology
Myeloproliferative diseases (myeloproliferative diseases, MPD) is, after myeloid tissue persistent anomaly is bred, to cause red corpuscle and hyperthrombocytemia, and the disease of myelofibrosis problem.According to WHO(World Health Organization in 2008, the World Health Organization) revision, change myeloproliferative diseases into bone marrow proliferative tumour (myeloproliferative, MPN).MPN comprises polycythemia vera (Polycythernia Vera, PV), primary thrombocytosis (Essential Thrombocytosis, and PMF (primary myelofibrosis ET), PMF), they are all diseases of hemocyte hyper-proliferative, if treatment not in time finally can develop into acute myeloblastic leukemia.Therefore, Rapid&Early diagnosis has developed key effect to patient disease's generation.
WHO in 2008 revision MPN Case definition according to being that most of patients all has JAK2 transgenation, be mainly that JAK2 gene V617F and JAK2 gene extron 12 suddenly change.Research shows that JAK2 gene V617F is the sensitive indicator that is diagnosed as PV, also sees in 50% ET and PMF.But can not get rid of JAK2 gene V617F feminine gender and but suffer from the possibility of MPN.In the MPN patient of JAK2V617F feminine gender, find subsequently thrombopoietin acceptor MPL transgenation, comprise MPL gene W515L site mutation and W515K site mutation.This gene is myeloproliferative leukemia virus homology oncogene, belong to the super family of erythropoietin, can impel thrombopoietin part (Thromboietin, TPO) whole body hematopoiesis and megakaryocyte growth and differentiation, this gene is positioned at karyomit(e) 1q34 and comprises 12 exons.
2006, human body MPL gene W515L site mutation was to be found in the PMF of JAK2 gene V617F feminine gender, and sudden change is arranged in exon10, is transformed into T at No. 1544 G of Nucleotide place, causes transmembrane region 515 codons to have tryptophane to be transformed into leucine.After this, find that again changing AA at 1543 and 1544 place's Nucleotide into by TG is MPL gene W515K site mutation.Although find again afterwards other sudden change in exon10, only have the sudden change in MPL gene W515 site can induce the function of imitating JAK2 gene V617F, develop into MPN.
The gain-of-function mutation rate in MPL gene W515L site and MPL gene W515K site is respectively 10% and 3% in PMF and ET, and never occurs in PV.Therefore, MPL gene W515L sudden change and W515K site mutation are mainly used in diagnosing PMF and ET.In the patient of JAK2 gene V617F feminine gender, detect the diagnosis that MPL gene W515L sudden change and W515K site mutation contribute to MPN.Use allele-specific quantitative fluorescent PCR (Polymerase Chain Reaction, polymerase chain reaction) detection technique, its susceptibility is significantly higher than direct Sequencing.Real-Time Fluorescent Quantitative PCR Technique realized PCR from qualitative to real meaning determine quantum leap, for the detection by quantitative of Human disease gene provides an effective testing tool, have compared with regular-PCR that specificity strengthens, sensitivity improves and detect fast, reduced the features such as pollutions, but not yet having the relevant report of real time fluorescence quantifying PCR method detection MPL gene W515 site mutation test kit at present.
Summary of the invention
In order to solve the problem of prior art, the embodiment of the present invention provides the test kit of a kind of MPL of detection gene W515L site mutation and MPL gene W515K site mutation.Described technical scheme is as follows:
The embodiment of the present invention provides a kind of test kit of the MPL of detection gene W515 site mutation, comprise and detecting with primer, fluorescent probe, quantitative fluorescent PCR mixed solution, negative control and positive control, described for detection primer, fluorescent probe comprise at least one and reference gene ABL primer and the Taqman fluorescent probe in MPL gene W515L site mutation Auele Specific Primer and MPL gene W515K site mutation Auele Specific Primer
described test kit also comprises: inner positive control sequence, inner positive the Taqman fluorescent probe of the primer of control sequence and inner positive control sequence,wherein:
MPL gene W515L site mutation specificity upstream primer sequence is as shown in SEQ ID NO:1 in sequence table;
MPL gene W515L site mutation specificity downstream primer sequence is as shown in SEQ ID NO:2 in sequence table;
MPL gene W515L site mutation specificity T aqman fluorescent probe is as shown in SEQ ID NO:3 in sequence table;
MPL gene W515K site mutation specificity upstream primer sequence is as shown in SEQ ID NO:4 in sequence table;
MPL gene W515K site mutation specificity downstream primer sequence is as shown in SEQ ID NO:5 in sequence table;
MPL gene W515K site mutation specificity T aqman fluorescent probe is as shown in SEQ ID NO:6 in sequence table;
Abl gene upstream primer sequence is as shown in SEQ ID NO:7 in sequence table;
Abl gene downstream primer sequence is as shown in SEQ ID NO:8 in sequence table;
Abl gene Taqman fluorescent probe is as shown in SEQ ID NO:9 in sequence table;
Inner positive control sequence is as shown in SEQ ID NO:10 in sequence table;
The upstream primer of inner positive control sequence is as shown in SEQ ID NO:11 in sequence table;
The downstream primer of inner positive control sequence is as shown in SEQ ID NO:12 in sequence table;
The Taqman fluorescent probe of inner positive control sequence is as shown in SEQ ID NO:13 in sequence table.
Further, 5 ' end of the Taqman fluorescent probe of described MPL gene W515L site mutation specificity T aqman fluorescent probe, MPL gene W515K site mutation specificity T aqman fluorescent probe and abl gene is connected with fluorescence report group FAM, and 3 ' end is all connected with fluorescent quenching group TAMRA; 5 ' end of the positive control sequence Taqman fluorescent probe in described inside is connected with fluorescence report group TET, and 3 ' end is connected with fluorescent quenching group TAMRA.
Particularly, the nucleotide sequence of described reference gene ABL is as shown in SEQ ID NO:14 in sequence table.
Particularly, described negative control is deionized water; Described positive control is the genome DNA sample that contains MPL gene W515L site mutation and MPL gene W515K site mutation.
Particularly, the mixed solution of described quantitative fluorescent PCR (representing taking reaction system final concentration) as: 1 × PCR premix (stoste is 2 × PCR Premix), the Mg of 2.5-4.0mM
2+, the Taq enzyme of dUTP, 0.2U/ μ L of dNTPs, 0.3-0.6mM of 0.2-0.4mM and the UNG enzyme of 0.01-0.05U/ μ L and without RNase deionized water.
Advantage and the effect of test kit of the present invention are as follows:
(1) susceptibility is high: can repeat susceptibility is 0.01%, in 10000 cells, has one just can be detected containing MPL gene W515L site mutation and MPL gene W515K site mutation.
(2) high specificity: use specific probe to identify quantitative molecular, accuracy is high.Meanwhile, target sequence is by primer and the dual control of probe, and specificity is good, false positive is low.
(3) handy and safe: simple to operate, safety, level of automation is high and prevent from polluting.Amplification and detection can detect in same pipe, do not need to uncap, and are difficult for contaminated; Increasing simultaneously and detecting a step completes, and does not need post-processed, need not worry radiocontamination.
(4) complete monitoring: the test kit that the embodiment of the present invention provides has been introduced the inner positive quality control system of controlling, and testing process is carried out to Complete Quality Supervision, effectively avoids false positive or false negative.
(5) quick: speed is fast, high-throughput, can complete at 3-4 hour.
Test kit of the present invention can be fast, accurately, detection by quantitative MPL gene W515L site mutation and MPL gene W515K site mutation level, false positive and false-negative generation are effectively stopped, for the diagnosis of bone marrow proliferative tumour and the monitoring of therapeutic process minimal residual disease, for diagnosis, formulation treatment plan and therapeutic evaluation and the prognosis of bone marrow proliferative tumour provide important detection means.
Brief description of the drawings
In order to be illustrated more clearly in the technical scheme in the embodiment of the present invention, below the accompanying drawing of required use during embodiment is described is briefly described, apparently, accompanying drawing in the following describes is only some embodiments of the present invention, for those of ordinary skill in the art, do not paying under the prerequisite of creative work, can also obtain according to these accompanying drawings other accompanying drawing.
Fig. 1 is the fluorescence curve figure of the bone marrow proliferative tumour patient of the fluorescence real-time quantitative PCR reaction system amplification MPL gene W515L site mutation positive of the test kit that provides of the embodiment of the present invention 2, and the fluorescence curve figure of the fluorescence curve figure of ABL standard substance amplification and inner positive control sequence standard substance amplification;
Fig. 2 is the fluorescence curve figure of the bone marrow proliferative tumour patient of the fluorescence real-time quantitative PCR reaction system amplification MPL gene W515K site mutation positive of the test kit that provides of the embodiment of the present invention 2, and the fluorescence curve figure of the fluorescence curve figure of ABL standard substance amplification and inner positive control sequence standard substance amplification.
In figure: 1-MPL gene W515L site mutation amplification curve, 2-ABL standard substance amplification curve, the inner positive control product amplification curve of 3-, 4-MPL gene W515K site mutation amplification curve.
Embodiment
For making the object, technical solutions and advantages of the present invention clearer, below in conjunction with accompanying drawing, embodiment of the present invention is described further in detail.
The preparation of embodiment 1. test kits
1, the design of specific primer and fluorescent probe
According to gene order, (abl gene sequence and MPL gene order derive from American National biotechnology information center nucleic acid database, and wherein abl gene ID is respectively 25, and reference sequences number is NM_005157.4; MPL gene I/D is respectively 4352, and reference sequences number is NG_007525.1) design respectively primer and the fluorescent probe special to above-mentioned each gene order.
2, according to each component of the composition reagent preparation box of following test kit
Test kit of the present invention is composed as follows:
1. extracting genome DNA reagent: adopt tissue gene group DNA extraction test kit (Qiagen company, article No.: 69504) rapid extraction 0.5ml bone marrow proliferative tumour patient myeloid tissue genomic dna.
2. primer, probe and standard substance: comprise MPL gene W515L site mutation Auele Specific Primer, MPL gene W515K site mutation Auele Specific Primer, inner positive control sequence sequence, inner positive control sequence primer and reference gene ABL primer, and the Taqman fluorescent probe of answering with primer pair, specific as follows:
MPL gene W515L site mutation specificity upstream primer: 5 '-GAAGTCTGACCCTTTTTGTCTCCTA-3 ' (sequence 1 in sequence table);
MPL gene W515L site mutation specificity downstream primer: 5 '-CTGGTCCACCGCCAGTCT-3 ' (sequence 2 in sequence table);
MPL gene W515L site mutation specificity T aqman fluorescent probe: sequence 3 in FAM5 '-CTGCTGAGGTTGC-3 ' TAMRA(sequence table);
MPL gene W515K site mutation specificity upstream primer: 5 '-TTTTTGTCTCCTAGCCTGGATCTC-3 ' (sequence 4 in sequence table);
MPL gene W515K site mutation specificity downstream primer: 5 '-GTCACAGAGCGAACCAAGAATG-3 ' (sequence 5 in sequence table);
MPL gene W515K site mutation specificity T aqman fluorescent probe: sequence 6 in FAM5 '-CTGCTGAGGAAGCA-3 ' TAMRA(sequence table);
Inner positive control sequence sequence is: 5 '-GAAGTCTGACCCTTTTTGTCTCCATGCCTGGATCTCCTTGGTGACCGCTCTGCATC TAGTGCTGGGCCTCAGCGCCGTCCTGGGCCTGCTGCTGCTGAGGTGGCAGTTTCCT GCACACTACAGGTACCGCCCCCGCCAGGCAGGACAGTGGCGGTGGACCAG-3 ' (sequence 10 in sequence table);
Inner positive control sequence upstream primer sequence is: 5 '-GAAGTCTGACCCTTTTTGTCTCCAT-3 ' (sequence 11 in sequence table);
Inner positive control sequence downstream primer sequence is: 5 '-CTGGTCCACCGCCACTGT-3 ' (sequence 12 in sequence table);
Inner positive control sequence Taqman fluorescent probe: sequence 13 in TET5 '-CTGCTGAGGTGGCA-3 ' TAMRA(sequence table);
Abl gene sequence is: 5 '-CAGGGTCTGAGTGAAGCCGCTCGTTGGAACTCCAAGGAAAACCTTCTCGCTGGACC CAGTGAAAATGACCCCAACCTTTTCGTTGCACTGTATGATTTTGTGGCCAGTGGAG ATAACACTCTAAGCATAACTAAAGGTGAAAAGCTCCGGGTCTTAGGCTATAATCAC AATGGGGAATGGTGTGAAGCCCAAACCAAAAATGGCCAAGGCTGGGTCCCAAGCAA CTACATCACGCCAGTCAACAGTCTGGAGAAACACTCCTGGTACCATGGGCCTGTGT CCCGCAATGCCGCTGAGTATCTGCTGAGCAGCGGGATCAATGGCAGCTTCTTGGTG CGTGAGAGTGAGAGCAGTCCTGGC-3 ' (sequence 14 in sequence table);
Abl gene upstream primer sequence is: 5 '-CCGGGTCTTAGGCTATAATCACA-3 ' (sequence 7 in sequence table);
Abl gene downstream primer sequence is: 5 '-GCCTTGGCCATTTTTGGTT-3 ' (sequence 8 in sequence table);
Abl gene Taqman fluorescent probe: sequence 9 in FAM5 '-TGGTGTGAAGCCC-3 ' TAMRA(sequence table);
Wherein, inner positive control sequence is artificial synthesized sequence, comprise MPL gene W515K/L mutational site gene order and the artificial composition sequence of a part; Inner positive control sequence and abl gene sequence are used separately as standard substance.
Above-mentioned primer sequence, control sequence, gene order and Taqman fluorescent probe sequence are synthesized by Shanghai Sangon Biological Engineering Technology And Service Co., Ltd.
3. negative control and positive control: with the negative contrast of deionized water, to contain the positive contrast of genome DNA sample of MPL gene W515L site mutation and MPL gene W515K site mutation, adopt the test kit of the present invention providing in above-described embodiment 1 to form 1. extracting genome DNA reagent, the 0.5ml myeloid tissue DNA of the bone marrow proliferative tumour patient that what rapid extraction had been made a definite diagnosis contain MPL gene W515L site mutation, as positive control.
4. MPL gene W515L site mutation fluorescence quantitative PCR reaction solution: by quantitative fluorescent PCR mixed solution and detect MPL gene W515L site mutation specific primer, probe forms: 1 × PCR premix (Qiagen company, product article No.: 210212,2 × PCR Premix), the Mg of 2.5-4.0mM
2+, the dNTPs of 0.2-0.4mM and the dUTP of 0.3-0.6mM, the UNG enzyme of the Taq enzyme of 0.2U/ μ L and 0.01-0.05U/ μ L, in the MPL gene W515L site mutation specificity of 0.25pmol/ μ L, downstream primer (sequence 1 in sequence table, 2) the MPL gene W515L site mutation specific probe (sequence 3 in sequence table) of 0.3pmol/uL, the positive control sequence upstream primer in inside (sequence 11 in sequence table) of 0.25pmol/ μ L, the positive control sequence downstream primer in inside (sequence 12 in sequence table) of 0.25pmol/ μ L, the positive control sequence Taqman fluorescent probe in inside (sequence 13 in sequence table) (above all concentration all refers to the final concentration of PCR reaction system) of 0.3pmol/ μ L.The template (comprising sample DNA and inner positive control sequence DNA) of conventionally getting 1-2 μ L, all the other are without RNase deionized water, reaction cumulative volume is generally 20 μ L.
5. MPL gene W515K site mutation fluorescence quantitative PCR reaction solution: by quantitative fluorescent PCR mixed solution and detect MPL gene W515L site mutation specific primer, probe forms: 1 × PCR premix (Qiagen company, product article No.: 210212,2 × PCR Premix), the Mg of 2.5-4.0mM
2+, the dNTPs of 0.2-0.4mM and the dUTP of 0.3-0.6mM, the UNG enzyme of the Taq enzyme of 0.2U/ μ L and 0.01-0.05U/ μ L, in the MPL gene W515K site mutation specificity of 0.25pmol/ μ L, downstream primer (sequence 4 in sequence table, 5) the MPL gene W515K site mutation specific probe (sequence 6 in sequence table) of 0.3pmol/uL, the positive control sequence upstream primer in inside (sequence 11 in sequence table) of 0.25pmol/ μ L, the positive control sequence downstream primer in inside (sequence 12 in sequence table) of 0.25pmol/ μ L, the positive control sequence Taqman fluorescent probe in inside (sequence 13 in sequence table) (above all concentration all refers to the final concentration of PCR reaction system) of 0.3pmol/ μ L.The template (comprising sample DNA and inner positive control sequence DNA) of conventionally getting 1-2 μ L, all the other are without RNase deionized water, reaction cumulative volume is generally 20 μ L.
6. ABL reference gene fluorescence quantitative PCR reaction solution: formed by quantitative fluorescent PCR mixed solution and the primer, the probe that detect ABL reference gene: 1 × PCR premix (Qiagen company, product article No.: 210212,2 × PCR Premix), the Mg of 2.5-4.0mM
2+, the Taq enzyme of dUTP, 0.2U/ μ L of dNTPs, 0.3-0.6mM of 0.2-0.4mM and the UNG enzyme of 0.01-0.05U/ μ L, the ABL reference gene upstream primer (sequence 7 in sequence table) of 0.25pmol/ μ L, the ABL reference gene downstream primer (sequence 8 in sequence table) of 0.25pmol/ μ L, the abl gene Taqman fluorescent probe (sequence 9 in sequence table) (above all concentration all refers to the final concentration of PCR reaction system) of 0.3pmol/ μ L.When detection, add abl gene standard substance template 2 μ L, reaction cumulative volume is generally 20 μ L.
7. inner positive control sequence fluorescence quantitative PCR reaction solution: by quantitative fluorescent PCR mixed solution with detect primer, the probe of inner positive control sequence and form: 1 × PCR premix (Qiagen company, product article No.: 210212,2 × PCR Premix), the Mg of 2.5-4.0mM
2+, the dNTPs of 0.2-0.4mM and the Taq enzyme of the dUTP of 0.3-0.6mM, 0.2U/ μ L and the UNG enzyme of 0.01-0.05U/ μ L, the positive control sequence upstream primer in inside (sequence 11 in sequence table) of 0.25pmol/ μ L, the positive control sequence downstream primer in inside (sequence 12 in sequence table) of 0.25pmol/ μ L, the positive control sequence Taqman fluorescent probe in inside (sequence 13 in sequence table) (above all concentration all refers to the final concentration of PCR reaction system) of 0.3pmol/ μ L.When detection, conventionally get the template (inner positive control sequence DNA) of 1-2 μ L, all the other are without RNase deionized water, and reaction cumulative volume is generally 20 μ L.
3, the setting of pcr amplification program: on lightcycler instrument first through 50 DEG C of 10s, 95 DEG C of 10min, and then through 95 DEG C of 15s, 60 DEG C of 1min, totally 40 circulations.
Test kit prepared by embodiment 2. use embodiment 1 detects MPL gene W515 site mutation
To detect 30 routine bone marrow proliferative tumour patient myeloid tissue sample results as example.
The testing process that detects MPL gene W515L site mutation and MPL gene W515K site mutation with test kit of the present invention is: first design specific primer and fluorescent probe according to gene order.Next obtains clinical bone marrow proliferative tumour patient myeloid tissue sample, rapid extraction tissue DNA; First prepare the fluorescence quantitative PCR reaction solution of ABL reference gene and inner positive control sequence, it is 1.0x10 that positive inside control sequence standard substance and ABL standard substance are diluted to respectively to copy number/mL
3, 1.0x10
4, 1.0x10
5and 1.0x10
6make respectively inner positive control sequence standard substance typical curve and ABL standard substance typical curve, and then the fluorescence quantitative PCR reaction solution of preparation MPL gene W515L site mutation and MPL gene W515K site mutation, carry out fluorescence quantitative PCR detection sample, in quantitative real time PCR Instrument data analysis system, read CT value result.After pcr amplification finishes, first analyze inner positive control sequence amplification, if its Ct value is less than 33, point out whole testing process effective, if its Ct value is greater than 35, prompting detects unsuccessfully, need to re-start detection, if its Ct value is between 33～35, need duplicate detection.In the time that the positive control sequence Ct value in inside is less than 33, real-time fluorescence quantitative PCR the data obtained is calculated, calculate respectively the Ct value of MPL W515L transgenation (as shown in Figure 1), MPL W515K transgenation (as shown in Figure 2) and abl gene, both differences are Δ Ct value.Finally, fluorescent quantitative PCR result adopts software analysis, and markization is calculated sampled data.
Concrete steps are as follows:
1. the extracting of bone marrow proliferative tumour patient myeloid tissue genomic dna: the method extracting bone marrow proliferative tumour patient myeloid tissue genomic dna of pressing DNA extracting and purifying.
2. the bone marrow proliferative tumour patient myeloid tissue genomic dna of said extracted is identified to its integrity through agarose gel electrophoresis, measure purity and the concentration of 260nm and 280nm optical density value calculating DNA by ultraviolet spectrophotometer, to same concentrations, put refrigerator-20 DEG C preservation with the DNA of aseptic deionized water adjusting extracting.
3. positive inside control sequence standard substance and ABL standard substance being diluted to respectively to copy number/mL is 1.0x10
3, 1.0x10
4, 1.0x10
5and 1.0x10
6, the positive control sequence in inside providing with embodiment 1 and the fluorescence quantitative PCR reaction solution of ABL reference gene, make respectively inner positive control sequence standard substance typical curve (as shown in Figure 1) and ABL standard substance typical curve (as shown in Figure 2).
4. MPL gene W515L site mutation fluorescent quantitative PCR: PCR reaction system is 20 μ L: containing 1 × PCR premix (Qiagen company, product article No.: 210212,2 × PCR Premix), 10.0 μ L, the Mg of 2.5-4.0mM
2+, the dNTPs of 0.2-0.4mM and the dUTP of 0.3-0.6mM, the UNG enzyme of the Taq enzyme of 0.2U/ μ L and 0.01-0.05U/ μ L, in MPL gene W515L site mutation specificity, downstream primer final concentration is 0.2 μ mol/L, MPL gene W515L site mutation specificity T aqman fluorescent probe final concentration 0.3 μ mol/L, the bone marrow proliferative tumour patient myeloid tissue genomic dna 2.0 μ L that are extracted, add in inner positive control sequence simultaneously, the final concentration of downstream primer is 0.2 μ mol/L, inner positive control sequence Taqman fluorescent probe concentration 0.3 μ mol/L, final concentration is the positive control sequence in the inside of 0.2 μ mol/L, add ultrapure water to cumulative volume 20 μ L.On lightcycler quantitative real time PCR Instrument, react: amplification condition: 95 DEG C of 10min denaturations, then 95 DEG C of 15s, 40 circulations of 60 DEG C of 1min amplifications, amplification procedure Instrumental is collected fluorescent signal automatically.
5. MPL gene W515K site mutation fluorescent quantitative PCR: PCR reaction system is 20 μ L: containing 1 × PCR premix (Qiagen company, product article No.: 210212,2 × PCR Premix), 10.0 μ L, the Mg of 2.5-4.0mM
2+, the dNTPs of 0.2-0.4mM and the dUTP of 0.3-0.6mM, the UNG enzyme of the Taq enzyme of 0.2U/ μ L and 0.01-0.05U/ μ L, in MPL gene W515K site mutation specificity, downstream primer final concentration is 0.2 μ mol/L, MPL gene W515K site mutation specificity T aqman fluorescent probe final concentration 0.3 μ mol/L, the bone marrow proliferative tumour patient myeloid tissue genomic dna 2.0 μ L that are extracted, add in inner positive control sequence simultaneously, the final concentration of downstream primer is 0.2 μ mol/L, inner positive control sequence Taqman fluorescent probe concentration 0.3 μ mol/L, final concentration is the positive control sequence in the inside of 0.2 μ mol/L, add ultrapure water to cumulative volume 20 μ L.On lightcycler quantitative real time PCR Instrument, react: amplification condition: 95 DEG C of 10min denaturations, then 95 DEG C of 15s, 40 circulations of 60 DEG C of 1min amplifications, amplification procedure Instrumental is collected fluorescent signal automatically.
6. ABL reference gene fluorescent quantitative PCR: PCR reaction system is 20 μ L: containing 2 × PCR mixed solution (Qiagen company, product article No.: 210212,2 × PCR Premix), 10.0 μ L, the Mg of 2.5-4.0mM
2+, the dNTPs of 0.2-0.4mM and the Taq enzyme of the dUTP of 0.3-0.6mM, 0.2U/ μ L and the UNG enzyme of 0.01-0.05U/ μ L, abl gene primer final concentration 0.2 μ mol/L, probe final concentration 0.2 μ mol/L, abl gene DNA1.0 μ L, adds without RNase deionized water and mends to cumulative volume 20 μ L.On lightcycler quantitative real time PCR Instrument, react: amplification condition is 95 DEG C of 10min denaturations, then 95 DEG C of 15s, 40 circulations of 60 DEG C of 1min amplifications, amplification procedure Instrumental is collected fluorescent signal automatically.
7. positive control, negative control fluorescent quantitative PCR: PCR reaction system is 20 μ L: containing 2 × PCR mixed solution (Qiagen company, product article No.: 210212,2 × PCR Premix), 10.0 μ L, the Mg of 2.5-4.0mM
2+, the dNTPs of 0.2-0.4mM and the Taq enzyme of the dUTP of 0.3-0.6mM, 0.2U/ μ L and the UNG enzyme of 0.01-0.05U/ μ L, MPL gene W515K site mutation specificity and the specific primer final concentration of MPL gene W515L site mutation are 0.2 μ mol/L, MPL gene W515K site mutation specificity and the specific Taqman fluorescent probe of MPL gene W515L site mutation final concentration are 0.3 μ mol/L, the DNA1.0 μ L of positive control or negative control deionized water 1.0 μ L, add without RNase deionized water and mend to cumulative volume 20 μ L.On lightcycler quantitative real time PCR Instrument, react: amplification condition is 95 DEG C of 10min denaturations, then 95 DEG C of 15s, 40 circulations of 60 DEG C of 1min amplifications, amplification procedure Instrumental is collected fluorescent signal automatically.
8. data collection process and analysis: after pcr amplification finishes, first analyze inner positive control sequence amplification, if its Ct value is less than 33, point out whole testing process effective, if its Ct value is greater than 35, need to re-start detection.In the time that the positive control sequence Ct value in inside is less than 33, real-time fluorescence quantitative PCR the data obtained is calculated, draw after MPL gene W515L site mutation (as shown in Figure 1) and MPL gene W515K site mutation (as shown in Figure 2) are with respect to the relative expression quantity of ABL reference gene and carry out again statistical study, be more than or equal to 0.0001 positive expression with ratio, be less than 0.0001 negative expression (specifically referring to table 1):
Table 1 is the expression of quantitative fluorescent PCR analyzing gene MPL gene W515 site mutation in bone marrow proliferative tumour
Numerical value in above-mentioned table in MPL gene W515 site mutation template and ABL template all represents fluorescence aggregate-value.
Test kit detectivity is evaluated:
With qualitative PCR detection method as a comparison, above-mentioned 30 routine bone marrow proliferative tumour patient myeloid tissue samples are detected simultaneously, comparative result shows, adopt this test kit of the present invention to detect its susceptibility, specificity and sensitivity more accurate compared with Immunohistochemical Method, meet current clinic diagnosis real requirement (specifically referring to table 2) completely:
Table 2 is the comparison that two kinds of different methods detect MPL gene W515 site mutation in bone marrow proliferative tumour patient
As seen from the above table, by qualitative PCR method inspection fluorescent quantitation, qualitative PCR method is as reference, fluorescent quantitation and qualitative PCR method detect that 14 examples are positive simultaneously, and qualitative PCR method has detected that 2 examples are negative, draw thus, the positive predictive value of fluorescent quantitation is 85.7%; Fluorescent quantitation and qualitative PCR method detect that 14 examples are negative simultaneously, all do not detect the positive, draw thus, and the negative predictive value of fluorescent quantitation is 100%.
Wherein:
1. specificity: 87.5%;
2. sensitivity: 100%;
3. positive predictive value: positive predictive value reaches 87.5%;
4. negative predictive value: negative predictive value reaches 100%;
5. repeatability: repeatedly repeat experimental result consistent;
6. consuming time: be about 4h the detection time of a clinical samples, consuming time short, and Immunohistochemical Method about 72h consuming time.
Above-mentioned experiment can illustrate, adopt all higher fluorescence quantitative PCR detection MPL gene W515L site mutation and MPL gene W515K site mutation expression levels of susceptibility and specificity, adopt artificial design and the positive control sequence in synthetic inside, the whole process that monitoring leukemia MPL gene W515L site mutation and MPL gene W515K site mutation real-time fluorescence quantitative PCR detect, can effectively solve the false positive in current leukemia fusion gene MPL gene W515L site mutation and MPL gene W515K site mutation real-time fluorescence quantitative PCR testing process, Problem of False Negative, make detected result more reliable, specificity and the susceptibility of its detected result are significantly increased, this test kit is diagnosis and the somatotype of myeloproliferative diseases, recurrence monitoring provides a kind of brand-new fast and convenient gene diagnosis technology.
The foregoing is only preferred embodiment of the present invention, in order to limit the present invention, within the spirit and principles in the present invention not all, any amendment of doing, be equal to replacement, improvement etc., within all should being included in protection scope of the present invention.
Sequence table
<160>14
<210>1
<211>25
<212>DNA
<213> artificial sequence
<400>1
gaagtctgac cctttttgtc tccta 25
<210>2
<211>18
<212>DNA
<213> artificial sequence
<400>2
ctggtccacc gccagtct 18
<210>3
<211>13
<212>DNA
<213> artificial sequence
<400>3
ctgctgaggt tgc 13
<210>4
<211>24
<212>DNA
<213> artificial sequence
<400>4
tttttgtctc ctagcctgga tctc 24
<210>5
<211>22
<212>DNA
<213> artificial sequence
<400>5
gtcacagagc gaaccaagaa tg 22
<210>6
<211>14
<212>DNA
<213> artificial sequence
<400>6
ctgctgagga agca 14
<210>7
<211>23
<212>DNA
<213> artificial sequence
<400>7
ccgggtctta ggctataatc aca 23
<210>8
<211>19
<212>DNA
<213> artificial sequence
<400>8
gccttggcca tttttggtt 19
<210>9
<211>13
<212>DNA
<213> artificial sequence
<400>9
tggtgtgaag ccc 13
<210>10
<211>162
<212>DNA
<213> artificial sequence
<400>10
gaagtctgac cctttttgtc tccatgcctg gatctccttg gtgaccgctc tgcatctagt 60
gctgggcctc agcgccgtcc tgggcctgct gctgctgagg tggcagtttc ctgcacacta 120
caggtaccgc ccccgccagg caggacagtg gcggtggacc ag 162
<210>11
<211>25
<212>DNA
<213> artificial sequence
<400>11
gaagtctgac cctttttgtc tccat 25
<210>12
<211>18
<212>DNA
<213> artificial sequence
<400>12
ctggtccacc gccactgt 18
<210>13
<211>14
<212>DNA
<213> artificial sequence
<400>13
ctgctgaggt ggca 14
<210>14
<211>360
<212>DNA
<213> artificial sequence
<400>14
cagggtctga gtgaagccgc tcgttggaac tccaaggaaa accttctcgc tggacccagt 60
gaaaatgacc ccaacctttt cgttgcactg tatgattttg tggccagtgg agataacact 120
ctaagcataa ctaaaggtga aaagctccgg gtcttaggct ataatcacaa tggggaatgg 180
tgtgaagccc aaaccaaaaa tggccaaggc tgggtcccaa gcaactacat cacgccagtc 240
aacagtctgg agaaacactc ctggtaccat gggcctgtgt cccgcaatgc cgctgagtat 300
ctgctgagca gcgggatcaa tggcagcttc ttggtgcgtg agagtgagag cagtcctggc 360
Claims (5)
1. one kind is detected the test kit of MPL gene W515 site mutation, comprise detection primer, fluorescent probe, quantitative fluorescent PCR mixed solution, negative control and positive control, it is characterized in that, described detection primer, fluorescent probe comprises at least one and reference gene ABL primer and Taqman fluorescent probe in MPL gene W515L site mutation Auele Specific Primer and MPL gene W515K site mutation Auele Specific Primer, described test kit also comprises: inner positive control sequence, the inner primer of positive control sequence and the Taqman fluorescent probe of inner positive control sequence, wherein:
MPL gene W515L site mutation specificity upstream primer sequence is as shown in SEQ ID NO:1 in sequence table;
MPL gene W515L site mutation specificity downstream primer sequence is as shown in SEQ ID NO:2 in sequence table;
MPL gene W515L site mutation specificity T aqman fluorescent probe is as shown in SEQ ID NO:3 in sequence table;
MPL gene W515K site mutation specificity upstream primer sequence is as shown in SEQ ID NO:4 in sequence table;
MPL gene W515K site mutation specificity downstream primer sequence is as shown in SEQ ID NO:5 in sequence table;
MPL gene W515K site mutation specificity T aqman fluorescent probe is as shown in SEQ ID NO:6 in sequence table;
Abl gene upstream primer sequence is as shown in SEQ ID NO:7 in sequence table;
Abl gene downstream primer sequence is as shown in SEQ ID NO:8 in sequence table;
Abl gene Taqman fluorescent probe is as shown in SEQ ID NO:9 in sequence table;
Inner positive control sequence is as shown in SEQ ID NO:10 in sequence table;
The upstream primer of inner positive control sequence is as shown in SEQ ID NO:11 in sequence table;
The downstream primer of inner positive control sequence is as shown in SEQ ID NO:12 in sequence table;
The Taqman fluorescent probe of inner positive control sequence is as shown in SEQ ID NO:13 in sequence table.
2. test kit according to claim 1, it is characterized in that, 5 ' end of the Taqman fluorescent probe of described MPL gene W515L site mutation specificity T aqman fluorescent probe, the specific Taqman fluorescent probe of MPL gene W515K site mutation and abl gene is connected with fluorescence report group FAM, and 3 ' end is all connected with fluorescent quenching group TAMRA; 5 ' end of the Taqman fluorescent probe of the positive control sequence in described inside is connected with fluorescence report group TET, and 3 ' end is connected with fluorescent quenching group TAMRA.
3. test kit according to claim 1, is characterized in that, the nucleotide sequence of described reference gene ABL is as shown in SEQ ID NO:14 in sequence table.
4. test kit according to claim 1, is characterized in that, described negative control is deionized water; Described positive control is the genome DNA sample that contains MPL gene W515L site mutation and MPL gene W515K site mutation.
5. test kit according to claim 1, is characterized in that, the mixed solution of described quantitative fluorescent PCR is: 1 × PCR premix, the Mg of 2.5-4.0mM
2+, 0.2-0.4mM dNTPs, 0.3-0.6mM dUTP, 0.2U/ μ L Taq enzyme, 0.01-0.05U/ μ L UNG enzyme and without RNase deionized water.
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| CN103757100A (en) * | 2013-12-20 | 2014-04-30 | 郑州艾迪康医学检验所（普通合伙） | Method and primer for detecting mutation type of 515th locus of MPL (myeloproliferative leukemia) gene |
| CN105154545A (en) * | 2015-09-09 | 2015-12-16 | 广州金域医学检验中心有限公司 | Primers and method for detecting MPL gene mutation |
| CN112941171A (en) * | 2021-03-30 | 2021-06-11 | 迈杰转化医学研究(苏州)有限公司 | Primer probe for detecting mutations of W515L, W515S and W515A of MPL gene and application thereof |
| CN114525339A (en) * | 2022-02-11 | 2022-05-24 | 求臻医学科技（北京）有限公司 | Specific probe and detection method for MPL gene mutation detection |
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Effective date of registration: 20160615 Address after: 510663 Guangdong city of Guangzhou province high tech Industrial Development Zone of Guangzhou city science and technology innovation base on Road No. 80 C District second layer 204-206, 207-211 unit, D unit area sixth 615-617 Patentee after: Guangzhou Bao Bao Biotechnology Co., Ltd. Address before: 430060 No. 99 Zhang Zhidong Road, Wuchang District, Hubei, Wuhan Patentee before: Tong Yongqing Patentee before: Li Yan |