CN108624687B - Primer, primer combination, kit and its application - Google Patents
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Abstract
The present invention relates to field of biotechnology, in particular to primer, primer combination, kit and its application.Using real-time fluorescence quantitative PCR of a variety of mix primers (real-time PCR), specificity detection at least 15 kinds of NPM1 in cDNA level are mutated the present invention.Mainly for the detection of microresidual disease after AML treatment, it can also be used to which whether there is or not NPM1 mutation for first visit patient identification.
Description
Technical field
The present invention relates to field of biotechnology, in particular to primer, primer combination, kit and its application.
Background technique
Microresidual disease (MRD) refers to that hematological system tumor obtains tectology after receiving treatment and alleviates, and nothing is faced
Bed symptom but still the sub- microscope lesion of presence in vivo.Multi-purpose PCR clinical at present and flow cytometer are detected.
Acute myelocytic leukemia (AML) is a maximum type in adult leukemia, accounts for about 50%-70%, is important
Fatal disease.Recently as continuously improving for treatment method, the patient rate for obtaining morphology alleviation is higher and higher, but
Most patients still recur after being alleviated.Understand in the morphology reduction of patient body obtained under microscope with the presence or absence of swollen
Oncocyte, and then decide whether further to treat, it is the committed step of prevention recurrence.However, due to the heterogeneity of AML,
MRD is carried out for the AML patient of paracmasis and detects ununified method.Most common method is to utilize melting in AML cell
Gene is closed to be detected.But the patient of all fusion (such as PML-RARA, AML1-ETO) positives is added together the ratio for accounting for AML
Rate is 30%, most of AML not specific fusion.Whether other gene mutations can be used as the mark of AML tracking
Arguement is always existed, because some mutation (such as DNMT3A) still have in the bone marrow cell of patient for obtaining long-term remission.
In addition, need to also have certain positive frequency in AML patient as the mark of MRD tracking.To after 2016, NPM1 is acknowledged as
A few can be used as the mark of AML MRD tracking, because it persistently exists in AML patient tumors cell, and delay for a long time
It is very low to solve recall rate in patient, it is main gene mutation form in AML that NPM1 mutation positive patient, which accounts for the 30% of AML,.It is special
It is not in accounting for more than half karyotype normal patient of AML, NPM1 is mutated up to 50%-70%, and this kind of patient is swollen
Oncocyte does not have other good biomarkers.
NPM1 gene is present on No. 5 chromosomes, is positioned at 5q35.1.It is a variety of heavy to participate in DNA reparation and cell cycle etc.
Want vital movement.NPM1 mutation occurs mainly in acute myelocytic leukemia, at least 27 kinds mutation (see Fig. 1, Fig. 2).
It concentrates on the 12nd exon, and site is 1 to 2 tetrad nucleotides inserteds or 4-5 nucleotide deletion between 956 to 971
With 9 nucleotides inserteds.It leads to missense mutation, so as to shorten reading frame, the combined area DNA is made to lose function.
At present clinically there are many method of detection NPM1 mutation, main purpose concentrates on discovery first visit or the white blood of recurrence
Whether NPM1 mutation is had in patient.Method includes PCR next generation (Sanger) sequencing and common fluorescent quantitative PCR.PCR is latter
There are sensitivity problems for generation (Sanger) sequencing approach.Since the NPM1 mutation in leukaemia cell is heterozygote, even if sample
In 100% be that sequencing reading result is not also especially clear after leukaemia cell PCR, normal and mutating alkali yl weave in (see
Fig. 3 (A)~Fig. 3 (C) and Fig. 4).When the leukaemia cell in entire sample is 10%, mutation is not almost just seen.And white
In blood patient, leukaemia cell is much less than 30% in peripheral blood.As most of acute promyelocytic leukemic (APL) suffers from
Person.Therefore, the peripheral blood that detects or even cannot be used for first visit patient that Sanger sequencing approach is mutated NPM1 after PCR.As for
Contain that common fluorescent quantitative PCR rule for tri- Primary mutations of A, B, D is cumbersome and applicable surface is narrow.Because it is same
When do three quantitative fluorescent PCRs, and be once mutated not that (but one of other tens kinds, account for about 15%-20% at three kinds
NPM1 mutation) within, then result is false negative.
As for the detection of microresidual disease, PCR next generation (Sanger) sequencing approach is certain due to the relationship of sensitivity
Be not suitable for, because the paracmasis patient overwhelming majority cell of the first visit NPM1 mutation positive contains only wild type NPM1, be sequenced after PCR
As a result the signal of saltant type can be covered.Though and can be used for MRD inspection for the fluorescence quantitative PCR method of three Primary mutations
It surveys, but it must know that NPM1 is mutated sequencing result first, otherwise troublesome (can at least do three independent realities as described above
Test) and it is unreliable, because negative findings can not rule out patient, there are other NPM1 mutation outside three kinds of Primary mutations.In addition, people
Also carry out MRD detection with ASO-PCR method, i.e., it is numerous according to the primer or probe of each patient's sequencing result design specificity
It is trivial and expensive.
The prior art also once attempt simultaneously utilize more than 15 pairs or more primer mixing to once expand 15 kinds or more dash forward
Become.But practical operation discovery seriously affects amplification due to factors, the non-specific amplifications of generation such as interference between a variety of primers
Sensitivity and specificity.Therefore, the key of this detection is to amplify as much as possible dash forward with primer combination as few as possible
Become type, while guaranteeing the sensitivity and specificity (i.e. primer optimal combination) of PCR amplification.
Need in the prior art it is a kind of it is simple and convenient, sensitivity is high, high specificity, the detection reliable detection side NPM1 of result
Method.
Summary of the invention
For above-mentioned defect in the prior art, the present invention provides primer and combinations thereof, kit and its applications.This hair
The bright specificity inspection in cDNA level using real-time fluorescence quantitative PCR of a few mix primer (real-time PCR)
At least 17 kinds of NPM1 mutation out.Mainly for the detection of microresidual disease after AML treatment, it can also be used to which first visit patient identification has
No NPM1 mutation.
In order to achieve the above-mentioned object of the invention, the present invention the following technical schemes are provided:
The present invention provides primers, with any one in nucleotide sequence as follows:
I, there is nucleotide sequence shown in SEQ ID NO:1；
II, there is nucleotide sequence shown in SEQ ID NO:1 to be modified, replace, miss or add one or more alkali
The nucleotide sequence that base obtains；
III, the sequence with nucleotide sequence shown in SEQ ID NO:1 at least 80% homology；
The complementary series of IV, the sequence as shown in I, II or III.
In some specific embodiments of the invention, the nucleotide sequence as shown in SEQ ID NO:1 are as follows:
NCNTCCNCNNCNNNNNNNNNNN；
Wherein, N (1)=A or T；N (3)=T or C；N (7)=A or T；N (9)=T or C；N (10)=A or
G；N (12)=T or C；N (13)=A or G；N (14)=A or T or C or G；N (15)=C or G；N (16)=A
or C；N (17)=A or T or G；N (18)=A or G；N (19)=A or C；N (20)=A or*or G；N (21)=*
or G；N (22)=* or A.
In some specific embodiments of the invention, the primer has shown in SEQ ID NO:2~6 any one
Nucleotide sequence.
In some specific embodiments of the invention, primer includes NPM1type A primer (reverse), has such as SEQ
Nucleotide sequence shown in ID NO:2: ACTTCCTCCACTGCCAGACAGA.
In some specific embodiments of the invention, primer includes NPM1type B primer (reverse), has such as SEQ
Nucleotide sequence shown in ID NO:3: TCCTCCACTGCCATGCAGAG.
In some specific embodiments of the invention, primer includes NPM1type D primer (reverse), has such as SEQ
Nucleotide sequence shown in ID NO:4: TCCTCCACTGCCAGGCAGA.
In some specific embodiments of the invention, primer includes NPM1type DD1 primer (reverse), is had such as
Nucleotide sequence shown in SEQ ID NO:5: TCCTCCACTGCCAAGCAGAG.
In some specific embodiments of the invention, primer includes NPM1typeE primer (reverse), has such as SEQ
Nucleotide sequence shown in ID NO:6: ACTTCCTCCACTGCCATACAGA.
The present invention also provides primer sets, and including the primer, forward universal primer and/or internal reference gene draw
Object；The forward universal primer has any one in nucleotide sequence as follows:
V, there is nucleotide sequence shown in SEQ ID NO:7；
VI, there is nucleotide sequence shown in SEQ ID NO:7 to be modified, replace, miss or add one or more alkali
The nucleotide sequence that base obtains；
VII, the sequence with nucleotide sequence shown in SEQ ID NO:7 at least 80% homology；
The complementary series of VIII, the sequence as shown in V, VI or VII.
In some specific embodiments of the invention, primer sets further include NPM1 forward universal primer, have such as SEQ
Nucleotide sequence shown in ID NO:7: GAAGAATTGCTTCCGGATGACTG.
Primer provided by the invention can be applied in combination with the forward universal primer, such as with such as SEQ ID NO:7
Shown in nucleotide sequence NPM1 forward universal primer respectively and have the nucleotide sequence as shown in NO:2~6 SEQ ID
Primer combination, can expand NPM1A, B, D respectively, DD1, the mutation of E type.
In some specific embodiments of the invention, primer combination is as follows:
In some specific embodiments of the invention, the primer of the internal reference gene has nucleotide as follows
Any one in sequence:
IX, there is nucleotide sequence shown in SEQ ID NO:8 or 9；
X, there is nucleotide sequence shown in SEQ ID NO:8 or 9 to be modified, replace, miss or add one or more
The nucleotide sequence that base obtains；
XI, the sequence with nucleotide sequence shown in SEQ ID NO:8 or 9 at least 80% homology；
The complementary series of XII, the sequence as shown in IX, X or XI.
In some specific embodiments of the invention, the primer of internal reference gene includes ABL1 primer (forward), tool
Just like nucleotide sequence shown in SEQ ID NO:8: GTGGAGATAACACTCTAAGCATAACTAAAGGT.
In some specific embodiments of the invention, the primer of internal reference gene further includes ABL1 primer (reverse),
With the nucleotide sequence as shown in SEQ ID NO:9: TGATGTAGTTGCTTGGGACCCA.
The present invention also provides the combinations of primer sets and probe, and including the primer sets and probe, the probe has
Any one in nucleotide sequence as follows:
XII, there is nucleotide sequence shown in SEQ ID NO:10 or 11；
XIII, have SEQ ID NO:10 or 11 shown in nucleotide sequence modified, replace, miss or add one or
The nucleotide sequence that multiple bases obtain；
XIV, the sequence with nucleotide sequence shown in SEQ ID NO:10 or 11 at least 80% homology；
The complementary series of XV, the sequence as shown in XII, XIII or XIV.
In some specific embodiments of the invention, probe includes NPM1 general probe and ABL1 probe.Wherein, NPM1
General probe is FAM-ACCAAGAGGCTATTCAAG-MGB, has the nucleotide sequence as shown in SEQ ID NO:10:
ACCAAGAGGCTATTCAAG.ABL1 probe is VIC-CATTTTTGGTTTGGGCTTCA-MGB, has such as SEQ ID NO:11
Shown in nucleotide sequence: CATTTTTGGTTTGGGCTTCA.
On this basis, the present invention also provides the primer or the primer sets or the primer sets and probes
Combination amplification NPM1 gene or its mutation in application.
The present invention also provides the combinations of the primer or the primer sets or the primer sets and probe to make
Application in the kit of standby detection acute myelocytic leukemia.
The present invention also provides the combinations of the primer or the primer sets or the primer sets and probe to make
Application in the kit of the microresidual disease of standby detection acute myelocytic leukemia.
The present invention also provides kits, including the primer or the primer sets or the primer sets and probe
Combination and acceptable reagent.
In some specific embodiments of the invention, the acceptable reagent includes reaction solution, negative control reagent
Or positive control agent.
In some specific embodiments of the invention, the concentration of the primer of the primer or the internal reference gene is
4~8pmol/ μ l；The concentration of the forward universal primer is 20~40pmol/ μ l；The concentration of the probe is 4~8pmol/
μl。
The present invention also provides a kind of methods of detection NPM1 mutation, include the following steps:
Step 1: the nucleotide for obtaining sample to be tested (is in some embodiments of the invention cDNA)；
Step 2: taking the cDNA to mix with the reagent in kit as claimed in claim 8, expand, tied according to amplification
Fruit obtains testing result.
In some specific embodiments of the invention, the program of the amplification are as follows:
1. 50.0℃2min
2. 95.0℃2min
3. 95.0℃1s
4. 60℃20s
5.GOTO step3,39X
Wherein the temperature ramp rate of each step is 2.74 DEG C/s.
In some specific embodiments of the invention, amplification are as follows:
According to 50,5x103、5x105、5x107The NPM1 positive plasmid of copies/uL and 102、104、106、108copies/
The standard curve that the ABL1 Plasmid samples of uL are drawn respectively reads the copy number testing result of each sample NPM1 and ABL1.Report
Announcement value (RQ) calculation formula are as follows: copy number/10 RQ=NPM1mut4ABL1 copy number.
In some specific embodiments of the invention, the amplification is PCR.
In some specific embodiments of the invention, PCR is real-time PCR.
Whether there is or not MRD and detection first visit patient, whether there is or not related mutations using after detection in Gene Mutation leukemia treating by the present invention
Difference is: method must be that discontinuity height is special, avoid that wild type gene can be detected.Suffer from because alleviating in morphology
In person's body, most of related gene is wild type, the only presence of only a few mutant.If this method can equally detect
Wild type gene, then wild type signal will cover jump signal after PCR amplification (see Fig. 2).So the present invention devises needle
The primer special to each wild type.The strategy of design of primers are as follows: mutation upstream include subregion design one it is public
Upstream primer, after upstream primer design a common probe in the same direction, mutated site design be directed to each wild type
Specific downstream primer.It contains the normal sequence of saltant type insertion base (4) and its left and right ends, and every downstream is drawn
Object length is between 20-23nt.
In view of various NPM1 mutation does not have special difference to the treatment and prognosis of patient, general testing goal is only
Whether there is or not mutation to exist for solution, so It is not necessary to detecting one by one.It is especially even more so when MRD is detected.So as far as possible one
Various mutations are detected in a system.However, a variety of different primers necessarily interfere with each other in same PCR system, thus shadow
Ring the sensitivity and specificity (specificity that specificity here refers to difference mutation and wild type) of PCR.So key problem in technology
It is to detect mutation type as much as possible using primer combination as few as possible.
It is found by the applicant that as shown in Figure 1, known NPM1 mutation in, at least 18 kinds of insertion points are identical, and be inserted into sequence
It partially overlaps between column.There are at least 15 bases in not mutated region between the primer that the present invention designs in mutated site two sides
It is at least a kind of with other classes in the mutation insetion sequence for being consistent, and being separately included in 5 pairs of primers designing of the present invention
The difference of the insetion sequence of type mutation is not more than 2 bases, and most of difference are 1 base.Through multiple repetition test, as a result table
Bright 5 pairs of primers being mutated using above-mentioned amplification NPM1A, B, D, DD1, E type can make being optimal of system.Even more important
It is that above-mentioned primer is put into a PCR system, in the case where sensitivity is constant, it not only can be above-mentioned five kinds with specific amplification
Mutation, moreover it is possible to expand other at least ten kinds of mutation, achieve unexpected technical effect (15 kinds of amplification Yu Benfa of mutation above
The plasmid of bright building specifically sports first 15 in Fig. 1 mutation list, mutation building detailed in Example 1).Further benefit
Highly significant (see embodiment 5) has also been achieved the effect that the test of at least 24 clinical patients in this way, and has passed through survey
Sequence finds that the combination of this primer can also be amplified except other two kinds of mutation in above-mentioned 15 kinds mutation.
Beneficial effects of the present invention:
1) present invention is using real-time fluorescence quantitative PCR of a few mix primer (real-time PCR) in cDNA
Specificity detection up to 17 kinds of NPM1 mutation (specificity that specificity here refers to difference mutation and wild type), side in level
Method is simple and convenient, and whether there is or not NPM1 mutation etc. to have for the detection and first visit patient identification for microresidual disease after AML treatment
Very significant clinical meaning, value is great, makes up the deficiency in existing detection method.
2) detection method high sensitivity of the invention can detect in a variety of NPM1 abrupt climatic changes down to 102Copy and 10-5A cell；
3) detection method of the invention is good for saltant type detection specificity, and primer of the invention cannot expand wild type
NPM1, but the NPM1 mutation that at least 17 kinds are more than 95% can be detected, effectively avoid false positive results.
4) detection method of the invention tracks the microresidual disease after the leukaemic's treatment being mutated with NPM1
It has a clear superiority.Due to the microresidual disease tracking that current acute granulocytic leukemia (AML) is not unified, often
That sees utilizes fusion tracking, and every kind of fusion can only track most 10%AML patients, and the NPM1 mutation positive is suffered from
Person accounts for the 30% of AML, and particularly, NPM1 mutation accounts for the 60%-70% of the normal AML of chromosome of no too many biological marker.
Therefore, his applicable surface is wider, with strong points.
Detailed description of the invention
In order to more clearly explain the embodiment of the invention or the technical proposal in the existing technology, to embodiment or will show below
There is attached drawing needed in technical description to be briefly described.
Fig. 1 shows 27 kinds of mutation of NPM1；
Fig. 2 shows normal and abnormal NPM1 gene and protein schematic diagram, mutated site in the 12nd exon of * *；
Fig. 3 shows Sanger sequencing result after the PCR for NPM1 gene；OCI-AML3 is white to be mutated containing NPM1A type
Blood disease cell line；Insertion base is CAGA；HL-60 is the Leukemia Cell Lines of the wild type gene containing NPM1；Fig. 3 (A) shows OCI-
AML3, sequencing result show that mutation result is also not very clear even if in OCI-AML3 cell line；OCI- is worked as in Fig. 3 (B) display
After AML3 and HL-60 1:9 mixing, mutation has been difficult to debate not；Fig. 3 (C) shows HL-60 (negative control)；
Fig. 4 shows Sanger sequencing result (instrument: ABI Prism 3700) after PCR；
Fig. 5 shows the building map of the plasmid of 15 kinds of mutation；
Fig. 6 shows the detection specificity for the mutant that each primer is directed to；
Fig. 7 shows that mix primer expands the specificity of saltant type not of the same race: mix primer can not only for show saltant type A, B,
D, DD1, E are expanded, while can also show that the present invention is set for the effectively amplification such as other saltant types F, G, H, I, J, K, L, M, N and O
The mix primer of meter is the very special (spy for specifically referring to difference mutation and wild type herein for the detection of mutant
It is different)；
Fig. 8 shows the sensitivity of mix primer amplification saltant type A, B, D, DD1, E；
Fig. 9 shows the sensitivity of mix primer amplification saltant type F, G, H, I, J；
Figure 10 shows the sensitivity of mix primer amplification saltant type K, L, M, N, O；
Figure 11, which shows, detects different gradient typeA mutant cell systems using kit provided by the invention；
Figure 11 (A) shows until mutant proportion reaches 10-6, the mutant proportion value of kit test of the present invention can be right with feminine gender
It is significantly distinguished according to the detected value of (complete wild type NPM1 cell line), therefore, the Monitoring lower-cut of kit of the present invention
It is 10-5-10-6Between；
Figure 11 (B) shows takes logarithm with detected value and theoretical value respectively, in 1-10-5Mutant proportion section in, detected value with
The linear dependence of theoretical value is very good (relative coefficient r=0.99782)；
Figure 12 shows clinical case testing result；
Figure 13 shows the position view of NPM1 primer and probe.
Specific embodiment
The invention discloses primer and combinations thereof, kit and its application, those skilled in the art can be used for reference in this paper
Hold, is suitably modified realization of process parameters.In particular, it should be pointed out that all similar substitutions and modifications are to those skilled in the art
For be it will be apparent that they are considered as being included in the present invention.Method and application of the invention, which has passed through, preferably to be implemented
Example is described, related personnel obviously can not depart from the content of present invention, in spirit and scope to method described herein and
Using being modified or appropriate changes and combinations, carry out implementation and application the technology of the present invention.
The technical solution adopted in the present invention is as follows:
6 specific primers of different mutant forms and Proportionality design and 1 of the present invention according to NPM1 gene in AML patient
Specific probe, while design has internal reference Gene A BL1 specific primer 2 and specific probe 1.See Table 1 for details.
Table 1
The position view of NPM1 primer and probe is as shown at 13:
As shown in figure 13, upstream primer design is on 11 exons, and probe is across 11,12 exons, downstream primer
Positioned at 12 exon upstreams, include insertion mutation site.
The present invention provides a kind of reagents with NPM1 gene mutation in Real-Time Fluorescent Quantitative PCR Technique quantitative detection human body
Box, kit contents include:
Negative control reagent: original plasmid DNA of the building containing mutant fragments.
Positive control agent: the Plasmid DNA containing mutant fragments.
Primer mixture: No.2~6 SEQ ID, SEQ ID No.7, No.8~9 SEQ ID；
Probe 1:NPM1 probe: SEQ ID No.10；
Probe 2:ABL1 probe: SEQ ID No.11.
Reaction solution.
Experiment specifically comprises the following steps:
(1) RNA is extracted: extracting the RNA of sample to be tested, the sample to be tested is to contain leucocyte or bone in clinical patient body
Myelocytic sample；
(2) reverse transcription reaction: reaction system: oligo dT (50 μM) 1 μ l, dNTP (10mM) 1 μ l, mould is prepared in PCR pipe
Plate RNA 1-8 μ l (RNA total amount is in 100ng-1 μ g), with sterile deionized water polishing to 10 μ l, lid upper tube cap, by above-mentioned reaction
4 μ l, RNase inhibitor (40U/ μ L) of 5x buffer, 0.5 μ l is added into reaction tube in 65 DEG C of reaction 5min for pipe, inverse
Transcriptase (200U/ μ L) 1 μ l, with sterile deionized water polishing to 20 μ l；It is put into PCR instrument, is reversed according to following procedure
Record: 30 DEG C of 10min；42℃30-60min；95℃5min.
(3) real-time PCR reacts: reaction system is prepared in PCR pipe: commercialization Taq enzyme PCR Mix (2x) 10 μ l,
1.6 μ l of primer mixed liquor, 10.5 μ l of probe, 2 0.5 μ l of probe, template cDNA/ feminine gender quality-control product/positive quality control product dilute respectively
For 50,5x103、5x105、5x107Copy (NPM1 positive plasmid)/be diluted to 10 respectively2、104、106、108copies/uL
(ABL1 plasmid) 2 μ l, with sterile deionized water polishing to 20 μ l；
Above-mentioned primer mixed liquor contains above-mentioned 8 species-specific primer (SEQ ID No.7, No.2~6 SEQ ID, SEQ ID
No.8~9), wherein the concentration of primer 3~9 (No.2~6 SEQ ID, No.8~9 SEQ ID) is 4~8pmol/ μ l, primer
The concentration of 1 (SEQ ID No.7) is 20~40pmol/ μ l, the concentration of probe 1,2 (SEQ ID No.10, SEQ ID No.11)
For 4~8pmol/ μ l.
Above-mentioned reaction tube is put into real-time fluorescence quantitative PCR instrument and is expanded and detected reaction according to following procedure:
1. 50.0℃2min
2. 95.0℃2min
3. 95.0℃1s
4. 60℃20s
5.GOTO step3,39X
Wherein the temperature ramp rate of each step is 2.74 DEG C/s
(4) result judges:
According to 50,5x103、5x105、5x107The NPM1 positive plasmid of copies/uL and 102、104、106、108copies/
The standard curve that the ABL1 Plasmid samples of uL are drawn respectively reads the copy number testing result of each sample NPM1 and ABL1.Report
Announcement value (RQ) calculation formula are as follows: copy number/10 RQ=NPM1mut4ABL1 copy number.The judgement of experimental result and Prognostic significance is not
The standard for being suitble to oneself laboratory can be established according to the actual situation with laboratory.
Note: result above determines to establish under the premise of yin and yang attribute control is normal, if it find that positive and negative control is abnormal,
It is detected again after then needing to exclude the reasons such as degradation or pollution.
The present invention is using real-time fluorescence quantitative PCR of a variety of mix primers (real-time PCR) in cDNA level
Specificity detection at least 15 kinds of NPM1 mutation.Mainly for the detection of microresidual disease after AML treatment, it can also be used to which first visit is suffered from
Whether there is or not NPM1 mutation for person's identification.
Raw materials used, auxiliary material and reagent are available on the market in primer provided by the invention, kit and its application.
Below with reference to embodiment, the present invention is further explained:
The building of the plasmid of 1 15 kinds of embodiment mutation
(1) expand the NPM1 segment containing insertion mutation: the both ends in Insert Fragment as shown in Figure 5, which separately design, draws
Object designs the two-way primer containing deletion mutation segment in mutational site, contains insertion mutation using the method building of bridging PCR
NPM1 segment；
(2) connection, conversion reaction: NPM1 mutant fragments and PMD19-T carrier are connected with commercial kit and is converted extremely
In DH5 α Escherichia coli；
(3) picking positive colony, conservation: Escherichia coli are coated on the LB agarose media containing ampicillin
37 DEG C are incubated overnight, picking single colonie, take the primer amplification of two end-specificity in part of Insert Fragment, can expand
It is saved with 25% glycerol at -20 DEG C after the colonies of purpose band size send sequencing to be confirmed as the positive out；
(4) it extracts plasmid: extracting plasmid with commercialized kit.
Plasmid construction map is shown in Fig. 5.
Embodiment 2 using only each mutant of each primer detection specificity
Grouping:
Experimental group: the plasmid being mutated in the 15 of the building acquisition of embodiment 1；
Negative control is wild type NPM1；Blank control is water.
(1) plasmid dilutes: by wild type (WT), typeA, typeB, typeD, typeDD1, typeE saltant type NPM1 matter
Grain is diluted to 2.7x10 respectively5copies/uL。
(2) real-time PCR reacts: prepare reaction system in PCR pipe: commercialization Taq enzyme PCRMix (2x) 10 μ l draws
0.8 μ l of object X, 1 0.8 μ l of primer, 1 0.5 μ l of probe, the wild type after the middle dilution in mutant plasmids/(1) in (1) after dilution
1 μ l of plasmid/water, the cDNA1uL extracted from HL60 cell line, with sterile deionized water polishing to 20 μ l；
Wherein, primer X be the concentration of corresponding primer (No.2~6 SEQ ID) when detecting different mutant plasmids be 4~
8pmol/ μ l, the concentration of primer 1 (SEQ ID No.7) are 4~8pmol/ μ l, the concentration of probe 1 (SEQ ID No.10) is 4~
8pmol/μl。
Above-mentioned reaction tube is put into real-time fluorescence quantitative PCR instrument and is expanded and detected reaction according to following procedure:
1. 50.0℃2min
2. 95.0℃2min
3. 95.0℃1s
4. 60℃20s
5.GOTO step3,39X。
Wherein the temperature ramp rate of 1,2 steps is 2.74 DEG C/s, the temperature ramp rates of 3,4,5 steps is 2.12 DEG C/
s。
(4) result judges:
The Ct value of the amplification curve of corresponding mutant plasmids and wild plasmid is detected by comparing different mutant primers
Difference evaluation primer specificity.
Test result shows (Fig. 6), Ct value when different mutant primers detect corresponding mutant plasmids 20-22 it
Between, and the Ct > 37 of the water of the wild plasmid and blank control as negative control, illustrate under the template of same concentrations, dashes forward
The amplification efficiency of modification template is at least the 2 of the amplification efficiency of wild type15(32768) again.Therefore, each primer that we design
For the detection of specified mutant be very special.
Embodiment 3 detects 15 kinds of mutant using mix primer
Grouping:
Experimental group: the plasmid being mutated in the 15 of the building acquisition of embodiment 1；
Negative control is wild type NPM1；Blank control is water.
(1) plasmid dilute: by wild type (WT), typeA, typeB, typeD, typeDD1, typeE, typeF, typeG,
TypeH, typeI, typeJ, typeK, typeL, typeM, typeN, typeO saltant type NPM1 plasmid are diluted to 5 ladders respectively
Degree: 2.7x105copies/uL、2.7x104copies/uL、2.7x103copies/uL、2.7x102copies/uL、
2.7x102copies/uL、2.7x101copies/uL。
(2) real-time PCR reacts: prepare reaction system in PCR pipe: commercialization Taq enzyme PCRMix (2x) 10 μ l draws
1.6 μ l of object mixed liquor, probe 1 0.5 μ l, template cDNA/ negative control reagent/2 μ l of positive control agent, with sterilizing deionization
Water polishing is to 20 μ l；
Above-mentioned primer mixed liquor contains above-mentioned 6 species-specific primer (SEQ ID No.7, No.2~6 SEQ ID), wherein
The concentration of primer 3~9 (No.2~6 SEQ ID) is 4~8pmol/ μ l, the concentration of primer 1 (SEQ ID No.7) is 20~
40pmol/ μ l, probe 2SEQ ID No.10) concentration be 4~8pmol/ μ l.
Above-mentioned reaction tube is put into real-time fluorescence quantitative PCR instrument and is expanded and detected reaction according to following procedure:
1. 50.0℃2min
2. 95.0℃2min
3. 95.0℃1s
4. 60℃20s
5.GOTO step3,39X。
Wherein the temperature ramp rate of 1,2 steps is 2.74 DEG C/s, the temperature ramp rates of 3,4,5 steps is 2.12 DEG C/
s。
(4) result judges:
The difference of the Ct value of the amplification curve of corresponding mutant plasmids and wild plasmid is detected by comparing mix primer
Evaluate the specificity of primer.The Ct of the amplification curve of gradient mutant plasmids and wild plasmid is detected by comparing mix primer
The sensitivity for whether having significant difference evaluation primer of value
Test result shows (table 2 and Fig. 7) that mix primer detects Ct value major part when corresponding mutant plasmids
(typeA、typeB、typeD、typeDD1、typeE、typeF、typeG、typeH、typeI、typeJ、typeL、typeM、
TypeO) between 20-22, for typeK between 25-27, typeN shows mix primer of the invention not only between 22-24
It can be expanded for saltant type A, B, D, DD1, E, while can also be for other saltant types F, G, H, I, J, K, L, M, N and O etc. effectively
Amplification shows that the mix primer has the good characteristic for the amplification of various mutations type.In addition, as the wild of negative control
The Ct > 35 of type plasmid and the water of blank control.Illustrate under the template of same concentrations, the saltant type for most of saltant type
The amplification efficiency of template is at least the 2 of the amplification efficiency of wild type13(8196) again.Therefore, the mix primer needle that the present invention designs
Pair mutant detection be very it is special (herein specifically refer to difference mutation and wild type it is special).
Test result shows (table 3 and Fig. 8-10) that mix primer detects Ct value major part when corresponding mutant plasmids
(typeA、typeB、typeD、typeDD1、typeE、typeF、typeG、typeH、typeI、typeJ、typeL、typeM、
TypeO sensitivity) at least can reach 2.7x102Copies/uL, and the sensitivity for detecting typeK and typeN at least can reach
2.7x103copies/uL。
2 mix primer of table expands the specificity (corresponding diagram 7) of saltant type not of the same race
The sensitivity (corresponding diagram 8,9,10) of the amplification of 3 mix primer of table saltant type A, B, D, DD1, E
4 sensitivity experiment of embodiment
Grouping:
Experimental group: the plasmid for 15 kinds of mutation that the building of embodiment 1 obtains；
Negative control is wild type NPM1；Blank control is water
(1) RNA is extracted: extraction sample to be tested (with 1:0,1:10,1:100,1:1000,1:10000,1:100000,1:
1000000,1:10000000,0:1 mixing NPM1 mutation cell line and wild type NPM1 cell line) RNA, it is described to
Test sample sheet is the sample containing leucocyte or bone marrow cell in clinical patient body；
(2) reverse transcription reaction: reaction system: oligo dT (50 μM) 1 μ l, dNTP (10mM) 1 μ l, mould is prepared in PCR pipe
Plate RNA 1-8 μ l (RNA total amount is in 100ng-1 μ g), with sterile deionized water polishing to 10 μ l, lid upper tube cap, by above-mentioned reaction
4 μ l, RNase inhibitor (40U/ μ L) of 5x buffer, 0.5 μ l is added into reaction tube in 65 DEG C of reaction 5min for pipe, inverse
Transcriptase (200U/ μ L) 1 μ l, with sterile deionized water polishing to 20 μ l；It is put into PCR instrument, is reversed according to following procedure
Record: 30 DEG C of 10min；42℃30-60min；95℃5min.
(3) real-time PCR reacts: reaction system is prepared in PCR pipe: commercialization Taq enzyme PCR Mix (2x) 10 μ l,
1.6 μ l of primer mixed liquor, 1 0.5 μ l of probe, probe 2 0.5 μ l, template cDNA/ddH2O2 μ l, with sterile deionized water polishing
To 20 μ l；
Above-mentioned primer mixed liquor contains above-mentioned 8 species-specific primer (SEQ ID No.7, No.2~6 SEQ ID, SEQ ID
No.8~9), wherein the concentration of primer 3~9 (No.2~6 SEQ ID, No.8~9 SEQ ID) is 4~8pmol/ μ l, primer
The concentration of 1 (SEQ ID No.7) is 20~40pmol/ μ l, the concentration of probe 1,2 (SEQ ID No.10, SEQ ID No.11)
For 4~8pmol/ μ l.
Above-mentioned reaction tube is put into real-time fluorescence quantitative PCR instrument and is expanded and detected reaction according to following procedure:
1. 50.0℃2min
2. 95.0℃2min
3. 95.0℃1s
4. 60℃20s
5.GOTO step3,39X。
Wherein the temperature ramp rate of 1,2 steps is 2.74 DEG C/s, the temperature ramp rates of 3,4,5 steps is 2.12 DEG C/
s。
(4) result judges:
The difference of the Ct value of the sample of different mutant proportions and the amplification curve of negative sample is detected by comparing kit
The sensitivity of kits for evaluation.
Figure 11 (A) the result shows that, until mutant proportion reach 10-6, the mutant proportion value of kit test of the present invention can
It is significantly distinguished with the detected value of negative control (complete wild type NPM1 cell line), therefore, kit of the present invention
Monitoring lower-cut is 10-5-10-6Between.
As shown in Figure 11 (B), logarithm is taken respectively with detected value and theoretical value, in 1-10-5Mutant proportion section in, detection
The linear dependence of value and theoretical value is very good (relative coefficient r=0.99782).
Embodiment 5 examines 3 normal person's samples and 24 patient clinical samples containing different proportion NPM1 mutation
It surveys
(1) myeloid tissue processing and extraction
The extraction of myeloid tissue takes TRIzol to extract, and specific extraction operation step refers to that " molecular cloning refers to
South ".Mentioned RNA is dissolved in DEPC processing water, through nanodrop ultramicron UV spectrophotometer measuring quality, determines that its is dense
Degree.
(2) reverse transcription (concentration)
Reaction system is prepared in 200 μ l PCR pipes: Oligo dT (50 μM) 1 μ l, dNTP (10mM) 1 μ l, template ribonucleic acid
1-8 μ l (RNA total amount is in 100ng-1 μ g), with sterile deionized water polishing to 10 μ l；By sample in 65 DEG C of reaction 5min；To anti-
5x buffer 4 μ l, RNase inhibitor (40U/ μ L) 0.5 μ l, reverse transcriptase (200U/ μ L) 1 μ l is added in Ying Guanzhong, uses
Sterile deionized water polishing is to 20 μ l；Sample is put into PCR instrument, carries out reverse transcription: 30 DEG C of 10min according to following procedure；42℃
30-60min；95℃5min.
(3) pcr amplification reaction system is established
CDNA template obtained in (2)/feminine gender quality-control product/positive quality control product/ddH2O/ is diluted to 50 respectively,
5x103、5x105、5x107The NPM1 positive plasmid of copies/uL/be diluted to 10 respectively2、104、106、108Copies/uL's
ABL1 plasmid is used as the template of real-time fluorescent PCR amplification, carries out PCR amplification according to following amplification system:
It is commercialized Taq enzyme PCR Mix (2x) 10 μ l
1.6 μ l of primer mixed liquor
10.5 μ l of probe
20.5 μ l of probe
cDNA 2μl
With sterile deionized water polishing to 20 μ l.
Above-mentioned primer mixed liquor contains above-mentioned 8 species-specific primer (SEQ ID No.7, No.2~6 SEQ ID, SEQ ID
No.8~9), wherein the concentration of primer 3~9 (No.2~6 SEQ ID, No.8~9 SEQ ID) is 4~8pmol/ μ l, primer
The concentration of 1 (SEQ ID No.7) is 20~40pmol/ μ l, the concentration of probe 1,2 (SEQ ID No.10, SEQ ID No.11)
For 4~8pmol/ μ l.
PCR reaction condition is as follows:
1. 50.0℃2min
2. 95.0℃2min
3. 95.0℃1s
4. 60℃20s
5.GOTO step3,39X。
Wherein the temperature ramp rate of 1,2 steps is 2.74 DEG C/s, the temperature ramp rates of 3,4,5 steps is 2.12 DEG C/
s。
The collection of fluorescence signal is set to FAM, and data acquisition is scheduled on 60 DEG C.
After experiment, follows the steps below analysis, determines:
According to 50,5x103、5x105、5x107The NPM1 positive plasmid of copies/uL and 102、104、106、108copies/
The standard curve that the ABL1 Plasmid samples of uL are drawn respectively reads the copy number testing result of each sample NPM1 and ABL1.Report
Announcement value (RQ) calculation formula are as follows: copy number/10 RQ=NPM1mut4ABL1 copy number.It is generally to determine AMLMRD with RQ value number
The standard of state, different experiments room can establish the standard for being suitble to oneself laboratory according to the actual situation.
As a result referring to table 4:
Table 4
As shown in table 2 and Figure 12, using OCI-AML3 cell line as positive control, HL-60 cell line be negative control, 3
In normal person's sample, testing result (RQ value) is below general positive cutoff value (200), and testing result is in normal range (NR).Through it
Its Molecular Detection confirms in 24 positive patients of NPM1 mutation, kit test result of the present invention be determined as it is positive (RQ >=
200) patient has 23, and wherein two generation of No.11267 patient, which is sequenced, confirms that NPM1 mutation rate is 1.07%, RQ value of the invention
Being sequenced for two generation of 8745, No.1881 patient confirms that NPM1 mutation rate is 9.43%, our RQ value is 286477.No.2474 and
Although the sequencing of 13513 rear two generations confirm mutation rates also 10% hereinafter, but RQ value be all larger than of the invention sensitive of 100000. proofs
It spends very high.Only 1 (No.2079) testing result is feminine gender, and two generation sequencing analysis show that the mutation type of this case does not exist after
Within the scope of the mutation type of kit covering detection of the present invention.In addition, sample number is two samples of No.13894 and No.2569
Show that its mutation type not among the mutational range type that has detected of the present invention, but still can be detected through generation sequencing analysis,
Thus " this method can expand the mutation of more NPM1 in addition to the mutation type having verified that " before the verifying present invention.It comes from
CDNA (negative control) detected value of HL60 cell line is 1, well below general positive cutoff value (200), it is seen that the kit
Sensitivity and specificity can fully meet clinical detection demand.
The above is only a preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
For member, various improvements and modifications may be made without departing from the principle of the present invention, these improvements and modifications are also answered
It is considered as protection scope of the present invention.
Sequence table
<110>Nanjing first sign medical test Co., Ltd；Jiangsu first sign medical diagnosis Co., Ltd；Beijing first sign medicine
Examine Laboratories, Inc；
<120>primer, primer combination, kit and its application
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Claims (8)
1. a kind of composition of primer and probe, which is characterized in that such as SEQ ID NO of the nucleotide sequence including reverse primer:
Shown in 2-6；
Forward the nucleotide sequence of universal primer is as shown in SEQ ID NO:7；
The nucleotide sequence of probe is as shown in SEQ ID NO:10.
2. the composition of primer and probe described in claim 1 further comprises internal reference primer, the primer sequence
Nucleotide sequence is as shown in SEQ ID NO.8 and 9.
3. the composition of primer and probe as claimed in claim 1 or 2 is in preparation detection acute myelocytic leukemia or acute
The application in kit that microresidual disease after myelocytic leukemia treatment is mutated whether there is or not NPM1.
4. the composition of primer and probe as claimed in claim 1 or 2 is after preparation detection acute myelocytic leukemia treatment
The kit that is mutated whether there is or not NPM1 of microresidual disease first visit patient in application.
5. kit, which is characterized in that composition including primer and probe as claimed in claim 1 or 2 and acceptable
Reagent.
6. kit according to claim 5, which is characterized in that the concentration of reverse primer is 4 ~ 8pmol/ μ l.
7. kit according to claim 6, which is characterized in that the concentration of the forward universal primer is 20 ~ 40pmol/
µl；The concentration of the probe is 4 ~ 8pmol/ μ l.
8. according to any kit of claim 5-7, which is characterized in that the kit is real-time fluorescence quantitative PCR
(real-time PCR) kit.
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| CN102181564A (en) * | 2011-05-12 | 2011-09-14 | 北京英茂盛业生物科技有限公司 | Real-time fluorescence quantitative PCR method for detecting NPM1 genic mutation |
| CN102382896A (en) * | 2011-12-06 | 2012-03-21 | 苏州苏大赛尔免疫生物技术有限公司 | NPM1 gene mutation detection method and reagent box thereof |
| WO2017084027A1 (en) * | 2015-11-17 | 2017-05-26 | 安诺优达基因科技(北京)有限公司 | Kit for prognostic stratification of acute myelocytic leukemia and testing method therefor |
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Effective date of registration: 20190605 Address after: 210042 32 Xuanwu Road, Xuanwu District, Nanjing, Jiangsu, 699-18 Co-patentee after: Jiangsu pre medical diagnosis Co., Ltd. Patentee after: Nanjing pioneer medical laboratory Co., Ltd. Co-patentee after: Beijing pre Medical Laboratory Laboratory Co., Ltd. Co-patentee after: Jiangsu Xiansheng Medical Devices Co., Ltd. Address before: 210042 32 Xuanwu Road, Xuanwu District, Nanjing, Jiangsu, 699-18 Co-patentee before: Jiangsu pre medical diagnosis Co., Ltd. Patentee before: Nanjing pioneer medical laboratory Co., Ltd. Co-patentee before: Beijing pre Medical Laboratory Laboratory Co., Ltd. |