CN108823310A - It is a kind of detect DPEP1 gene expression dose reagent set and its application - Google Patents
It is a kind of detect DPEP1 gene expression dose reagent set and its application Download PDFInfo
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Abstract
The invention discloses a kind of reagent set for detecting DPEP1 gene expression dose and its applications.Reagent set includes primer pair DPEP1 and probe DPEP1-probe；Specific DNA fragment shown in sequence 7 of the target sequence of the primer pair DPEP1 containing ordered list；The probe DPEP1-probe is the single strand dna of 20-30 nucleotide composition, identical or complementary as the partial sector in the specific DNA fragment first.Using the expression of reagent set detection DPEP1 gene, and then generation and/or the disease progression of the therapeutic effect for the risk, auxiliary prediction acute lymphoblastic leukemia for assisting identification acute lymphoblastic leukemia, auxiliary to predict that object to be measured suffers from acute lymphoblastic leukemia and auxiliary prediction acute lymphoblastic leukemia.The present invention has important application value.
Description
Technical field
The invention belongs to field of biotechnology, and in particular to it is a kind of detect DPEP1 gene expression dose reagent set and
It is applied.
Background technique
DPEP1 (Dipeptidase 1) is a kind of zinc metallopetidase on people's 16q24.3 chromosome, also referred to as
Film dipeptidase, microsomal dipeptidase or kidney dipeptidase are a kind of zinc gold that can be hydrolyzed a variety of dipeptides and participate in glutathione metabolism
Belong to peptase, plays an important role in the regulation of glutathione and the relevant pro-inflammatory molecular leukotriene of tumour.
Studies have shown that DPEP1 abnormal expression in kinds of tumors, but its expression and prognosis meaning are different types of
Dispute is still had in tumour, the expression deletion of DPEP1 in Wilms tumour, the expression of DPEP1 in breast cancer and ductal adenocarcinoma of pancreas
It reduces, the expression of DPEP1 is increased in colon cancer, and the high expression of DPEP1 is related to poor prognosis.It is swollen in colon cancer, Wilms
In tumor, breast cancer and ductal adenocarcinoma of pancreas patient, the unconventionality expression of DPEP1 affects tumour cell to the sensibility of chemotherapeutics
And invasiveness ability, and have substantial connection with the prognosis of tumour patient.DPEP1 is had not yet to see in Hematological Malignancies
Correlative study.
Summary of the invention
The purpose of the present invention is the generation of auxiliary prediction acute lymphoblastic leukemia and/or disease progressions.
The present invention protects a kind of reagent set first.The reagent set may include primer pair DPEP1 and probe DPEP1-
probe；The primer pair DPEP1 can be made of primer DPEP1-F and primer DPEP1-R；The target sequence of the primer pair DPEP1
Specific DNA fragment first can be contained；The specific DNA fragment first can be following y1) or y2)：
Y1) single strand dna shown in the sequence 7 of sequence table；
Y2) there is phase by sequence 7 by the substitution and/or deletion and/or addition of one or several nucleotide and with sequence 7
The DNA molecular of congenerous；
The probe DPEP1-probe can be the single strand dna of 20-30 nucleotide composition, with the specific DNA piece
The partial sector of Duan Jiazhong is identical or complementary.
The primer DPEP1-F can be following a1) or a2)：
A1) single strand dna shown in sequence 1 in sequence table；
A2) there is phase by sequence 1 by the substitution and/or deletion and/or addition of one or several nucleotide and with sequence 1
The single strand dna of congenerous.
The primer DPEP1-R can be following b1) or b2)：
B1) single strand dna shown in sequence 2 in sequence table；
B2) there is phase by sequence 2 by the substitution and/or deletion and/or addition of one or several nucleotide and with sequence 2
The single strand dna of congenerous.
The probe DPEP1-probe can be following c1) or c2)：
C1) single strand dna shown in sequence 3 in sequence table；
C2) there is phase by sequence 3 by the substitution and/or deletion and/or addition of one or several nucleotide and with sequence 3
The single strand dna of congenerous.
The end of the probe DPEP1-probe can have fluorescent marker.5 ' the ends of the probe DPEP1-probe
And/or 3 ' end can have fluorescent marker.5 ' the ends of the probe DPEP1-probe can have FAM fluorescent marker, 3 ' ends
There can be BHQ fluorescent marker.
In the reagent set, the primer DPEP1-F, the primer DPEP1-R and the probe DPEP1-probe's
Molar ratio concretely 40:40:25.
Any of the above-described reagent set may also include primer pair ABL1 and probe ABL1-probe；The primer pair ABL1
It can be made of primer ABL1-F and primer ABL1-R；The target sequence of the primer pair ABL1 can contain specific DNA fragment second；It is described
Specific DNA fragment second can be following z1) or z2)：
Z1) single strand dna shown in the sequence 8 of sequence table；
Z2) there is phase by sequence 8 by the substitution and/or deletion and/or addition of one or several nucleotide and with sequence 8
The DNA molecular of congenerous；
The probe ABL1-probe can be the single strand dna of 20-30 nucleotide composition, with the specific DNA piece
The partial sector of Duan Yizhong is identical or complementary.
The primer ABL1-F can be following d1) or d2)：
D1) single strand dna shown in sequence 4 in sequence table；
D2) there is phase by sequence 4 by the substitution and/or deletion and/or addition of one or several nucleotide and with sequence 4
The single strand dna of congenerous.
The primer ABL1-R can be following e1) or e2)：
E1) single strand dna shown in sequence 5 in sequence table；
E2) there is phase by sequence 5 by the substitution and/or deletion and/or addition of one or several nucleotide and with sequence 5
The single strand dna of congenerous.
The probe ABL1-probe is following f1) or f2)：
F1) single strand dna shown in sequence 6 in sequence table；
F2) there is phase by sequence 6 by the substitution and/or deletion and/or addition of one or several nucleotide and with sequence 6
The single strand dna of congenerous.
The end of the probe ABL1-probe can have fluorescent marker.5 ' the ends of the probe ABL1-probe and/
Or 3 ' end can have fluorescent marker.5 ' the ends of the probe ABL1-probe can have FAM fluorescent marker, and 3 ' ends can have
There is TAMRA fluorescent marker.
In the reagent set, the molar ratio of primer ABL1-F, primer ABL1-R and probe ABL1-probe can be 40:
40:25。
The reagent set may also include positive control plasmid and/or internal reference control plasmid；
The positive control plasmid is the double-stranded DNA shown in sequence 7 into cloning vector or expression vector insetion sequence table
Molecule, obtained recombinant plasmid；The internal reference control plasmid is 8 institute of sequence into cloning vector or expression vector insetion sequence table
The double chain DNA molecule shown, obtained recombinant plasmid.
The cloning vector concretely pMD18-T carrier.
The reagent set specifically can be by primer pair DPEP1, probe DPEP1-probe, primer pair ABL1, probe ABL1-
Probe, positive control plasmid and internal reference control plasmid composition.
The preparation method of any of the above-described reagent set also belongs to protection scope of the present invention.It is any of the above-described described complete
The preparation method of reagent can be by primer DPEP1-F and/or primer DPEP1-R and/or probe DPEP1-probe and/or primer
ABL1-F and/or primer ABL1-R and/or probe ABL1-probe and/or positive control plasmid and/or internal reference control plasmid point
It does not pack not individually.
Any of the above-described reagent set is preparing the application in product；The function of the product can be following h1)-h8)
At least one of：
H1) auxiliary identification acute lymphoblastic leukemia；
H2) auxiliary identifies whether person under test is Patients With Acute Lymphoblastic Leukemia；
H3) auxiliary identifies whether cell to be measured is acute lymphoblastic leukemia tumour cell；
H4 DPEP1 gene) is detected；
H5 the expression of DPEP1 gene) is detected；
H6) auxiliary predicts the risk that object to be measured suffers from acute lymphoblastic leukemia；
H7) the therapeutic effect of auxiliary prediction acute lymphoblastic leukemia；
H8) generation of auxiliary prediction acute lymphoblastic leukemia and/or disease progression.
The present invention also protects a kind of product comprising any of the above-described reagent set；The function of the product can be for such as
At least one of lower h1)-h8)：
H1) auxiliary identification acute lymphoblastic leukemia；
H2) auxiliary identifies whether person under test is Patients With Acute Lymphoblastic Leukemia；
H3) auxiliary identifies whether cell to be measured is acute lymphoblastic leukemia tumour cell；
H4 DPEP1 gene) is detected；
H5 the expression of DPEP1 gene) is detected；
H6) auxiliary predicts the risk that object to be measured suffers from acute lymphoblastic leukemia；
H7) the therapeutic effect of auxiliary prediction acute lymphoblastic leukemia；
H8) generation of auxiliary prediction acute lymphoblastic leukemia and/or disease progression.
DPEP1 gene also belongs to this as application of the marker in the reagent of exploitation diagnosis acute lymphoblastic leukemia
The protection scope of invention.
The substance for detecting the expression of DPEP1 gene also belongs to protection model of the invention in the application prepared in product
It encloses；The function of the product can be following h1) or h2) or h3) h6) or h7) or at least one of h8)：
H1) auxiliary identification acute lymphoblastic leukemia；
H2) auxiliary identifies whether person under test is Patients With Acute Lymphoblastic Leukemia；
H3) auxiliary identifies whether cell to be measured is acute lymphoblastic leukemia tumour cell；
H6) auxiliary predicts the risk that object to be measured suffers from acute lymphoblastic leukemia；
H7) the therapeutic effect of auxiliary prediction acute lymphoblastic leukemia；
H8) generation of auxiliary prediction acute lymphoblastic leukemia and/or disease progression.
In above-mentioned application, the substance of the expression of the detection DPEP1 gene includes above-mentioned reagent set.
Any of the above-described product may also include with following f1) or f2) or f3) f4) or f5) or data f6) at
The device of reason and conclusion display function：
F1) compare the expression of DPEP1 gene in the cDNA of the person under test and cDNA of normal person, if person under test
In cDNA the expression of DPEP1 gene be higher than normal person cDNA, then person under test is or candidate is the white blood of acute lymphoblastic
Patient, if the cDNA or less that the expression of DPEP1 gene in the cDNA of person under test is normal person, if person under test be not or
Candidate is not Patients With Acute Lymphoblastic Leukemia；
F2) compare relative expression's water of DPEP1 gene reference reference gene in the cDNA of the person under test and cDNA of normal person
It is flat, if the relative expression levels of DPEP1 gene reference reference gene in the cDNA of person under test be higher than normal person cDNA, if to
Survey person is or candidate is Patients With Acute Lymphoblastic Leukemia, if DPEP1 gene reference reference gene in the cDNA of person under test
Relative expression levels be normal person cDNA or less, then person under test be not or candidate be not acute lymphoblastic leukemia suffer from
Person；
F3) in the cDNA of the cell more to be measured and cDNA of normal cell DPEP1 gene expression, if to be measured thin
In the cDNA of born of the same parents the expression of DPEP1 gene be higher than normal cell cDNA, then cell to be measured is or candidate is acute lymphoblastic
Chronic myeloid leukemia tumour cell, if in the cDNA of cell to be measured DPEP1 gene expression be normal cell cDNA with
Under, then cell to be measured is not or candidate is not acute lymphoblastic leukemia tumour cell；
F4) in the cDNA of the cell more to be measured and cDNA of normal cell DPEP1 gene reference reference gene opposite table
Up to level, if the relative expression levels of DPEP1 gene reference reference gene are higher than normal cell in the cDNA of cell to be measured
CDNA, then cell to be measured are or candidate is acute lymphoblastic leukemia tumour cell, if DPEP1 in the cDNA of cell to be measured
The relative expression levels of gene reference reference gene are the cDNA or less of normal cell, then cell to be measured is not or candidate is not anxious
Property lymphocytic leukemia tumour cell；
F5 the expression for) detecting DPEP1 gene, including using the cDNA of sample to be tested as template, using any of the above-described institute
State the step of primer pair DPEP1 and probe DPEP1-probe carries out RQ-PCR amplification in reagent set；
F6 the relative expression levels for) detecting DPEP1 gene reference reference gene, including using the cDNA of sample to be tested as mould
Plate draws using the step of the primer pair DPEP1 and probe DPEP1-probe progress RQ-PCR amplification and using described
The step of object carries out RQ-PCR amplification to the ABL1 and probe ABL1-probe.
The relative expression levels (or relative expression levels of DPEP1 gene reference reference gene) of DPEP1 gene are higher, then
The risk that object to be measured suffers from acute lymphoblastic leukemia is higher.DPEP1 gene relative expression levels (or DPEP1 gene ginseng
Than the relative expression levels of reference gene) it is higher, then the therapeutic effect of acute lymphoblastic leukemia is poorer.DPEP1 gene
Relative expression levels (or relative expression levels of DPEP1 gene reference reference gene) are higher, and acute lymphoblastic leukemia is got over
It is easy to recur.
Following g1) or g2) or g3) or g4) be also the scope of protection of the invention：
G1) auxiliary identify person under test whether be Patients With Acute Lymphoblastic Leukemia method, include the following steps：Detection
The relative expression levels of DPEP1 gene reference reference gene in the cDNA of the person under test and cDNA of normal person, if person under test
In cDNA the relative expression levels of DPEP1 gene reference reference gene be higher than normal person cDNA, then person under test is or candidate is
Patients With Acute Lymphoblastic Leukemia, if in the cDNA of person under test DPEP1 gene reference reference gene relative expression levels
For the cDNA or less of normal person, then person under test is not or candidate is not Patients With Acute Lymphoblastic Leukemia；
G2) auxiliary identifies whether cell to be measured is the method for acute lymphoblastic leukemia tumour cell, including walks as follows
Suddenly：The relative expression levels of DPEP1 gene reference reference gene in the cDNA of the cell to be measured and cDNA of normal cell are detected, such as
In the cDNA of fruit cell to be measured the relative expression levels of DPEP1 gene reference reference gene be higher than normal cell cDNA, then to
Survey cell is or candidate is acute lymphoblastic leukemia tumour cell, if DPEP1 gene reference in the cDNA of cell to be measured
The relative expression levels of reference gene are the cDNA or less of normal cell, then cell to be measured is not or candidate is not that acute lymphoblastic is thin
Born of the same parents' leukemia tumor cells；
G3 the method for) detecting the expression of DPEP1 gene, includes the following steps：Using the cDNA of sample to be tested as template,
RQ-PCR amplification is carried out using the primer pair DPEP1 and probe DPEP1-probe；
G4 the relative expression levels for) detecting DPEP1 gene reference reference gene, include the following steps：With sample to be tested
CDNA is template, carries out RQ-PCR amplification using the primer pair DPEP1 and probe DPEP1-probe and using described
The primer pair ABL1 and probe ABL1-probe carries out RQ-PCR amplification.
The expression of DPEP1 gene is higher than the cDNA of normal person in the cDNA of any of the above-described person under test, specifically may be used
It is significantly higher than the cDNA of normal person for the expression of DPEP1 gene in the cDNA of person under test.
The relative expression levels of DPEP1 gene reference reference gene are higher than normal in the cDNA of any of the above-described person under test
The cDNA of people, concretely the relative expression levels of DPEP1 gene reference reference gene are significantly higher than just in the cDNA of person under test
The cDNA of ordinary person.
The expression of DPEP1 gene is higher than the cDNA of normal cell, tool in the cDNA of any of the above-described cell to be measured
Body can be significantly higher than the cDNA of normal cell for the expression of DPEP1 gene in the cDNA of cell to be measured.
The relative expression levels of DPEP1 gene reference reference gene are higher than just in the cDNA of any of the above-described cell to be measured
The cDNA of normal cell, concretely the relative expression levels of DPEP1 gene reference reference gene are significant in the cDNA of cell to be measured
Higher than the cDNA of normal cell.
The table of the relative expression levels of any of the above-described DPEP1 gene reference reference gene concretely DPEP1 gene
Up to the ratio between the expression quantity of amount and reference gene.
The expression quantity of the expression quantity of any of the above-described DPEP1 gene and any of the above-described reference gene can according to
The copy number that standard curve and CT value obtain.
The reference gene can be people ABL1 gene.
The NCBI GenBank sequences number of any of the above-described DPEP1 gene are XM_024450173.It is any of the above-described described
The NCBI GenBank sequences number of ABL1 gene are NM_005157.
It is demonstrated experimentally that can detecte DPEP1 gene expression dose using reagent set provided by the invention, and then for auxiliary
Risk, the auxiliary for helping identification acute lymphoblastic leukemia, auxiliary to predict that object to be measured suffers from acute lymphoblastic leukemia are predicted
The therapeutic effect of acute lymphoblastic leukemia and generation and/or the disease progression of auxiliary prediction acute lymphoblastic leukemia.
The present invention has important application value in medical science.
Detailed description of the invention
Fig. 1 is the RQ-PCR fluorescence standard curve of positive control plasmid.
Fig. 2 is the result for detecting the relative expression levels of DPEP1 gene in acute lymphatic leukaemia patient.
Specific embodiment
Embodiment below facilitates a better understanding of the present invention, but does not limit the present invention.Experiment in following embodiments
Method is unless otherwise specified conventional method.The experimental materials used in the following example is unless otherwise specified certainly
What routine biochemistry reagent shop was commercially available.Quantitative experiment in following embodiment is respectively provided with three repeated experiments, as a result makes even
Mean value.
Fluorescence real-time quantitative PCR instrument is the product of American AB I company, model 7500-FAST type.2 × TaqMan is general
PCR public system is the product of American AB I company.
Embodiment 1, detect DPEP1 gene relative expression levels reagent set preparation
1, according to DPEP1 gene (NCBI GenBank sequences number：XM_024450173 nucleotide sequence) designs and synthesizes
Primer pair DPEP1 and probe DPEP1-probe.Primer pair DPEP1 is by primer DPEP1-F：5'-
CAACAATTACATTTCCTGCACCAA-3 ' (sequence 1 in sequence table) and primer DPEP1-R：5'-
CCACCTCCTTGATGTGATCCA-3 ' (sequence 2 in sequence table) composition.Probe DPEP1-probe is：5'-FAM-
CAACCTGTCCCAAGTGGCCGACC-BHQ-3 ' (sequence 3 in sequence table).5 ' the ends of probe DPEP1-probe have FAM
Fluorescent marker, 3 ' ends have BHQ fluorescent marker.
2, according to ABL1 gene (as reference gene, NCBI GenBank sequences number：NM_005157 nucleotide sequence) is set
It counts and synthetic primer is to ABL1 and probe ABL1-probe.Primer pair ABL1 is by primer ABL1-F：5'-
TGGAGATAACACTCTAAGCATAACTAAAGGT-3 ' (sequence 4 in sequence table) and primer ABL1-R：5'-
GATGTAGTTGCTTGGGACCCA-3 ' (sequence 5 in sequence table) composition.Probe ABL1-probe is：5'-FAM-
CCATTTTTGGTTTGGGCTTCACACCATT-TAMRA-3 ' (sequence 6 in sequence table).5 ' the ends of probe ABL1-probe have
There is FAM fluorescent marker, 3 ' ends have TAMRA fluorescent marker.
3,7 institute of sequence into insetion sequence table at the digestion recognition site of the restriction enzyme EcoRV of pMD18-T plasmid
The double chain DNA molecule shown, obtains positive control plasmid.
The target sequence of primer DPEP1-F and primer DPEP1-R are as shown in sequence 7 in sequence table.
PMD18-T plasmid is the product of Shanghai Hao Jia company, catalog number D101A.
4,8 institute of sequence into insetion sequence table at the digestion recognition site of the restriction enzyme EcoRV of pMD18-T plasmid
The double chain DNA molecule shown obtains internal reference control plasmid.
The target sequence of primer ABL1-F and primer ABL1-R are as shown in sequence 8 in sequence table.
The reagent set of DPEP1 gene relative expression levels is detected by primer pair DPEP1, probe DPEP1-probe, primer
ABL1, probe ABL1-probe, positive control plasmid and internal reference control plasmid are formed.
Embodiment 2, sensitivity technique
Ct value (Cycle threshold) is experienced when the fluorescence signal in each reaction tube reaches given threshold follows
Number of rings.10 times of the standard deviation of the fluorescence signal of fluorescence thresholding 3~15 circulations are set as the default of fluorescence thresholding, fluorescence sheet
Bottom signal is the fluorescence signal of preceding 15 circulations of RQ-PCR reaction.
1, positive control plasmid is taken, gradient dilution is carried out with the double waters for injection that steam of sterilizing, obtains dilution.With the dilution
For template, RQ-PCR is carried out on fluorescence real-time quantitative PCR instrument using primer pair DPEP1 and probe DPEP1-probe.
Reaction system (10 μ L)：The general PCR public system of 52 × TaqMan of μ L, 1 μ L dilution (contain in 1 μ L dilution
The copy number of positive control plasmid is 105It is a, 104It is a, 103It is a, 102It is a, 101It is a or 100It is a), 1 μ L primer DPEP1-F, 1 μ L
Primer DPEP1-R, 1 μ L probe DPEP1-probe, moisturizing to 10 μ L.In reaction system, primer DPEP1-F and primer DPEP1-R
Concentration be 0.4 μM, the concentration of probe DPEP1-probe is 0.25 μM.
Reaction condition：50 DEG C of 2min, 1 circulation；95 DEG C of 10min, 1 circulation；95 DEG C of 15s, 60 DEG C of 1min, 40
Circulation.
Using the copy number of positive control plasmid as abscissa, Ct value is ordinate, draws RQ-PCR fluorescence standard curve.Sun
Property control plasmid RQ-PCR fluorescence standard curve be specifically shown in Fig. 1 (threshold value 0.082), function be log10DPEP1=(Ct-
36.985)/- 3.418, related coefficient reaches 0.99 or more.The result shows that primer pair DPEP1 and probe DPEP1-probe inspection
The sensitivity for surveying positive control plasmid is 10 copy/reaction systems.
2, internal reference control plasmid is taken, gradient dilution is carried out with the double waters for injection that steam of sterilizing, obtains dilution.With the dilution
For template, RQ-PCR is carried out on fluorescence real-time quantitative PCR instrument using primer pair ABL1 and probe ABL1-probe.
Reaction system (10 μ L)：The general PCR public system of 52 × TaqMan of μ L, 1 μ L dilution (contain in 1 μ L dilution
The copy number of positive control plasmid is 105It is a, 104It is a, 103It is a, 102It is a, 101It is a or 100It is a), 1 μ L primer ABL1-F, 1 μ L draw
Object ABL1-R, 1 μ L probe ABL1-probe, moisturizing to 10 μ L.In reaction system, the concentration of primer ABL1-F and primer ABL1-R
It is 0.4 μM, the concentration of probe ABL1-probe is 0.25 μM.
Reaction condition：50 DEG C of 2min, 1 circulation；95 DEG C of 10min, 1 circulation；95 DEG C of 15s, 60 DEG C of 1min, 40
Circulation.
Using the copy number of internal reference control plasmid as abscissa, Ct value is ordinate, draws RQ-PCR fluorescence standard curve.It is interior
Join control plasmid RQ-PCR fluorescence standard curve threshold value be 0.082, function be log10ABL1=(Ct-38.47)/-
3.22, related coefficient reaches 0.99 or more.The result shows that primer pair ABL1 and probe ABL1-probe detection internal reference compares matter
The sensitivity of grain is 10 copy/reaction systems.
Embodiment 3, using embodiment 1 prepare reagent set detection blood tumor cell system in DPEP1 gene it is opposite
Expression
The title of blood tumor cell system is shown in Table in 1 the 1st column, sell company's information, cell type and original disease according to
It is secondary to be shown in Table the 2nd column to the 4th column in 1.
Threshold value is fixed as 0.082 when analyzing RQ-PCR result.
Experiment, which is repeated twice, to be averaged, and duplicate every time steps are as follows：
1, the total serum IgE of each blood tumor cell system is extracted respectively, and then reverse transcription is cDNA.
2, the cDNA obtained respectively using step 1 is template, using primer pair DPEP1 and probe DPEP1-probe in fluorescence
RQ-PCR is carried out on real-time PCR, then the RQ-PCR according to the positive control plasmid that step 1 obtains in embodiment 2 is glimmering
Cursor directrix curve obtains the copy number of DPEP1 gene in each blood tumor cell system.
Reaction system (10 μ L)：The cDNA that the general PCR public system of 52 × TaqMan of μ L, 1 μ L step 1 obtain is (about
100ng), 1 μ L primer DPEP1-F, 1 μ L primer DPEP1-R, 1 μ L probe DPEP1-probe, moisturizing to 10 μ L.Reaction system
In, the concentration of primer DPEP1-F and primer DPEP1-R are 0.4 μM, and the concentration of probe DPEP1-probe is 0.25 μM.
Reaction condition：50 DEG C of 2min, 1 circulation；95 DEG C of 10min, 1 circulation；95 DEG C of 15s, 60 DEG C of 1min, 40
Circulation.
3, the cDNA obtained respectively using step 1 is template, using primer pair ABL1 and probe ABL1-probe in fluorescence reality
When quantitative PCR apparatus on carry out RQ-PCR, the RQ-PCR fluorescence of the internal reference control plasmid then obtained according to step 2 in embodiment 2
Standard curve obtains the copy number of ABL1 gene in each blood tumor cell system.
Reaction system (10 μ L)：The cDNA that the general PCR public system of 52 × TaqMan of μ L, 1 μ L step 1 obtain is (about
100ng), 1 μ L primer ABL1-F, 1 μ L primer ABL1-R, 1 μ L probe ABL1-probe, moisturizing to 10 μ L.In reaction system, draw
The concentration of object ABL1-F and primer ABL1-R are 0.4 μM, and the concentration of probe ABL1-probe is 0.25 μM.
Reaction condition：50 DEG C of 2min, 1 circulation；95 DEG C of 10min, 1 circulation；95 DEG C of 15s, 60 DEG C of 1min, 40
Circulation.
4, after completing step 2 and 3, the relative expression levels of DPEP1 gene in each blood tumor cell system are calculated；Certain blood
In liquid tumor cell line in the relative expression levels of the DPEP1 gene=blood tumor cell system DPEP1 gene copy number/should
Copy number × 100% of ABL1 gene in blood tumor cell system.
Experimental result is shown in Table the 5th column in 1.DPEP1 gene is expressed in acute lymphoblastic leukemia cell system BV173 high,
It is expressed in other blood tumor cell systems lower.
The essential information of 1. blood tumor cell system of table and the expression of DPEP1 gene
Embodiment 4 detects DPEP1 gene in acute lymphatic leukaemia patient using reagent set prepared by embodiment 1
Relative expression levels
The experiment of the present embodiment is to carry out under the premise of the informed consent of patient, and sign informed consent form.
Acute lymphatic leukaemia patient includes 132 B-ALL first visit persons, 50 alleviation persons and 19 recidivists.
132 B-ALL first visit persons form first visit group.50 alleviation persons form alleviation group.19 recidivists form recurrence group.
28 Healthy Peoples form normal healthy controls.
Threshold value is fixed as 0.082 when analyzing RQ-PCR result.
1, it extracts person under test respectively using TRIzol kit (product of Invitrogen company of the U.S.) (acute lymphoblastic is white
Blood patient or Healthy People) marrow specimen or periphery blood specimen total serum IgE, then reverse transcription is cDNA.
2, the cDNA obtained respectively using step 1 is template, using primer pair DPEP1 and probe DPEP1-probe in fluorescence
RQ-PCR is carried out on real-time PCR, then the RQ-PCR according to the positive control plasmid that step 1 obtains in embodiment 2 is glimmering
Cursor directrix curve obtains the copy number of DPEP1 gene in each person under test.
Reaction system (10 μ L)：The cDNA that the general PCR public system of 52 × TaqMan of μ L, 1 μ L step 1 obtain is (about
100ng), 1 μ L primer DPEP1-F, 1 μ L primer DPEP1-R, 1 μ L probe DPEP1-probe, moisturizing to 10 μ L.Reaction system
In, the concentration of primer DPEP1-F and primer DPEP1-R are 0.4 μM, and the concentration of probe DPEP1-probe is 0.25 μM.
Reaction condition：50 DEG C of 2min, 1 circulation；95 DEG C of 10min, 1 circulation；95 DEG C of 15s, 60 DEG C of 1min, 40
Circulation.
3, the cDNA obtained respectively using step 1 is template, using primer pair ABL1 and probe ABL1-probe in fluorescence reality
When quantitative PCR apparatus on carry out RQ-PCR, the RQ-PCR fluorescence of the internal reference control plasmid then obtained according to step 2 in embodiment 2
Standard curve obtains the copy number of ABL1 gene in each person under test.
Reaction system (10 μ L)：The cDNA that the general PCR public system of 52 × TaqMan of μ L, 1 μ L step 1 obtain is (about
100ng), 1 μ L primer ABL1-F, 1 μ L primer ABL1-R, 1 μ L probe ABL1-probe, moisturizing to 10 μ L.In reaction system, draw
The concentration of object ABL1-F and primer ABL1-R are 0.4 μM, and the concentration of probe ABL1-probe is 0.25 μM.
Reaction condition：50 DEG C of 2min, 1 circulation；95 DEG C of 10min, 1 circulation；95 DEG C of 15s, 60 DEG C of 1min, 40
Circulation.
4, after completing step 2 and 3, the relative expression levels of DPEP1 gene in each person under test are calculated；In certain person under test
In the relative expression levels of the DPEP1 gene=person under test in the copy number of the DPEP1 gene/person under test ABL1 gene copy
Number × 100%.
Experimental result is shown in Fig. 2：Relative expression levels' range of DPEP1 gene is 0.4-21.7%, middle position in normal healthy controls
Value is 6.9%；Relative expression levels' range of DPEP1 gene is 0.1-22596.1%, median 217.4% in first visit person；
Relative expression levels' range of DPEP1 gene is 0.4-16.1%, median 2.7% in alleviation group；DPEP1 base in recurrence group
Relative expression levels' range of cause is 35.8%-943.6%, median 223.1%.The result shows that first visit group and recurrence group
The relative expression levels of middle DPEP1 gene are significantly higher than normal healthy controls (p<0.01), in alleviation group DPEP1 gene relative expression
It is horizontal to be substantially less than first visit group and recurrence group (p<0.01).Therefore, according to relative expression's water of DPEP1 gene in person under test
It is flat, generation and the disease progression of acute lymphatic leukaemia can be predicted.
<110>The People's Hospital of Peking University
<120>It is a kind of detect DPEP1 gene expression dose reagent set and its application
<160> 8
<170> PatentIn version 3.5
<210> 1
<211> 24
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 1
caacaattac atttcctgca ccaa 24
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<213>Artificial sequence
<220>
<223>
<400> 2
ccacctcctt gatgtgatcc a 21
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<213>Artificial sequence
<220>
<223>
<400> 3
caacctgtcc caagtggccg acc 23
<210> 4
<211> 31
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 4
tggagataac actctaagca taactaaagg t 31
<210> 5
<211> 21
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<213>Artificial sequence
<220>
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<400> 5
gatgtagttg cttgggaccc a 21
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<213>Artificial sequence
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ccatttttgg tttgggcttc acaccatt 28
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<213>Artificial sequence
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<400> 7
caacaattac atttcctgca ccaacaaggc caacctgtcc caagtggccg accatctgga 60
tcacatcaag gaggtgg 77
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<212> DNA
<213>Artificial sequence
<220>
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tggagataac actctaagca taactaaagg tgaaaagctc cgggtcttag gctataatca 60
caatggggaa tggtgtgaag cccaaaccaa aaatggccaa ggctgggtcc caagcaacta 120
catc
Claims (10)
1. reagent set, including primer pair DPEP1 and probe DPEP1-probe；The primer pair DPEP1 is by primer DPEP1-F
It is formed with primer DPEP1-R；The target sequence of the primer pair DPEP1 contains specific DNA fragment first；The specific DNA fragment first
For following y1) or y2)：
Y1) single strand dna shown in the sequence 7 of sequence table；
Y2 sequence 7 by the substitution and/or deletion and/or addition of one or several nucleotide and) had into identical function with sequence 7
The DNA molecular of energy；
The probe DPEP1-probe is the single strand dna of 20-30 nucleotide composition, in the specific DNA fragment first
Partial sector it is identical or complementary.
2. reagent set as described in claim 1, it is characterised in that：
The primer DPEP1-F is following a1) or a2)：
A1) single strand dna shown in sequence 1 in sequence table；
A2 sequence 1 by the substitution and/or deletion and/or addition of one or several nucleotide and) had into identical function with sequence 1
The single strand dna of energy；
The primer DPEP1-R is following b1) or b2)：
B1) single strand dna shown in sequence 2 in sequence table；
B2 sequence 2 by the substitution and/or deletion and/or addition of one or several nucleotide and) had into identical function with sequence 2
The single strand dna of energy；
The probe DPEP1-probe is following c1) or c2)：
C1) single strand dna shown in sequence 3 in sequence table；
C2 sequence 3 by the substitution and/or deletion and/or addition of one or several nucleotide and) had into identical function with sequence 3
The single strand dna of energy.
3. reagent set as claimed in claim 1 or 2, it is characterised in that：The reagent set further includes primer pair ABL1 and spy
Needle ABL1-probe；The primer pair ABL1 is made of primer ABL1-F and primer ABL1-R；The target sequence of the primer pair ABL1
Column contain specific DNA fragment second；
The specific DNA fragment second is following z1) or z2)：
Z1) single strand dna shown in the sequence 8 of sequence table；
Z2 sequence 8 by the substitution and/or deletion and/or addition of one or several nucleotide and) had into identical function with sequence 8
The DNA molecular of energy；
The probe ABL1-probe is the single strand dna of 20-30 nucleotide composition, in the specific DNA fragment second
Partial sector it is identical or complementary.
4. reagent set as claimed in claim 3, it is characterised in that：
The primer ABL1-F is following d1) or d2)：
D1) single strand dna shown in sequence 4 in sequence table；
D2 sequence 4 by the substitution and/or deletion and/or addition of one or several nucleotide and) had into identical function with sequence 4
The single strand dna of energy；
The primer ABL1-R is following e1) or e2)：
E1) single strand dna shown in sequence 5 in sequence table；
E2 sequence 5 by the substitution and/or deletion and/or addition of one or several nucleotide and) had into identical function with sequence 5
The single strand dna of energy；
The probe ABL1-probe is following f1) or f2)：
F1) single strand dna shown in sequence 6 in sequence table；
F2 sequence 6 by the substitution and/or deletion and/or addition of one or several nucleotide and) had into identical function with sequence 6
The single strand dna of energy.
5. the reagent set as described in Claims 1-4 is any, it is characterised in that：The reagent set further includes positive control matter
Grain and/or internal reference control plasmid；
The positive control plasmid is the double chain DNA molecule shown in sequence 7 into cloning vector or expression vector insetion sequence table,
Obtained recombinant plasmid；The internal reference control plasmid is shown in the sequence 8 into cloning vector or expression vector insetion sequence table
Double chain DNA molecule, obtained recombinant plasmid.
6. the preparation method of any reagent set of claim 1 to 5, for by any reagent set of claim 1 to 5
In primer DPEP1-F and/or primer DPEP1-R and/or probe DPEP1-probe and/or primer ABL1-F and/or primer
ABL1-R and/or probe ABL1-probe and/or positive control plasmid and/or internal reference control plasmid are individually packed.
7. any reagent set of claim 1 to 5 is preparing the application in product；The function of the product is following h1)-
At least one of h8)：
H1) auxiliary identification acute lymphoblastic leukemia；
H2) auxiliary identifies whether person under test is Patients With Acute Lymphoblastic Leukemia；
H3) auxiliary identifies whether cell to be measured is acute lymphoblastic leukemia tumour cell；
H4 DPEP1 gene) is detected；
H5 the expression of DPEP1 gene) is detected；
H6) auxiliary predicts the risk that object to be measured suffers from acute lymphoblastic leukemia；
H7) the therapeutic effect of auxiliary prediction acute lymphoblastic leukemia；
H8) generation of auxiliary prediction acute lymphoblastic leukemia and/or disease progression.
8. a kind of product comprising any reagent set of claim 1 to 5；The function of the product is following h1)-h8)
At least one of：
H1) auxiliary identification acute lymphoblastic leukemia；
H2) auxiliary identifies whether person under test is Patients With Acute Lymphoblastic Leukemia；
H3) auxiliary identifies whether cell to be measured is acute lymphoblastic leukemia tumour cell；
H4 DPEP1 gene) is detected；
H5 the expression of DPEP1 gene) is detected；
H6) auxiliary predicts the risk that object to be measured suffers from acute lymphoblastic leukemia；
H7) the therapeutic effect of auxiliary prediction acute lymphoblastic leukemia；
H8) generation of auxiliary prediction acute lymphoblastic leukemia and/or disease progression.
Application of the 9.DPEP1 gene as marker in the reagent of exploitation diagnosis acute lymphoblastic leukemia.
10. the substance for detecting the expression of DPEP1 gene is preparing the application in product；The function of the product is as follows
H1) or h2) or h3) h6) or h7) or at least one of h8)：
H1) auxiliary identification acute lymphoblastic leukemia；
H2) auxiliary identifies whether person under test is Patients With Acute Lymphoblastic Leukemia；
H3) auxiliary identifies whether cell to be measured is acute lymphoblastic leukemia tumour cell；
H6) auxiliary predicts the risk that object to be measured suffers from acute lymphoblastic leukemia；
H7) the therapeutic effect of auxiliary prediction acute lymphoblastic leukemia；
H8) generation of auxiliary prediction acute lymphoblastic leukemia and/or disease progression.
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Non-Patent Citations (2)
| Title |
|---|
| JIA-MIN ZHANG等: "DPEP1 expression promotes proliferation and survival of leukaemia cells and correlates with relapse in adults with common B cell acute lymphoblastic leukaemia", 《BRITISH JOURNAL OF HAEMATOLOGY》 * |
| 许开宇等: "肾二肽酶基因mRNA 在左右半结肠腺癌组织中的表达及其临床意义", 《结直肠肛门外科》 * |
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| CN111100907A (en) * | 2019-12-02 | 2020-05-05 | 郑州大学第一附属医院 | Primer probe set, kit and method for quantitatively detecting GNA15 gene expression quantity |
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