WO2019214058A1 - 引物、引物组合、试剂盒及其应用 - Google Patents

引物、引物组合、试剂盒及其应用 Download PDF

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WO2019214058A1
WO2019214058A1 PCT/CN2018/095601 CN2018095601W WO2019214058A1 WO 2019214058 A1 WO2019214058 A1 WO 2019214058A1 CN 2018095601 W CN2018095601 W CN 2018095601W WO 2019214058 A1 WO2019214058 A1 WO 2019214058A1
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primer
seq
nucleotide sequence
npm1
kit
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PCT/CN2018/095601
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English (en)
French (fr)
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王阳
刘进
马冉冉
崔欢喜
任用
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南京先声医学检验有限公司
江苏先声医学诊断有限公司
北京先声医学检验实验室有限公司
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Priority claimed from CN201810447833.0A external-priority patent/CN108624687B/zh
Priority claimed from CN201810731209.3A external-priority patent/CN108753976B/zh
Application filed by 南京先声医学检验有限公司, 江苏先声医学诊断有限公司, 北京先声医学检验实验室有限公司 filed Critical 南京先声医学检验有限公司
Publication of WO2019214058A1 publication Critical patent/WO2019214058A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence

Definitions

  • the present invention relates to the field of biotechnology, and in particular to primers, primer combinations, kits and applications thereof.
  • MRD minimal residual disease
  • Acute myeloid leukemia is the largest species of adult leukemia, accounting for about 50%-70%, and is an important fatal disease.
  • AML Acute myeloid leukemia
  • the proportion of patients with morphological remission has become higher and higher, but most patients still relapse after remission.
  • Understanding the morphology under the microscope to alleviate the presence of tumor cells in patients and then decide whether further treatment is needed is a key step in preventing recurrence.
  • due to the heterogeneity of AML there is no uniform method for MRD testing in patients with remission of AML. The most common method is to use the fusion gene in AML cells for detection.
  • NPM1 is recognized as one of the few markers that can be tracked as AML MRD because it persists in tumor cells of AML patients and has a low detection rate in patients with long-term remission, and NPM1 mutation-positive patients account for 30% of AML is the main form of genetic mutation in AML. Especially in normal karyotype patients who account for more than half of AML, NPM1 mutations can reach 50%-70%, and tumor cells of these patients have no other good biomarkers.
  • NPM1 gene is present on chromosome 5 and is located at 5q35.1. Participate in a variety of important life activities such as DNA repair and cell cycle. NPM1 mutations occur mainly in acute myeloid leukemia with at least 27 mutations (see Figure 1, Figure 2). It is concentrated in the 12th exon, with a position between 956 and 971, with 1 to 2 tetranucleotide insertions or 4-5 nucleotide deletions with 9 nucleotide insertions. It causes a missense mutation that shortens the reading frame and renders the DNA binding region inactive.
  • NPM1 mutations There are a number of clinically available methods for detecting NPM1 mutations, the main purpose of which is to find out whether there is a NPM1 mutation in patients with newly diagnosed or relapsed leukemia. Methods include post-PCR (Sanger) sequencing and common fluorescent quantitative PCR. There is a sensitivity problem in the post-PCR (Sanger) sequencing method. Since the NPM1 mutation in leukemia cells is heterozygous, even if 100% of the samples are leukemia cells, the results of sequencing after PCR are not particularly clear, and the normal and the mutated bases are intertwined (see Fig. 3(A) to Fig. 3(C)). And Figure 4). When the white blood cells in the whole sample were at 10%, almost no mutation was observed.
  • the post-PCR Sanger sequencing method is certainly not suitable due to the sensitivity, because the majority of cells in the remission phase of the newly diagnosed NPM1 mutation only contain wild-type NPM1, and the sequencing results will cover the mutant after PCR. signal of.
  • the real-time PCR method for the three major mutations can also be used for MRD detection, but it must first know the sequencing result of NPM1 mutation, otherwise it will be cumbersome (at least three independent experiments) and unreliable as described above. Because the negative results cannot rule out other NPM1 mutations in the patient other than the three major mutations.
  • the prior art has also attempted to simultaneously employ more than 15 pairs of primer mixes in order to amplify more than 15 mutations at a time.
  • the actual operation found that non-specific amplification caused by the interference between various primers seriously affects the sensitivity and specificity of amplification. Therefore, the key to this assay is to amplify as many mutation types as possible with as few primer combinations as possible while ensuring the sensitivity and specificity of PCR amplification (ie, optimal combination of primers).
  • the present invention provides primers and combinations thereof, kits and uses thereof.
  • the present invention utilizes a few mixed primers for one-time quantitative PCR to specifically detect at least 16 NPM1 mutations at the cDNA level. It is mainly used to detect the detection of minimal residual disease after AML treatment, and can also be used to identify the presence or absence of NPM1 mutation in newly diagnosed patients.
  • the present invention provides the following technical solutions:
  • the invention provides primers having any of the nucleotide sequences shown below:
  • nucleotide sequence obtained by modifying, substituting, deleting or adding one or more bases of the nucleotide sequence represented by SEQ ID NO: 1;
  • nucleotide sequence set forth in SEQ ID NO: 1 is:
  • the primer has the nucleotide sequence set forth in any one of SEQ ID NOs: 2-6.
  • the primer comprises an NPM1 type A primer (reverse) having the nucleotide sequence set forth in SEQ ID NO: 2: ACTTCCTCCACTGCCAGACAGA.
  • the primer comprises an NPM1 type B primer (reverse) having the nucleotide sequence set forth in SEQ ID NO: 3: TCCTCCACTGCCATGCAGAG.
  • the primer comprises an NPM1 type D primer (reverse) having the nucleotide sequence set forth in SEQ ID NO: 4: TCCTCCACTGCCAGGCAGA.
  • the primer comprises an NPM1 type DD1 primer (reverse) having the nucleotide sequence set forth in SEQ ID NO: 5: TCCTCCACTGCCAAGCAGAG.
  • the primer comprises an NPM1 typeE primer (reverse) having the nucleotide sequence set forth in SEQ ID NO: 6: ACTTCCTCCACTGCCATACAGA.
  • the invention also provides a primer set comprising the primers, the forward universal primers and/or the primers of the internal control gene; the forward universal primers having any one of the nucleotide sequences shown below:
  • nucleotide sequence obtained by modifying, substituting, deleting or adding one or more bases of the nucleotide sequence represented by SEQ ID NO: 7;
  • the primer set further comprises an NPM1 forward universal primer having the nucleotide sequence set forth in SEQ ID NO: 7: GAAGAATTGCTTCCGGATGACTG.
  • the primers provided by the present invention can be used in combination with the forward universal primers, for example, NPM1 forward universal primers having a nucleotide sequence as shown in SEQ ID NO: 7 and having SEQ ID NOS: 2 to 6 respectively.
  • NPM1A, B, D, DD1, E-type mutations can be amplified, respectively, by any one or a portion or all of the primer combinations of the nucleotide sequences.
  • the primer combinations are as follows (but are not limited to the following):
  • the primer of the internal control gene has any one of the nucleotide sequences shown below:
  • nucleotide sequence obtained by modifying, substituting, deleting or adding one or more bases of the nucleotide sequence represented by SEQ ID NO: 8 or 9;
  • XI a sequence having at least 80% homology to the nucleotide sequence set forth in SEQ ID NO: 8 or 9;
  • the primer for the internal control gene comprises an ABL1 primer (forward) having the nucleotide sequence set forth in SEQ ID NO: 8: GTGGAGATAACACTCTAAGCATAACTAAAGGT.
  • the primer for the internal control gene further comprises an ABL1 primer (reverse) having the nucleotide sequence set forth in SEQ ID NO: 9: TGATGTAGTTGCTTGGGACCCA.
  • the invention also provides a combination of a primer set and a probe, comprising the primer set and probe, the probe having any one of the nucleotide sequences shown below:
  • XIII a nucleotide sequence obtained by modifying, substituting, deleting or adding one or more bases of the nucleotide sequence shown by SEQ ID NO: 10 or 11;
  • XIV a sequence having at least 80% homology to the nucleotide sequence set forth in SEQ ID NO: 10 or 11;
  • a complementary sequence of XV such as the sequence shown by XII, XIII or XIV.
  • the probe comprises an NPM1 universal probe and an ABL1 probe.
  • the NPM1 universal probe is FAM-ACCAAGAGGCTATTCAAG-MGB having the nucleotide sequence shown in SEQ ID NO: 10: ACCAAGAGGCTATTCAAG.
  • the ABL1 probe is VIC-CATTTTTGGTTTGGGCTTCA-MGB having the nucleotide sequence shown as SEQ ID NO: 11: CATTTTTGGTTTGGGCTTCA.
  • the present invention also provides the use of the primer or the primer set or the combination of the primer set and the probe for amplifying the NPM1 gene or a mutation thereof.
  • the invention also provides the use of the primer or the primer set or the combination of the primer set and the probe in the preparation of a kit for detecting acute myeloid leukemia.
  • the invention also provides the use of the primer or the primer set or the combination of the primer set and the probe in the preparation of a kit for detecting minimal residual disease of acute myeloid leukemia.
  • kits comprising said primers or said primer sets or combinations of said primer sets and probes and acceptable reagents.
  • the acceptable reagent comprises a reaction solution, a negative control reagent, or a positive control reagent.
  • the primer or the primer of the internal control gene has a concentration of 4 to 8 pmol/ ⁇ l; and the concentration of the forward universal primer is 20 to 40 pmol/ ⁇ l; The concentration of the needle is 4 to 8 pmol/ ⁇ l.
  • the kit is a real-time PCR kit or a ddPCR kit.
  • the invention also provides a method of detecting a NPM1 mutation, the method being based on real-time PCR amplification or ddPCR-based amplification.
  • Step 1 obtaining nucleotides of the sample to be tested (in some embodiments of the invention, cDNA);
  • Step 2 The cDNA is mixed with the reagent in the kit, amplified, and the detection result is obtained according to the amplification result.
  • the amplification procedure is:
  • the temperature rise and fall rate of each step was 2.74 ° C / s.
  • the amplification results are:
  • the assay is based on ddPCR amplification, it includes the following steps:
  • Step 1 obtaining nucleotides of the sample to be tested (in some embodiments of the invention, cDNA);
  • Step 2 The cDNA is mixed with the reagent in the kit, amplified, and the detection result is obtained according to the amplification result.
  • the amplification procedure is:
  • the temperature rise rate of each step is 2.5 ° C / s
  • the amplification results are:
  • RQ ⁇ 200 is used as a criterion for determining the recurrence of AML patients. Different laboratories can establish standards suitable for their own laboratories according to actual conditions. Note: The above results are judged on the premise that the negative positive control is normal. If the negative and positive control are found to be abnormal, it is necessary to eliminate the cause of degradation or contamination before testing.
  • the present invention utilizes gene mutations to detect the presence or absence of MRD after treatment of leukemia and to detect whether there is a related mutation in the newly diagnosed patient: the method must be highly specific to the mutation, and the wild type gene can be detected. Because in the morphological relief patients, most of the related genes are wild-type, and only a few mutants exist. If this method can also detect wild-type genes, the wild-type signal will cover the mutation signal after PCR amplification (see Figure 2). Therefore, the present invention has designed primers specific for each wild type.
  • the strategy of primer design is to design a common upstream primer in the intron region upstream of the mutation, design a common probe in the same direction after the upstream primer, and design specific downstream primers for each wild type at the mutation position. It contains a mutated insertion base (4) and its normal sequences at the left and right ends, each downstream primer being between 20-23 nt in length.
  • the general purpose of the test is simply to know whether or not there is a mutation, so there is no need to detect them one by one. This is especially true when detecting MRD. So try to detect multiple mutations in one system. However, a variety of different primers must interfere with each other in the same PCR system, thereby affecting the sensitivity and specificity of PCR (specificity here refers to the specificity of distinguishing mutations and wild type). So the key to the technology is to detect as many mutations as possible using as few primer combinations as possible.
  • Applicants have found that as shown in Figure 1, at least 18 insertion sites are identical in the known NPM1 mutations, and the insertion sequences are partially coincident.
  • the primers designed in the present invention have at least 15 bases in the non-mutated region on both sides of the mutation position, and at least one of the mutation insertion sequences respectively included in the 5 pairs of primers designed by the present invention is different from other types of mutations. Insertion sequences differ by no more than 2 bases, with most differences being 1 base. After repeated trials and tests, the results showed that the above five primers for amplifying NPM1A, B, D, DD1, and E mutations can optimize the system.
  • the present invention utilizes a few mixed primers for real-time PCR to specifically detect up to 17 NPM1 mutations at the cDNA level (the specificity herein refers to the specificity of the differential mutation and the wild type).
  • the method is simple and convenient; using ddPCR (Digital Droplet PCR, digital microdrop PCR) for real-time quantification, at least 17 traces of NPM1 mutations are specifically detected at the cDNA level.
  • ddPCR Digital Droplet PCR, digital microdrop PCR
  • the detection method of the invention has high sensitivity, and both real-time PCR and ddPCR can detect as low as 10 2 copies and 10 -5 cells in various NPM1 mutation detection;
  • the detection method of the present invention has good specificity for mutation detection, and none of the primers of the present invention can amplify wild-type NPM1, but at least 17 kinds of NPM1 mutations of more than 95% can be detected, thereby effectively avoiding false positive results.
  • the ddPCR provided by the invention absolutely quantifies the detected object, thereby more intuitively and accurately detecting the NPM1 mutation, especially the micro NPM1 mutation effect, and providing a more accurate and reliable method for detecting the minimal residual disease of leukemia.
  • the detection method of the present invention has obvious advantages for tracking minute residual lesions after treatment of leukemia patients with NPM1 mutation. Since there is no uniform micro-residual lesion tracking method in acute myeloid leukemia (AML), the common fusion gene tracking method can only track up to 10% of AML patients per fusion gene, while NPM1 mutation-positive patients account for 30% of AML. In particular, the NPM1 mutation accounts for 60%-70% of the normal AML of the chromosome without too many biomarkers. Therefore, he has a wide range of applications and is highly targeted.
  • AML acute myeloid leukemia
  • Figure 1 shows 27 mutations of NPM1
  • Figure 2 shows a schematic diagram of normal and abnormal NPM1 genes and proteins, ** mutation positions in the 12th exon;
  • Figure 3 shows the results of Sanger sequencing after PCR for NPM1 gene;
  • OCI-AML3 is a leukemia cell line containing NPM1A type mutation; the insertion base is CAGA;
  • HL-60 is a leukemia cell line containing NPM1 wild type gene;
  • Figure 3 (A OCI-AML3, sequencing results show that even in the OCI-AML3 cell line, the mutation results are not clear;
  • Figure 3 (B) shows that when OCI-AML3 and HL-60 1:9 are mixed (ie sensitivity is 10%) ), the mutation has been difficult to discriminate;
  • Figure 3 (C) shows HL-60 (negative control);
  • Figure 4 shows the results of Sanger sequencing after PCR (instrument: ABI Prism 3700);
  • Figure 5 shows the construction map of 15 mutant plasmids
  • Figure 6 shows the detection specificity of the mutants to which each primer is directed
  • Figure 7 shows the specificity of different types of mutants amplified by mixed primers based on real-time PCR: mixed primers can not only amplify mutants A, B, D, DD1, E, but also other mutants F, Efficient amplification of G, H, I, J, K, L, M, N and O, indicating that the mixed primer designed by the present invention is very specific for the detection of mutants (specificity here refers to differential mutation and wild type) Specific)
  • Figure 8 shows the sensitivity of amplifying primers A, B, D, DD1, E based on real-time PCR-based mixed primers
  • Figure 9 shows the sensitivity of the mixed primers based on real-time PCR amplification mutants F, G, H, I, J;
  • Figure 10 shows the sensitivity of amplifying mutant K, L, M, N, O by mixed primers based on real-time PCR
  • Figure 11 shows the detection of different gradient type A mutant cell lines using the real-time PCR kit provided by the present invention
  • Figure 11 (A) shows that until the mutation ratio reaches 10 -6 , the mutation ratio value tested by the kit of the present invention can be clearly distinguished from the detection value of the negative control (complete wild-type NPM1 cell line), therefore, the reagent of the present invention
  • the detection limit of the box is between 10 -5 -10 -6 ;
  • Fig. 11(B) shows that the logarithm of the detected value and the theoretical value are respectively taken.
  • Figure 12 shows the results of real-time PCR detection of clinical cases
  • Figure 13 is a schematic view showing the positions of NPM1 primers and probes
  • Figure 14 shows the specificity of amplification of different mutants by ddPCR-based mixed primers
  • Figure 15 shows the sensitivity of ddPCR-based mixed primer amplification mutants A, B, D, DD1, E;
  • Figure 16 shows the sensitivity of ddPCR-based mixed primer amplification mutants F, G, H, I, J;
  • Figure 17 shows the sensitivity of ddPCR-based mixed primers to amplify mutant K, L, M, N, O;
  • Figure 18 shows the results of the kit sensitivity test provided by the present invention
  • Figure 18 (A) shows the results of sensitivity test of the NPM1 kit by ddPCR using different dilutions of the OCI-AML3 cell line
  • Figure 18 (B) shows the use The result of the sensitivity test of the kit for the gradient ratio of the type A mutant cell line;
  • Figure 19 shows the results of comparison of ddPCR and qPCR results of clinical samples.
  • the invention discloses primers and combinations thereof, kits and applications thereof, and those skilled in the art can learn from the contents of the paper and appropriately improve the process parameters. It is to be understood that all such alternatives and modifications are obvious to those skilled in the art and are considered to be included in the present invention.
  • the method and the application of the present invention have been described by the preferred embodiments, and it is obvious that the method and application described herein may be modified or appropriately modified and combined without departing from the scope of the present invention.
  • the technique of the present invention is applied.
  • six specific primers and one specific probe are designed according to different mutation forms and ratios of the NPM1 gene in AML patients, and two internal primer ABL1 specific primers and one specific probe are designed. See Table 1 for details.
  • the upstream primer was designed on exon 11 with the probe spanning the exons 11 and 12, and the downstream primer was located upstream of exon 12, containing the insertional mutation site.
  • the invention provides a kit for quantitatively detecting NPM1 gene mutation in human body by real-time fluorescent quantitative PCR technology, and the kit includes:
  • Negative control reagent Original plasmid DNA containing the mutant fragment.
  • Positive control reagent Plasmid DNA containing the mutant fragment.
  • Primer mixture SEQ ID No. 2-6, SEQ ID No. 7, SEQ ID No. 8-9;
  • Probe 1 NPM1 probe: SEQ ID No. 10;
  • Probe 2 ABL1 probe: SEQ ID No. 11.
  • the experiment specifically includes the following steps:
  • RNA extraction extracting RNA of a sample to be tested, wherein the sample to be tested is a sample containing leukocytes or bone marrow cells in a clinical patient;
  • Reverse transcription reaction The reaction system was prepared in a PCR tube: 1 ⁇ l of oligo dT (50 ⁇ M), 1 ⁇ l of dNTP (10 mM), 1-8 ⁇ l of template RNA (total amount of RNA was 100 ng-1 ⁇ g), and filled with sterile deionized water.
  • the above primer mixture contains the above eight specific primers (SEQ ID No. 7, SEQ ID No. 2 to 6, and SEQ ID Nos. 8 to 9), wherein primers 3 to 9 (SEQ ID Nos. 2 to 6, The concentration of SEQ ID No. 8 to 9) is 4 to 8 pmol/ ⁇ l, the concentration of primer 1 (SEQ ID No. 7) is 20 to 40 pmol/ ⁇ l, and probes 1 and 2 (SEQ ID No. 10, SEQ ID No) The concentration of .11) is 4 to 8 pmol/ ⁇ l.
  • the reaction tube was placed in a real-time PCR instrument and amplified according to the following procedure:
  • the temperature rise rate of each step is 2.74 ° C / s
  • the invention also provides a ddPCR kit.
  • the kit includes the primers provided by the present invention or the primer set or the combination of the primer set and the probe.
  • the ddPCR kit comprises:
  • Negative control cDNA containing the NPM1 mutation
  • Positive control product cDNA containing normal NPM1; primer set provided by the present invention
  • Probe 1 NPM1 probe provided by the present invention
  • Probe 2 ABL1 probe provided by the present invention
  • the test specifically includes the following steps:
  • RNA extraction extracting RNA of a sample to be tested, wherein the sample to be tested is a sample containing leukocytes or bone marrow cells in a clinical patient;
  • Reverse transcription reaction The reaction system was prepared in a PCR tube: 1 ⁇ l of oligo dT (50 ⁇ M), 1 ⁇ l of dNTP (10 mM), 1-8 ⁇ l of template RNA (total amount of RNA was 100 ng-1 ⁇ g), and filled with sterile deionized water.
  • reaction system Prepare reaction system in PCR tube: commercial ddPCR Mix for Probes (2x, No dUTP) 10 ⁇ l, primer mix 3.6 ⁇ l, probe 1 0.5 ⁇ l, probe 2 0.5 ⁇ l, template cDNA/ 2 ⁇ l of negative control reagent / positive control reagent, supplemented to 20 ⁇ l with sterile deionized water;
  • the above primer mixture contains the above eight specific primers (SEQ ID No. 7, SEQ ID No. 2 to 6, and SEQ ID Nos. 8 to 9), wherein primers 3 to 9 (SEQ ID Nos. 2 to 6, The concentration of SEQ ID No. 8 to 9) is 4 to 8 pmol/ ⁇ l, the concentration of primer 1 (SEQ ID No. 7) is 20 to 40 pmol/ ⁇ l, and probes 1 and 2 (SEQ ID No. 10, SEQ ID No) The concentration of .11) is 4-8 pmol/ ⁇ l;
  • Droplet formation According to the QX200 specification, the 20 uL reaction system in (3) was placed in a droplet generator together with 70 uL of droplets to generate droplets, and the final volume was about 40 uL. Transfer to a matching 96-well plate and seal with a heat seal film.
  • PCR amplification The above reaction tube was placed in a common PCR instrument to perform amplification and detection reaction according to the following procedure:
  • the temperature rise rate of each step is 2.5 ° C / s
  • Microdrop reading The 96-well plate after PCR is placed in a microdrop reader, and the operation is performed according to the instrument manual. After the program is finished, the instrument will automatically analyze the result.
  • RQ ⁇ 200 is used as a criterion for determining the recurrence of AML patients. Different laboratories can establish standards suitable for their own laboratories according to actual conditions. Note: The above results are judged on the premise that the negative positive control is normal. If the negative and positive control are found to be abnormal, it is necessary to eliminate the cause of degradation or contamination before testing.
  • primers, kits and the materials, adjuvants and reagents used in the application of the present invention are commercially available.
  • Escherichia coli was spread on LB agarose medium containing ampicillin overnight at 37 ° C, single colonies were picked, and some of them were specifically used in the inserts.
  • the primers were amplified, and the cloned colonies capable of amplifying the target band size were confirmed to be positive and then stored with 25% glycerol at -20 ° C;
  • the plasmid construction map is shown in Figure 5.
  • Example 2 The specificity of each mutant was determined by using each primer real-time PCR alone.
  • Example 1 constructs the obtained 15 mutated plasmid
  • the negative control was wild type NPM1; the blank control was water.
  • Plasmid dilution The wild type (WT), type A, type B, type D, type DD1, type E mutant NPM1 plasmids were each diluted to 2.7 x 10 5 copies/uL.
  • (2) Real-time PCR reaction Prepare the reaction system in PCR tube: commercial Taq PCR Mix (2x) 10 ⁇ l, primer X 0.8 ⁇ l, primer 1 0.8 ⁇ l, probe 1 0.5 ⁇ l, diluted in (1) 1 ⁇ L of the wild type plasmid/water diluted in the mutant plasmid/(1), and 1 ⁇ L of the cDNA extracted from the HL60 cell line, supplemented to 20 ⁇ l with sterile deionized water;
  • primer X is a primer (SEQ ID No. 2 to 6) having a concentration of 4 to 8 pmol/ ⁇ l and a primer 1 (SEQ ID No. 7) having a concentration of 4 to 8 pmol/ ⁇ l when detecting different mutant plasmids.
  • concentration of probe 1 (SEQ ID No. 10) was 4 to 8 pmol/ ⁇ l.
  • the reaction tube was placed in a real-time PCR instrument and amplified according to the following procedure:
  • the temperature rise and fall rate of the steps 1 and 2 is 2.74 ° C / s, and the temperature rise rate of the 3, 4, 5 steps is 2.12 ° C / s.
  • the specificity of the primers was evaluated by comparing the difference in the Ct values of the amplification curves of the corresponding mutant plasmids and the wild type plasmids by comparing different mutant primers.
  • Example 1 constructs the obtained 15 mutated plasmid
  • the negative control was wild type NPM1; the blank control was water.
  • Plasmid dilution The wild type (WT), type A, type B, type D, type DD1, type E, type F, type G, type H, type I, type J, type K, type L, type M, type N, type O mutant NPM1 plasmids were each diluted to 5 Gradient: 2.7x10 5 copies/uL, 2.7x10 4 copies/uL, 2.7x10 3 copies/uL, 2.7x10 2 copies/uL, 2.7x10 2 copies/uL, 2.7x10 1 copies/uL.
  • the above primer mixture contains the above six specific primers (SEQ ID No. 7, SEQ ID Nos. 2 to 6), wherein the concentrations of the primers 3 to 9 (SEQ ID No. 2 to 6) are 4 to 8 pmol/ ⁇ l.
  • the concentration of primer 1 (SEQ ID No. 7) was 20 to 40 pmol/ ⁇ l, and the concentration of probe 2 SEQ ID No. 10) was 4 to 8 pmol/ ⁇ l.
  • the reaction tube was placed in a real-time PCR instrument and amplified according to the following procedure:
  • the temperature rise and fall rate of the steps 1 and 2 is 2.74 ° C / s, and the temperature rise rate of the 3, 4, 5 steps is 2.12 ° C / s.
  • the specificity of the primers was evaluated by comparing the mixed primers to detect the difference in the Ct values of the amplification curves of the corresponding mutant plasmids and the wild type plasmids.
  • the sensitivity of the primers was evaluated by comparing the mixed primers to detect whether there is a significant difference in the Ct value of the amplification curve of the gradient mutant plasmid and the wild type plasmid.
  • the mixed primers detected most of the Ct values of the corresponding mutant plasmids (typeA, typeB, typeD, typeDD1, typeE, typeF, typeG, typeH, typeI, typeJ, typeL, typeM, typeO). Between 20-22, typeK is between 25-27, and typeN is between 22-24, indicating that the mixed primer of the present invention can not only amplify mutants A, B, D, DD1, E, but also Efficient amplification against other mutants F, G, H, I, J, K, L, M, N and O showed that the mixed primers have good properties for amplification of multiple mutants.
  • the wild type plasmid as a negative control and the blank control water had a Ct > 35. It is shown that under the same concentration of template, the amplification efficiency of the mutant template is at least 2 13 (8196) times that of the wild type for most mutants. Therefore, the detection of mutants against the mixed primers designed by the present invention is very specific (specificity here refers to the specificity of the differential mutation and the wild type).
  • the test results show that (Table 3 and Figure 8-10), the mixed primers detect most of the Ct values of the corresponding mutant plasmids (typeA, typeB, typeD, typeDD1, typeE, typeF, typeG, typeH, typeI, typeJ, typeL, typeM
  • the sensitivity of typeO is at least 2.7x10 2 copies/uL, while the sensitivity of detecting typeK and typeN is at least 2.7x10 3 copies/uL.
  • Example 1 constructs the obtained 15 mutant plasmids
  • the negative control was wild type NPM1; the blank control was water.
  • RNA extraction extracting the sample to be tested (NPM1 mixed at 1:0, 1:10, 1:100, 1:1000, 1:10000, 1:100,000, 1:1000000, 1:10000000, 0:1) RNA of the mutated cell line and the cell line of wild-type NPM1, wherein the sample to be tested is a sample containing leukocytes or bone marrow cells in a clinical patient;
  • Reverse transcription reaction The reaction system was prepared in a PCR tube: 1 ⁇ l of oligo dT (50 ⁇ M), 1 ⁇ l of dNTP (10 mM), 1-8 ⁇ l of template RNA (total amount of RNA was 100 ng-1 ⁇ g), and filled with sterile deionized water.
  • the above primer mixture contains the above eight specific primers (SEQ ID No. 7, SEQ ID No. 2 to 6, and SEQ ID Nos. 8 to 9), wherein primers 3 to 9 (SEQ ID Nos. 2 to 6, The concentration of SEQ ID No. 8 to 9) is 4 to 8 pmol/ ⁇ l, the concentration of primer 1 (SEQ ID No. 7) is 20 to 40 pmol/ ⁇ l, and probes 1 and 2 (SEQ ID No. 10, SEQ ID No) The concentration of .11) is 4 to 8 pmol/ ⁇ l.
  • the reaction tube was placed in a real-time PCR instrument and amplified according to the following procedure:
  • the temperature rise and fall rate of the steps 1 and 2 is 2.74 ° C / s, and the temperature rise rate of the 3, 4, 5 steps is 2.12 ° C / s.
  • the sensitivity of the kit was evaluated by comparing the difference between the Ct values of the amplification curves of the samples with different mutation ratios and the negative samples by the comparison kit.
  • Fig. 11(A) show that until the mutation ratio reaches 10 -6 , the mutation ratio value tested by the kit of the present invention can be clearly distinguished from the detection value of the negative control (complete wild-type NPM1 cell line), therefore,
  • the detection limit of the kit of the invention is between 10 -5 and 10 -6 .
  • Example 5 Real-time PCR was used to detect clinical samples of 3 normal human samples and 24 patients with different proportions of NPM1 mutations.
  • the extraction of bone marrow tissue was performed by TRIzol.
  • the specific extraction procedure please refer to the Guide to Molecular Cloning.
  • the extracted RNA was dissolved in DEPC-treated water, and the mass was determined by nanodrop ultra-micro UV spectrophotometer to determine its concentration.
  • the reaction system was prepared in a 200 ⁇ l PCR tube: Oligo dT (50 ⁇ M) 1 ⁇ l, dNTP (10 mM) 1 ⁇ l, template RNA 1-8 ⁇ l (total RNA in 100 ng-1 ⁇ g), supplemented with sterile deionized water to 10 ⁇ l; 5 min buffer 5 ⁇ l, RNase inhibitor (40 U / ⁇ L) 0.5 ⁇ l, reverse transcriptase (200 U / ⁇ L) 1 ⁇ l, supplemented with sterile deionized water to 20 ⁇ l; sample placed in PCR In the instrument, reverse transcription was carried out according to the following procedure: 30 ° C for 10 min; 42 ° C for 30-60 min; 95 ° C for 5 min.
  • the cDNA template/negative control/positive control/ddH2O/ obtained in (2) was diluted to 50, 5 ⁇ 10 3 , 5 ⁇ 10 5 , 5 ⁇ 10 7 copies/uL of NPM1 positive plasmid/diluted to 10 2 , respectively.
  • the ABL1 plasmid of 10 4 , 10 6 , 10 8 copies/uL was used as a template for real-time fluorescent PCR amplification, and PCR amplification was carried out according to the following amplification system:
  • the above primer mixture contains the above eight specific primers (SEQ ID No. 7, SEQ ID No. 2 to 6, and SEQ ID Nos. 8 to 9), wherein primers 3 to 9 (SEQ ID Nos. 2 to 6, The concentration of SEQ ID No. 8 to 9) is 4 to 8 pmol/ ⁇ l, the concentration of primer 1 (SEQ ID No. 7) is 20 to 40 pmol/ ⁇ l, and probes 1 and 2 (SEQ ID No. 10, SEQ ID No) The concentration of .11) is 4 to 8 pmol/ ⁇ l.
  • the PCR reaction conditions are as follows:
  • the temperature rise and fall rate of the steps 1 and 2 is 2.74 ° C / s, and the temperature rise rate of the 3, 4, 5 steps is 2.12 ° C / s.
  • the fluorescence signal was collected as FAM and the data collection was set at 60 °C.
  • the OCI-AML3 cell line was used as the positive control
  • the HL-60 cell line was the negative control
  • the test results (RQ value) were lower than the general positive judgment value in the 3 normal samples. ), the test results are in the normal range.
  • the 24 patients who were confirmed to be positive for NPM1 mutation by other molecular tests 23 patients were positive (RQ ⁇ 200) in the kit of the present invention, and the second-generation sequencing of No.11267 patients confirmed that the NPM1 mutation rate was 1.07%.
  • the RQ value of the present invention was 8745, and the second generation sequencing of No.1881 patients confirmed that the NPM1 mutation rate was 9.43%, and our RQ value was 286477. No. 2474 and 13513.
  • the cDNA (negative control) from the HL60 cell line has a detection value of 1, which is much lower than the general positive judgment value (200). It can be seen that the sensitivity and specificity of the kit can fully meet the clinical detection requirements.
  • the invention also establishes a set of ddPCR detection kits and detection methods based on the above primer system, and the specific detection steps are as follows:
  • RNA extraction extracting RNA of a sample to be tested, wherein the sample to be tested is a sample containing leukocytes or bone marrow cells in a clinical patient;
  • Reverse transcription reaction The reaction system was prepared in a PCR tube: 1 ⁇ l of oligo dT (50 ⁇ M), 1 ⁇ l of dNTP (10 mM), 1-8 ⁇ l of template RNA (total amount of RNA was 100 ng-1 ⁇ g), and filled with sterile deionized water.
  • reaction system Prepare reaction system in PCR tube: commercial ddPCR Mix for Probes (2x, No dUTP) 10 ⁇ l, primer mix 3.6 ⁇ l, probe 1 0.5 ⁇ l, probe 2 0.5 ⁇ l, template cDNA/ 2 ⁇ l of negative control reagent / positive control reagent, supplemented to 20 ⁇ l with sterile deionized water;
  • the above primer mixture contains the above eight specific primers (SEQ ID No. 7, SEQ ID No. 2 to 6, and SEQ ID Nos. 8 to 9), wherein primers 3 to 9 (SEQ ID Nos. 2 to 6, The concentration of SEQ ID No. 8 to 9) is 4 to 8 pmol/ ⁇ l, the concentration of primer 1 (SEQ ID No. 7) is 20 to 40 pmol/ ⁇ l, and probes 1 and 2 (SEQ ID No. 10, SEQ ID No) The concentration of .11) is 4-8 pmol/ ⁇ l;
  • Droplet formation According to the QX200 specification, the 20 uL reaction system in (3) was placed in a droplet generator together with 70 uL of droplets to generate droplets, and the final volume was about 40 uL. Transfer to a matching 96-well plate and seal with a heat seal film.
  • PCR amplification The above reaction tube was placed in a common PCR instrument to perform amplification and detection reaction according to the following procedure:
  • the temperature rise rate of each step is 2.5 ° C / s
  • Microdrop reading The 96-well plate after PCR is placed in a microdrop reader, and the operation is performed according to the instrument manual. After the program is finished, the instrument will automatically analyze the result.
  • RQ ⁇ 200 is used as a criterion for determining the recurrence of AML patients. Different laboratories can establish standards suitable for their own laboratories according to actual conditions. Note: The above results are judged on the premise that the negative positive control is normal. If the negative and positive control are found to be abnormal, it is necessary to eliminate the cause of degradation or contamination before testing.
  • the 15 mutations shown in Fig. 5 of the same amount were specifically detected by the kit of the present invention.
  • the experimental results showed that the number of copies when detecting different mutant plasmids was 1394-14800, which was much larger than that of the negative control 29 and the blank control 20, indicating that the detection of the mutants provided by the primer set of the present invention is very specific (eg, Figure 14).
  • the inconsistency between the measured copy number and the theoretical copy number also implies that there is a certain error in calculating the plasmid copy number and dilution to achieve the theoretical concentration based on the plasmid concentration (errors formed during the dilution process and degradation at low concentrations).
  • sensitivity detection was performed on the 15 mutations described in Fig. 5 using the kit provided by the present invention.
  • the experimental results show that the sensitivity of detecting different mutant plasmids is between 270-2700 copies, indicating that the detection of the mutants provided by the primer set of the present invention is very sensitive (as shown in FIGS. 15-17).
  • the cell line containing the NPM1 mutation (type A) (OCI-AML3) and the cell line of wild-type NPM1 (HL60) were mixed in different ratios, and the kit was tested using the kit provided by the present invention and the above experimental procedure to test the sensitivity of the kit. .
  • the mutation ratio values tested by our kit were significantly different from those of the negative control (complete wild-type NPM1 cell line).
  • the experimental results are shown in Fig. 18(A) until the mutation ratio is reduced to 10 -4 , and the gradient of the detection result is very good. Therefore, the detection limit provided by the present invention has a detection limit of at least 10 -7
  • the present invention detects 14 clinical samples of patients with different proportions of NPM1 mutations, and compares them with the results of the qPCR method. As shown in Table 8 and Figure 19, the results of the two detection methods are highly consistent.

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Abstract

涉及生物技术领域，特别涉及引物、引物组合、试剂盒及其应用。利用多种混合引物一次定量PCR在cDNA水平上特异性检出至少17种NPM1突变。主要针对AML治疗后微小残留病变的检测，也可用于初诊患者鉴定有无NPM1突变。

Description

引物、引物组合、试剂盒及其应用
本申请要求于2018年05月11日提交中国专利局、申请号为201810447833.0、发明名称为“引物、引物组合、试剂盒及其应用”以及于2018年07月05日提交中国专利局、申请号为201810731209.3、发明名称为“引物、引物组合、试剂盒及其应用”的中国专利申请的优先权，其全部内容通过引用结合在本申请中。
技术领域
本发明涉及生物技术领域，特别涉及引物、引物组合、试剂盒及其应用。
背景技术
微小残留病变(MRD)是指血液系统肿瘤在接受治疗后获得组织形态学缓解，无临床症状但仍然存在在体内的亚显微镜病变。目前临床多用PCR和流式细胞仪进行检测。
急性髓细胞白血病(AML)是成人白血病中最大一个种类，约占50％-70％，是重要的致死性疾病。近年来随着治疗方法的不断改进，获得形态学缓解的患者比率越来越高，但大部分病人在获得缓解后仍然复发。了解获得显微镜下的形态学缓解患者体内是否存在肿瘤细胞，进而决定是否需要进一步治疗，是预防复发的关键步骤。然而，由于AML的异质性，对于缓解期的AML患者进行MRD检测没有统一的方法。最常用的方法是利用AML细胞中的融合基因进行检测。但所有融合基因(如PML-RARA,AML1-ETO)阳性的患者加在一起占AML的比率为30％，大多数AML没有特异性的融合基因。其它基因突变是否可以作为AML跟踪的标志一直存在争论，因为一些突变(如DNMT3A)在获得长期缓解的患者的骨髓细胞中仍然存在。另外，做为MRD跟踪的标志也需在AML患者中有一定的阳性频率。至2016年后，NPM1被公认为少数几个可做为AML MRD跟踪的标志，因为它在AML患者肿瘤细胞中持续存在，且在长期缓解患者中检出率很低，NPM1突变阳性患者占到AML的30％，是AML中主要的基因突变形式。特别是在占AML一半以上的染色体核型正常患者中，NPM1突变可达50％-70％，而这类患者肿瘤细胞没有其它很好的生物标记。
NPM1基因存在于5号染色体上，定位于5q35.1。参与DNA修复和细胞周期等多种重要生命活动。NPM1突变主要发生在急性髓细胞性白血病中，至少有27种突变(见图1，图2)。它集中在第12外显子，位点在956至971之间，为1到2个四联核苷酸插入或4-5个核苷酸缺失伴随9个核苷酸插入。它导致错义突变，从而缩短阅读框，使DNA结合区失去功能。
目前临床上有多种检测NPM1突变的方法，主要目的均集中在发现初诊或复发白血病患者中是否有NPM1突变。方法包括PCR后一代(Sanger)测序和普通荧光定量PCR。PCR后一代(Sanger)测序方法存在灵敏度问题。由于白血病细胞中的NPM1突变是杂合子，即便样品中100％是白血病细胞PCR后测序阅读结果也不特别清晰，正常和突变碱基交织在一起(见图3(A)～图3(C)和图4)。当整个样本中的白血 病细胞在10％时，几乎就看不出突变。而在白血病患者中，外周血中白血病细胞少于30％并不少见。如大部分急性早幼粒白血病(APL)患者。因此，PCR后Sanger测序方法对NPM1突变的检测甚至不能用于初诊患者的外周血。至于包含了针对A、B、D三个主要突变的的普通荧光定量PCR法则较繁琐且适用面窄。因为它要同时做三个荧光定量PCR，且一旦突变不在三种(而是其它几十种中的一种，约占15％-20％NPM1突变)之内，则结果为假阴性。
至于微小残留病变的检测，PCR后一代(Sanger)测序方法由于灵敏度的关系当然不适合，因为初诊NPM1突变阳性的缓解期患者绝大多数细胞只含有野生型NPM1,PCR后测序结果会覆盖突变型的信号。而针对三个主要突变的的荧光定量PCR法虽也可以用于MRD检测，但它必须首先知道NPM1突变测序结果，否则如上所述会很繁琐(至少做三个独立的实验)且不可靠，因为阴性结果不能排除患者存在三种主要突变外的其它NPM1突变。另外，人们也用ASO-PCR方法进行MRD检测，即根据每个患者测序结果设计特异性的引物或探针，繁琐而昂贵。
现有技术也曾尝试同时采用超过15对以上的引物混合以期一次扩增15种以上突变。但实际操作发现由于多种引物之间的干扰等因素，产生的非特异性扩增严重影响扩增的灵敏度和特异性。因此，这一检测的关键是用尽可能少的引物组合扩增出尽可能多的突变类型，同时保证PCR扩增的灵敏度和特异性(即引物最佳组合)。
现有技术中亟需一种简捷方便、灵敏性高、特异性强、检出结果可靠的NPM1检测方法。
发明内容
针对上述现有技术中的缺陷，本发明提供了引物及其组合、试剂盒及其应用。本发明利用少数几种混合引物一次定量PCR在cDNA水平上特异性检出至少16种NPM1突变。主要针对AML治疗后微小残留病变的检测，也可用于初诊患者鉴定有无NPM1突变。
为了实现上述发明目的，本发明提供以下技术方案：
本发明提供了引物，具有如下所示的核苷酸序列中的任意一项：
Ⅰ、具有SEQ ID NO:1所示的核苷酸序列；
Ⅱ、具有SEQ ID NO:1所示的核苷酸序列经修饰、取代、缺失或添加一个或多个碱基获得的核苷酸序列；
III、与SEQ ID NO:1所示的核苷酸序列具有至少80％同源性的序列；
IV、如Ⅰ、Ⅱ或III所示序列的互补序列。
在本发明的一些具体实施方案中，如SEQ ID NO:1所示的核苷酸序列为：
NCNTCCNCNNCNNNNNNNNNNN；
其中，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。
在本发明的一些具体实施方案中，所述引物具有SEQ ID NO:2～6任意一项所示的核苷酸序列。
在本发明的一些具体实施方案中，引物包括NPM1 type A引物(逆向)，其具有如SEQ ID NO:2所示的核苷酸序列：ACTTCCTCCACTGCCAGACAGA。
在本发明的一些具体实施方案中，引物包括NPM1 type B引物(逆向)，其具有如SEQ ID NO:3所示的核苷酸序列：TCCTCCACTGCCATGCAGAG。
在本发明的一些具体实施方案中，引物包括NPM1 type D引物(逆向)，其具有如SEQ ID NO:4所示的核苷酸序列：TCCTCCACTGCCAGGCAGA。
在本发明的一些具体实施方案中，引物包括NPM1 type DD1引物(逆向)，其具有如SEQ ID NO:5所示的核苷酸序列：TCCTCCACTGCCAAGCAGAG。
在本发明的一些具体实施方案中，引物包括NPM1 typeE引物(逆向)，其具有如SEQ ID NO:6所示的核苷酸序列：ACTTCCTCCACTGCCATACAGA。
本发明还提供了引物组，包括所述的引物、顺向通用引物和/或内对照基因的引物；所述顺向通用引物具有如下所示的核苷酸序列中的任意一项：
V、具有SEQ ID NO:7所示的核苷酸序列；
VI、具有SEQ ID NO:7所示的核苷酸序列经修饰、取代、缺失或添加一个或多个碱基获得的核苷酸序列；
VII、与SEQ ID NO:7所示的核苷酸序列具有至少80％同源性的序列；
VIII、如V、VI或VII所示序列的互补序列。
在本发明的一些具体实施方案中，引物组还包括NPM1顺向通用引物，其具有如SEQ ID NO:7所示的核苷酸序列：GAAGAATTGCTTCCGGATGACTG。
本发明提供的引物可以与所述顺向通用引物组合使用，例如具有如SEQ ID NO:7所示的核苷酸序列的NPM1顺向通用引物分别与具有如SEQ ID NO:2～6所示的核苷酸序列的任一或部分或全部的引物组合，可以分别扩增NPM1A,B,D,DD1,E型突变。
在本发明的一些具体实施方案中，引物组合如下(但不限于如下)：
Figure PCTCN2018095601-appb-000001
Figure PCTCN2018095601-appb-000002
在本发明的一些具体实施方案中，所述内对照基因的引物具有如下所示的核苷酸序列中的任意一项：
IX、具有SEQ ID NO:8或9所示的核苷酸序列；
X、具有SEQ ID NO:8或9所示的核苷酸序列经修饰、取代、缺失或添加一个或多个碱基获得的核苷酸序列；
XI、与SEQ ID NO:8或9所示的核苷酸序列具有至少80％同源性的序列；
XII、如IX、X或XI所示序列的互补序列。
在本发明的一些具体实施方案中，内对照基因的引物包括ABL1引物(顺向)，其具有如SEQ ID NO:8所示的核苷酸序列：GTGGAGATAACACTCTAAGCATAACTAAAGGT。
在本发明的一些具体实施方案中，内对照基因的引物还包括ABL1引物(逆向)，其具有如SEQ ID NO:9所示的核苷酸序列：TGATGTAGTTGCTTGGGACCCA。
本发明还提供了引物组和探针的组合，包括所述的引物组和探针，所述探针具有如下所示的核苷酸序列中的任意一项：
XII、具有SEQ ID NO:10或11所示的核苷酸序列；
XIII、具有SEQ ID NO:10或11所示的核苷酸序列经修饰、取代、缺失或添加一个或多个碱基获得的核苷酸序列；
XIV、与SEQ ID NO:10或11所示的核苷酸序列具有至少80％同源性的序列；
XV、如XII、XIII或XIV所示序列的互补序列。
在本发明的一些具体实施方案中，探针包括NPM1通用探针和ABL1探针。其中，NPM1通用探针为FAM-ACCAAGAGGCTATTCAAG-MGB，具有如SEQ ID NO:10所示的核苷酸序列：ACCAAGAGGCTATTCAAG。ABL1探针为VIC-CATTTTTGGTTTGGGCTTCA-MGB，具有如SEQ ID NO:11所示的核苷酸序列： CATTTTTGGTTTGGGCTTCA。
在此基础上，本发明还提供了所述的引物或所述的引物组或所述的引物组和探针的组合在扩增NPM1基因或其突变中的应用。
本发明还提供了所述的引物或所述的引物组或所述的引物组和探针的组合在制备检测急性髓细胞白血病的试剂盒中的应用。
本发明还提供了所述的引物或所述的引物组或所述的引物组和探针的组合在制备检测急性髓细胞白血病的微小残留病变的试剂盒中的应用。
本发明还提供了试剂盒，包括所述的引物或所述的引物组或所述的引物组和探针的组合以及可接受的试剂。
在本发明的一些具体实施方案中，所述可接受的试剂包括反应液、阴性对照试剂或阳性对照试剂。
在本发明的一些具体实施方案中，所述的引物或所述内对照基因的引物的浓度为4～8pmol/μl；所述顺向通用引物的浓度为20～40pmol/μl；所述的探针的浓度为4～8pmol/μl。
在本发明的一些具体实施方案中，所述试剂盒为real-time PCR试剂盒或ddPCR试剂盒。
本发明还提供了一种检测NPM1突变的方法，所述方法基于real-time PCR扩增或基于ddPCR扩增。
当基于real-time PCR扩增时，其包括如下步骤：
步骤1：获得待测样本的核苷酸(在本发明的一些实施例中为cDNA)；
步骤2：取所述cDNA与所述的试剂盒中的试剂混合，扩增，根据扩增结果获得检测结果。
在本发明的一些具体实施方案中，所述扩增的程序为：
1. 50.0℃2min
2. 95.0℃2min
3. 95.0℃1s
4. 60℃20s
5.GOTO step3,39X
其中各个步骤的温度升降速率为2.74℃/s。
在本发明的一些具体实施方案中，扩增结果为：
根据50、5x10 3、5x10 5、5x10 7copies/uL的NPM1阳性质粒和10 2、10 4、10 6、10 8copies/uL的ABL1质粒样品分别绘制的标准曲线，读取每个样本NPM1及ABL1的拷贝数检测结果。报告值(RQ)计算公式为：RQ＝NPM1mut拷贝数/10 4ABL1拷贝数。
当所述检测基于ddPCR扩增时，其包括如下步骤：
步骤1：获得待测样本的核苷酸(在本发明的一些实施例中为cDNA)；
步骤2：取所述cDNA与所述的试剂盒中的试剂混合，扩增，根据扩增结果获得 检测结果。
在本发明的一些具体实施方案中，所述扩增的程序为：
1. 95.0℃10min
2. 95.0℃30s
3. 60.0℃1min
4.GOTO step2,39X5.98.0℃10min
6. 4.0℃Hold.
其中各个步骤的温度升降速率为2.5℃/s
在本发明的一些具体实施方案中，扩增结果为：
根据微滴分析仪(droplet reader)自动分析的结果，读取样本中突变型NPM1与对照基因ABL1分别的拷贝数浓度并计算的报告值(RQ)，计算公式为：RQ＝NPM1mut拷贝数/10 4ABL1拷贝数。一般以RQ≥200为判定AML病人复发的标准，不同实验室可以根据实际情况建立适合自己实验室的标准。注：以上结果判定建立在阴阳性对照正常的前提下，如果发现阴、阳性对照异常，则需要排除降解或污染等原因后再进行检测。
本发明利用基因突变检测白血病治疗后有无MRD和检测初诊患者有无相关突变的不同点在于：方法必须是突变高度特异的，避免可以检出野生型基因。因为在形态学缓解患者体内，大部分相关基因为野生型，只有极少数突变体的存在。如果这一方法同样可以检出野生型基因，那么PCR扩增后，野生型信号将会覆盖突变信号(见图2)。所以本发明设计了针对每一种野生型特异的引物。引物设计的策略为：在突变上游的内含子区域设计一条公用的上游引物，在上游引物后设计一条同向的公用探针，在突变位置设计针对每一种野生型的特异性下游引物。它包含了突变型插入碱基(4个)及其左右两端的正常序列，每条下游引物长度在20-23nt之间。
考虑到各种NPM1突变对患者的治疗和预后没有特别的差别，一般检测目的只是了解有无突变存在，所以没有必要一一检测。特别是在MRD检测时更是如此。所以尽可能在一个体系中检测出多种突变。然而，多种不同的引物在同一PCR体系中必然互相干扰，从而影响PCR的灵敏度和特异性(这里的特异性是指区别突变和野生型的特异性)。所以技术关键是使用尽可能少的引物组合检测出尽可能多的突变种类。
申请人发现如图1所示，在已知的NPM1突变中，至少18种插入位点相同，且插入序列之间有部分重合。本发明设计的引物之间在突变位置两侧的非突变区域有至少15个碱基是一致的，并且本发明设计的5对引物中分别包含的突变插入序列中至少有一种与其他类型突变的插入序列的差别不大于2个碱基，大多数差别为1个碱基。经多次反复试验，结果表明使用上述扩增NPM1A、B、D、DD1、E型突变的5对引物可以使体系达到最优化。更为重要的是，将上述引物放入一个PCR体系，在灵敏度不变的情况下它不仅可以特异性扩增上述五种突变，还能扩增其它至少10种突变，取得了预料不到的技术效果(以上突变15种扩增于本发明构建的质粒，具体突变为图1突变列表中的前十五个，突变构建详见实施例1)。进一步利用这种方法对至少 24例临床患者的测试也达到了非常显著的效果(见实施例5)，且通过测序发现这一引物组合还能扩增出除上述15种突变中的另外两种突变。
本发明的有益效果：
1)本发明利用少数几种混合引物一次实时荧光定量PCR(real-time PCR)在cDNA水平上特异性检出多至17种NPM1突变(这里的特异性是指区别突变和野生型的特异性)，方法简捷方便；利用ddPCR(Digital Droplet PCR，数字微滴PCR)一次实时定量，在cDNA水平上特异性检出至少17种微量NPM1突变。对于AML治疗后微小残留病变的检测，以及初诊患者鉴定有无NPM1突变等具有十分显著的临床意义，价值重大，弥补现有检测方法中的不足。
2)本发明的检测方法灵敏度高，无论是real-time PCR还是ddPCR，都在多种NPM1突变检测中，可检出低至10 2拷贝和10 -5个细胞；
3)本发明的检测方法针对突变型检测特异性好，本发明的引物均不能扩增野生型NPM1，但可检出至少17种超过95％的NPM1突变，有效避免假阳性结果。
4)、本发明提供的ddPCR对检测对象进行绝对定量，从而更加直观、精确的检测NPM1突变，尤其对微量NPM1突变效果更为明显，为白血病微小残留病变检测提供了更加准确，可靠的方法。
5)本发明的检测方法对具有NPM1突变的白血病患者治疗后的微小残留病变跟踪具有明显优势。由于目前急性粒细胞性白血病(AML)没有统一的微小残留病变跟踪方法，常见的利用融合基因跟踪方法，每种融合基因只能跟踪最多10％AML患者，而NPM1突变阳性患者占AML的30％，特别的，NPM1突变占没有太多生物标志的染色体正常的AML的60％-70％。因此，他适用面较广，针对性强。
附图说明
为了更清楚地说明本发明实施例或现有技术中的技术方案，下面将对实施例或现有技术描述中所需要使用的附图作简单地介绍。
图1示NPM1的27种突变；
图2示正常和异常NPM1基因和蛋白质示意图，**第12外显子中突变位置；
图3示针对NPM1基因的PCR后Sanger测序结果；OCI-AML3为含有NPM1A型突变的白血病细胞系；插入碱基为CAGA；HL-60为含NPM1野生型基因的白血病细胞系；图3(A)示OCI-AML3，测序结果显示即使在OCI-AML3细胞系中，突变结果也不甚清晰；图3(B)显示当OCI-AML3和HL-60 1：9混合后(即灵敏度为10％)，突变已很难辩别；图3(C)示HL-60(阴性对照)；
图4示PCR后Sanger测序结果(仪器：ABI Prism 3700)；
图5示15种突变的质粒的构建图谱；
图6示每个引物针对的突变体的检测特异性；
图7示基于real-time PCR的混合引物扩增不同种突变型的特异性：混合引物不仅能针对示突变型A、B、D、DD1、E扩增，同时还能针对其他突变型F、G、H、I、J、K、 L、M、N和O等有效扩增，表明本发明设计的混合引物针对突变体的检测是十分特异的(此处的特异是指区别突变和野生型的特异)；
图8示基于real-time PCR的混合引物扩增突变型A、B、D、DD1、E的灵敏度；
图9示基于real-time PCR的混合引物扩增突变型F、G、H、I、J的灵敏度；
图10示基于real-time PCR的混合引物扩增突变型K、L、M、N、O的灵敏度；
图11示利用本发明提供的real-time PCR试剂盒检测不同梯度typeA突变型细胞系；
图11(A)示直至突变比例到达10 -6，本发明试剂盒测试的突变比例值都能与阴性对照(完全的野生型NPM1细胞系)的检测值明显地区分开来，因此，本发明试剂盒的检测下限为10 -5-10 -6之间；
图11(B)示以检测值与理论值分别取对数，在1-10 -5的突变比例区间内，检测值与理论值的线性相关性非常好(相关性系数r＝0.99782)；
图12示临床病例的real-time PCR检测结果；
图13示NPM1引物及探针的位置示意图；
图14示基于ddPCR的混合引物扩增不同种突变型的特异性；
图15示基于ddPCR的混合引物扩增突变型A、B、D、DD1、E的灵敏度；
图16示基于ddPCR的混合引物扩增突变型F、G、H、I、J的灵敏度；
图17示基于ddPCR的混合引物扩增突变型K、L、M、N、O的灵敏度；
图18示本发明提供的试剂盒灵敏度测试结果；其中，图18(A)示使用不同稀释度的OCI-AML3细胞系通过ddPCR对NPM1试剂盒进行灵敏度测试的结果；图18(B)示使用A型突变细胞系的梯度比率对试剂盒进行灵敏度测试的结果；
图19示临床样品的ddPCR和qPCR检测结果的比较结果。
具体实施方式
本发明公开了引物及其组合、试剂盒及其应用，本领域技术人员可以借鉴本文内容，适当改进工艺参数实现。特别需要指出的是，所有类似的替换和改动对本领域技术人员来说是显而易见的，它们都被视为包括在本发明。本发明的方法及应用已经通过较佳实施例进行了描述，相关人员明显能在不脱离本发明内容、精神和范围内对本文所述的方法和应用进行改动或适当变更与组合，来实现和应用本发明技术。
本发明所采用的技术方案如下：
本发明根据NPM1基因在AML患者中的不同突变形式及比例设计6条特异性引物及1条特异性探针，同时设计有内对照基因ABL1特异性引物2条及特异性探针1条。详见表1。
表1
Figure PCTCN2018095601-appb-000003
Figure PCTCN2018095601-appb-000004
NPM1引物及探针的位置示意图如13所示：
如图13所示，上游引物设计在11号外显子上，探针横跨11、12号外显子，下游引物位于12号外显子上游、包含插入突变位点。
本发明提供了一种用实时荧光定量PCR技术定量检测人体内NPM1基因突变的试剂盒，试剂盒内容包括：
阴性对照试剂：含有突变片段的原始质粒DNA。
阳性对照试剂：含有突变片段的质粒DNA。
引物混合物：SEQ ID No.2～6、SEQ ID No.7、SEQ ID No.8～9；
探针1：NPM1探针：SEQ ID No.10；
探针2：ABL1探针：SEQ ID No.11。
反应液。
实验具体包括如下步骤：
(1)RNA提取：提取待测样品的RNA，所述待测样本为临床病人体内含有白细胞或骨髓细胞的样本；
(2)逆转录反应：在PCR管配制反应体系：oligo dT(50μM)1μl，dNTP(10mM)1μl，模板RNA 1-8μl(RNA总量在100ng-1μg)，用灭菌去离子水补齐到10μl，盖上管盖，将上述反应管于65℃反应5min，向反应管中加入5x buffer 4μl，RNase inhibitor(40U/μL)0.5μl，逆转录酶(200U/μL)1μl，用灭菌去离子水补齐到20μl；放入 PCR仪中，按照以下程序进行逆转录：30℃10min；42℃30-60min；95℃5min。
(3)real-time PCR反应：在PCR管配制反应体系：商业化Taq酶PCR Mix(2x)10μl，引物混合液1.6μl，探针1 0.5μl，探针2 0.5μl，模板cDNA/阴性质控品/阳性质控品分别稀释为50、5x10 3、5x10 5、5x10 7拷贝(NPM1阳性质粒)/分别稀释为10 2、10 4、10 6、10 8copies/uL(ABL1质粒)2μl，用灭菌去离子水补齐到20μl；
上述引物混合液含有上述8种特异性引物(SEQ ID No.7、SEQ ID No.2～6、SEQ ID No.8～9)，其中，引物3～9(SEQ ID No.2～6、SEQ ID No.8～9)的浓度为4～8pmol/μl，引物1(SEQ ID No.7)的浓度为20～40pmol/μl，探针1、2(SEQ ID No.10、SEQ ID No.11)的浓度为4～8pmol/μl。
将上述反应管放入实时荧光定量PCR仪按照以下程序进行扩增及检测反应：
1. 50.0℃2min
2. 95.0℃2min
3. 95.0℃1s
4. 60℃20s
5.GOTO step3,39X
其中各个步骤的温度升降速率为2.74℃/s
(4)结果判断：
根据50、5x10 3、5x10 5、5x10 7copies/uL的NPM1阳性质粒和10 2、10 4、10 6、10 8copies/uL的ABL1质粒样品分别绘制的标准曲线，读取每个样本NPM1及ABL1的拷贝数检测结果。报告值(RQ)计算公式为：RQ＝NPM1mut拷贝数/10 4ABL1拷贝数。实验结果和预后关系的判断不同实验室可以根据实际情况建立适合自己实验室的标准。
本发明还提供一种ddPCR试剂盒。
所述试剂盒包括本发明提供的所述的引物或所述的引物组或所述的引物组和探针的组合。
在本发明的一些具体实施方案中，ddPCR试剂盒包括：
阴性质控品：含有NPM1突变的cDNA；
阳性质控品：含有正常NPM1的cDNA；本发明提供的引物组；
探针1：本发明提供的NPM1探针；
探针2：本发明提供的ABL1探针；
反应液。
试验具体包括如下步骤：
(1)RNA提取：提取待测样品的RNA，所述待测样本为临床病人体内含有白细胞或骨髓细胞的样本；
(2)逆转录反应：在PCR管配制反应体系：oligo dT(50μM)1μl，dNTP(10mM)1μl，模板RNA 1-8μl(RNA总量在100ng-1μg)，用灭菌去离子水补齐到10μl，盖上管盖，将上述反应管于65℃反应5min，向反应管中加入5x buffer 4μl，RNase inhibitor(40U/μL)0.5μl，逆转录酶(200U/μL)1μl，用灭菌去离子水补齐到20μl；放入 PCR仪中，按照以下程序进行逆转录：30℃10min；42℃30-60min；95℃5min。
(3)配制PCR反应体系：在PCR管配制反应体系：商业化ddPCR Mix for Probes(2x,No dUTP)10μl，引物混合液3.6μl，探针1 0.5μl，探针2 0.5μl，模板cDNA/阴性对照试剂/阳性对照试剂2μl，用灭菌去离子水补齐到20μl；
上述引物混合液含有上述8种特异性引物(SEQ ID No.7、SEQ ID No.2～6、SEQ ID No.8～9)，其中，引物3～9(SEQ ID No.2～6、SEQ ID No.8～9)的浓度为4～8pmol/μl、引物1(SEQ ID No.7)的浓度为20～40pmol/μl，探针1、2(SEQ ID No.10、SEQ ID No.11)的浓度为4～8pmol/μl；
(4)微滴生成：根据QX200说明书将(3)中20uL反应体系与70uL微滴生成油一起放入微滴发生器中进行微滴生成，最终生成体积约为40uL。转入配套使用的96孔板中，用热封膜封口。
(5)PCR扩增：将上述反应管放入普通PCR仪按照以下程序进行扩增及检测反应：
1. 95.0℃10min
2. 95.0℃30s
3. 60.0℃1min
4.GOTO step2,39X5.98.0℃10min
6. 4.0℃Hold.
其中各个步骤的温度升降速率为2.5℃/s
(6)微滴读取：将PCR后的96孔板放入微滴读取仪中，根据仪器说明书进行操作，程序结束后仪器会自动分析结果。
(4)结果判断：
根据微滴分析仪(droplet reader)自动分析的结果，读取样本中突变型NPM1与对照基因ABL1分别的拷贝数浓度并计算的报告值(RQ)，计算公式为：RQ＝NPM1mut拷贝数/10 4ABL1拷贝数。一般以RQ≥200为判定AML病人复发的标准，不同实验室可以根据实际情况建立适合自己实验室的标准。注：以上结果判定建立在阴阳性对照正常的前提下，如果发现阴、阳性对照异常，则需要排除降解或污染等原因后再进行检测。
注：以上结果判定建立在阴阳性对照正常的前提下，如果发现阴、阳性对照异常，则需要排除降解或污染等原因后再进行检测。
本发明提供的引物、试剂盒及其应用中所用原料、辅料及试剂均可由市场购得。
下面结合实施例，进一步阐述本发明：
实施例1 15种突变的质粒的构建
(1)扩增含有插入突变的NPM1片段：在如图5所示的插入片段中的两端分别设计引物，在突变位点设计含有缺失突变片段的双向引物，利用搭桥PCR的方法构建含有插入突变的NPM1片段；
(2)连接、转化反应：用商业化试剂盒连接NPM1突变片段和PMD19-T载体并转化至DH5α大肠杆菌中；
(3)挑取阳性克隆、保种：将大肠杆菌涂布在含有氨苄青霉素的LB琼脂糖培养基上37℃过夜培养，挑取单菌落，取其中部分用插入片段中的两端特异性的引物扩增，能够扩增出目的条带大小的克隆菌落送测序确认为阳性后用25％的甘油在-20℃进行保存；
(4)提取质粒：用商业化的试剂盒提取质粒。
质粒构建图谱见图5。
实施例2单独利用每个引物real-time PCR检测每个突变体的特异性
分组：
实验组：实施例1构建获得的15中突变的质粒；
阴性对照为野生型NPM1；空白对照为水。
(1)质粒稀释：将野生型(WT)、typeA、typeB、typeD、typeDD1、typeE突变型NPM1质粒分别稀释至2.7x10 5copies/uL。
(2)real-time PCR反应：在PCR管配制反应体系：商业化Taq酶PCR Mix(2x)10μl，引物X 0.8μl，引物1 0.8μl，探针1 0.5μl，(1)中稀释后的突变型质粒/(1)中稀释后的野生型质粒/水1μl，从HL60细胞系提取的cDNA 1uL，用灭菌去离子水补齐到20μl；
其中，引物X为检测不同突变型质粒时对应的引物(SEQ ID No.2～6)的浓度为4～8pmol/μl，引物1(SEQ ID No.7)的浓度为4～8pmol/μl，探针1(SEQ ID No.10)的浓度为4～8pmol/μl。
将上述反应管放入实时荧光定量PCR仪按照以下程序进行扩增及检测反应：
1. 50.0℃2min
2. 95.0℃2min
3. 95.0℃1s
4. 60℃20s
5.GOTO step3,39X。
其中1、2步骤的温度升降速率为2.74℃/s，3、4、5步骤的温度升降速率为2.12℃/s。
(4)结果判断：
通过比较不同突变型引物检测相应突变型质粒与野生型质粒的扩增曲线的Ct值的差别评价引物的特异性。
试验结果表明(图6)，不同突变型引物检测相应突变型质粒时的Ct值在20-22之间，而作为阴性对照的野生型质粒与空白对照的水的Ct＞37，说明在相同浓度的模板下，突变型模板的扩增效率是野生型的扩增效率的至少2 15(32768)倍。因此，我们设计的每个引物针对的特定突变体的检测是十分特异的。
实施例3利用混合引物对十五种突变体进行real-time PCR检测
分组：
实验组：实施例1构建获得的15中突变的质粒；
阴性对照为野生型NPM1；空白对照为水。
(1)质粒稀释：将野生型(WT)、typeA、typeB、typeD、typeDD1、typeE、typeF、typeG、typeH、typeI、typeJ、typeK、typeL、typeM、typeN、typeO突变型NPM1质粒分别稀释至5个梯度：2.7x10 5copies/uL、2.7x10 4copies/uL、2.7x10 3copies/uL、2.7x10 2copies/uL、2.7x10 2copies/uL、2.7x10 1copies/uL。
(2)real-time PCR反应：在PCR管配制反应体系：商业化Taq酶PCR Mix(2x)10μl，引物混合液1.6μl，探针1 0.5μl，模板cDNA/阴性对照试剂/阳性对照试剂2μl，用灭菌去离子水补齐到20μl；
上述引物混合液含有上述6种特异性引物(SEQ ID No.7、SEQ ID No.2～6)，其中，引物3～9(SEQ ID No.2～6)的浓度为4～8pmol/μl，引物1(SEQ ID No.7)的浓度为20～40pmol/μl，探针2SEQ ID No.10)的浓度为4～8pmol/μl。
将上述反应管放入实时荧光定量PCR仪按照以下程序进行扩增及检测反应：
1. 50.0℃2min
2. 95.0℃2min
3. 95.0℃1s
4. 60℃20s
5.GOTO step3,39X。
其中1、2步骤的温度升降速率为2.74℃/s，3、4、5步骤的温度升降速率为2.12℃/s。
(4)结果判断：
通过比较混合引物检测相应突变型质粒与野生型质粒的扩增曲线的Ct值的差别评价引物的特异性。通过比较混合引物检测梯度突变型质粒与野生型质粒的扩增曲线的Ct值的是否有明显差别评价引物的灵敏度
试验结果表明(表2及图7)，混合引物检测相应突变型质粒时的Ct值大部分(typeA、typeB、typeD、typeDD1、typeE、typeF、typeG、typeH、typeI、typeJ、typeL、typeM、typeO)在20-22之间，typeK在25-27之间，typeN在22-24之间，表明本发明的混合引物不仅能针对突变型A、B、D、DD1、E扩增，同时还能针对其他突变型F、G、H、I、J、K、L、M、N和O等有效扩增，表明该混合引物具有良好的针对多种突变型扩增的特性。此外，作为阴性对照的野生型质粒与空白对照的水的Ct＞35。说明在相同浓度的模板下，对大部分突变型而言突变型模板的扩增效率是野生型的扩增效率的至少2 13(8196)倍。因此，本发明设计的混合引物针对的突变体的检测是十分特异的(此处的特异是指区别突变和野生型的特异)。
试验结果表明(表3及图8-10)，混合引物检测相应突变型质粒时的Ct值大部分(typeA、typeB、typeD、typeDD1、typeE、typeF、typeG、typeH、typeI、typeJ、typeL、 typeM、typeO)的灵敏度至少可达到2.7x10 2copies/uL，而检测typeK和typeN的灵敏度至少可达到2.7x10 3copies/uL。
表2混合引物扩增不同种突变型的特异性(对应图7)
Figure PCTCN2018095601-appb-000005
表3混合引物扩增突变型A、B、D、DD1、E的灵敏度(对应图8、9、10)
Figure PCTCN2018095601-appb-000006
Figure PCTCN2018095601-appb-000007
实施例4基于real-time PCR的灵敏度实验
分组：
实验组：实施例1构建获得的15种突变的质粒；
阴性对照为野生型NPM1；空白对照为水.
(1)RNA提取：提取待测样品(以1:0、1:10、1:100、1:1000、1:10000、1:100000、1:1000000、1:10000000、0:1混合的NPM1突变的细胞系与野生型NPM1的细胞系)的RNA，所述待测样本为临床病人体内含有白细胞或骨髓细胞的样本；
(2)逆转录反应：在PCR管配制反应体系：oligo dT(50μM)1μl，dNTP(10mM)1μl，模板RNA 1-8μl(RNA总量在100ng-1μg)，用灭菌去离子水补齐到10μl，盖上管盖，将上述反应管于65℃反应5min，向反应管中加入5x buffer 4μl，RNase inhibitor(40U/μL)0.5μl，逆转录酶(200U/μL)1μl，用灭菌去离子水补齐到20μl；放入PCR仪中，按照以下程序进行逆转录：30℃10min；42℃30-60min；95℃5min。
(3)real-time PCR反应：在PCR管配制反应体系：商业化Taq酶PCR Mix(2x) 10μl，引物混合液1.6μl，探针1 0.5μl，探针2 0.5μl，模板cDNA /ddH 2O 2μl，用灭菌去离子水补齐到20μl；
上述引物混合液含有上述8种特异性引物(SEQ ID No.7、SEQ ID No.2～6、SEQ ID No.8～9)，其中，引物3～9(SEQ ID No.2～6、SEQ ID No.8～9)的浓度为4～8pmol/μl，引物1(SEQ ID No.7)的浓度为20～40pmol/μl，探针1、2(SEQ ID No.10、SEQ ID No.11)的浓度为4～8pmol/μl。
将上述反应管放入实时荧光定量PCR仪按照以下程序进行扩增及检测反应：
1. 50.0℃2min
2. 95.0℃2min
3. 95.0℃1s
4. 60℃20s
5.GOTO step3,39X。
其中1、2步骤的温度升降速率为2.74℃/s，3、4、5步骤的温度升降速率为2.12℃/s。
(4)结果判断：
通过比较试剂盒检测不同突变比例的样本与阴性样本的扩增曲线的Ct值的差别评价试剂盒的灵敏度。
图11(A)结果表明，直至突变比例到达10 -6，本发明试剂盒测试的突变比例值都能与阴性对照(完全的野生型NPM1细胞系)的检测值明显地区分开来，因此，本发明试剂盒的检测下限为10 -5-10 -6之间。
如图11(B)所示，以检测值与理论值分别取对数，在1-10 -5的突变比例区间内，检测值与理论值的线性相关性非常好(相关性系数r＝0.99782)。
实施例5基于real-time PCR对3例正常人样本及24例含有不同比例NPM1突变的患者临床样本进行检测
(1)骨髓组织处理与提取
骨髓组织的提取采取TRIzol进行提取，具体提取操作步骤请参见《分子克隆指南》。所提RNA溶于DEPC处理水中，经nanodrop超微量紫外分光光度计检测质量，确定其浓度。
(2)逆转录(浓度)
在200μl PCR管中配制反应体系：Oligo dT(50μM)1μl，dNTP(10mM)1μl，模板RNA 1-8μl(RNA总量在100ng-1μg)，用灭菌去离子水补齐到10μl；将样品于65℃反应5min；向反应管中加入5x buffer 4μl，RNase inhibitor(40U/μL)0.5μl，逆转录酶(200U/μL)1μl，用灭菌去离子水补齐到20μl；样品放入PCR仪中，按照以下程序进行逆转录：30℃10min；42℃30-60min；95℃5min。
(3)建立PCR扩增反应体系
将(2)中所得到的cDNA模板/阴性质控品/阳性质控品/ddH2O/分别稀释为50、5x10 3、5x10 5、5x10 7copies/uL的NPM1阳性质粒/分别稀释为10 2、10 4、10 6、10 8copies/uL 的ABL1质粒用作实时荧光PCR扩增的模板，按照以下扩增体系进行PCR扩增：
商业化Taq酶PCR Mix(2x)10μl
引物混合液1.6μl
探针1 0.5μl
探针2 0.5μl
cDNA 2μl
用灭菌去离子水补齐到20μl。
上述引物混合液含有上述8种特异性引物(SEQ ID No.7、SEQ ID No.2～6、SEQ ID No.8～9)，其中，引物3～9(SEQ ID No.2～6、SEQ ID No.8～9)的浓度为4～8pmol/μl，引物1(SEQ ID No.7)的浓度为20～40pmol/μl，探针1、2(SEQ ID No.10、SEQ ID No.11)的浓度为4～8pmol/μl。
PCR反应条件如下：
1. 50.0℃2min
2. 95.0℃2min
3. 95.0℃1s
4. 60℃20s
5.GOTO step3,39X。
其中1、2步骤的温度升降速率为2.74℃/s，3、4、5步骤的温度升降速率为2.12℃/s。
荧光信号的收集定为FAM，数据采集定在60℃。
实验结束后，按照以下步骤进行分析、判定：
根据50、5x10 3、5x10 5、5x10 7copies/uL的NPM1阳性质粒和10 2、10 4、10 6、10 8copies/uL的ABL1质粒样品分别绘制的标准曲线，读取每个样本NPM1及ABL1的拷贝数检测结果。报告值(RQ)计算公式为：RQ＝NPM1mut拷贝数/10 4ABL1拷贝数。一般以RQ值多寡为判定AMLMRD状态的标准，不同实验室可以根据实际情况建立适合自己实验室的标准。
结果参见表4：
表4
Figure PCTCN2018095601-appb-000008
Figure PCTCN2018095601-appb-000009
如表4及图12所示，以OCI-AML3细胞系为阳性对照，HL-60细胞系为阴性对照，3例正常人样本中，检测结果(RQ值)均低于一般阳性判断值(200)，检测结果在正常范围。经其它分子检测确认NPM1突变阳性的24例病人中，本发明试剂盒检测结果判定为阳性的(RQ≥200)的病人有23例，其中No.11267患者二代测序证实NPM1突变率为1.07％，本发明的RQ值为8745，No.1881患者二代测序证实NPM1 突变率为9.43％，我们的RQ值为286477。No.2474和13513虽然后二代测序证实突变率也在10％以下，但RQ值均大于100000.证明本发明的灵敏度很高。仅1例(No.2079)检测结果为阴性，经后二代测序分析表明此病例的突变类型不在本发明试剂盒覆盖检测的突变类型范围内。另外，样本编号为No.13894和No.2569两个样本经一代测序分析表明其突变类型不在本发明已检测的突变范围类型之中，但仍可检测到，由此验证本发明之前“这一方法能扩增除已验证的突变类型以外的更多的NPM1突变”。来自HL60细胞系的cDNA(阴性对照)检测值为1，远远低于一般阳性判断值(200)，可见该试剂盒的灵敏度及特异性能够完全满足临床检测需求。
实施例6基于上述引物体系的ddPCR检测试验
本发明基于上述引物体系还建立一套ddPCR检测试剂盒以及检测方法，具体检测步骤如下:
(1)RNA提取：提取待测样品的RNA，所述待测样本为临床病人体内含有白细胞或骨髓细胞的样本；
(2)逆转录反应：在PCR管配制反应体系：oligo dT(50μM)1μl，dNTP(10mM)1μl，模板RNA 1-8μl(RNA总量在100ng-1μg)，用灭菌去离子水补齐到10μl，盖上管盖，将上述反应管于65℃反应5min，向反应管中加入5x buffer 4μl，RNase inhibitor(40U/μL)0.5μl，逆转录酶(200U/μL)1μl，用灭菌去离子水补齐到20μl；放入PCR仪中，按照以下程序进行逆转录：30℃10min；42℃30-60min；95℃5min。
(3)配制PCR反应体系：在PCR管配制反应体系：商业化ddPCR Mix for Probes(2x,No dUTP)10μl，引物混合液3.6μl，探针1 0.5μl，探针2 0.5μl，模板cDNA/阴性对照试剂/阳性对照试剂2μl，用灭菌去离子水补齐到20μl；
上述引物混合液含有上述8种特异性引物(SEQ ID No.7、SEQ ID No.2～6、SEQ ID No.8～9)，其中，引物3～9(SEQ ID No.2～6、SEQ ID No.8～9)的浓度为4～8pmol/μl、引物1(SEQ ID No.7)的浓度为20～40pmol/μl，探针1、2(SEQ ID No.10、SEQ ID No.11)的浓度为4～8pmol/μl；
(4)微滴生成：根据QX200说明书将(3)中20uL反应体系与70uL微滴生成油一起放入微滴发生器中进行微滴生成，最终生成体积约为40uL。转入配套使用的96孔板中，用热封膜封口。
(5)PCR扩增：将上述反应管放入普通PCR仪按照以下程序进行扩增及检测反应：
1. 95.0℃10min
2. 95.0℃30s
3. 60.0℃1min
4.GOTO step2,39X5.98.0℃10min
6. 4.0℃Hold.
其中各个步骤的温度升降速率为2.5℃/s
(6)微滴读取：将PCR后的96孔板放入微滴读取仪中，根据仪器说明书进行操作，程序结束后仪器会自动分析结果。
(7)结果判断：
根据微滴分析仪(droplet reader)自动分析的结果，读取样本中突变型NPM1与对照基因ABL1分别的拷贝数浓度并计算的报告值(RQ)，计算公式为：RQ＝NPM1mut拷贝数/10 4ABL1拷贝数。一般以RQ≥200为判定AML病人复发的标准，不同实验室可以根据实际情况建立适合自己实验室的标准。注：以上结果判定建立在阴阳性对照正常的前提下，如果发现阴、阳性对照异常，则需要排除降解或污染等原因后再进行检测。
利用本发明试剂盒对同等量(理论拷贝数为27000)的图5所示15种突变分别进行了特异性检测。实验结果表明，检测不同突变型质粒时的拷贝数在1394-14800，远远大于阴性对照的29及空白对照的20，说明本发明提供的引物组针对的突变体的检测是十分特异的(如图14所示)。注，测量拷贝数与理论拷贝数的不一致也暗示根据质粒浓度计算质粒拷贝数及稀释以达到理论浓度会有一定的误差(质粒在稀释过程中形成的误差及低浓度下的降解)。
表5引物组扩增不同种突变型的特异性(对应图14)
Figure PCTCN2018095601-appb-000010
进一步，利用本发明提供的试剂盒，对图5所述15种突变进行了灵敏度检测。 实验结果表明，检测不同突变型质粒时的灵敏度在270-2700拷贝之间，说明本发明提供的引物组针对的突变体的检测十分灵敏(如图15～17所示)。
表6混合引物扩增不同突变型的灵敏度(对应图15-17)
Figure PCTCN2018095601-appb-000011
Figure PCTCN2018095601-appb-000012
Figure PCTCN2018095601-appb-000013
将含有NPM1突变(typeA)的细胞系(OCI-AML3)与野生型NPM1的细胞系(HL60)按照不同比例混合，利用本发明提供的试剂盒和上述实验步骤进行检测，以测试试剂盒的灵敏度。直至突变比例到达10 -7，我们试剂盒测试的突变比例值都能与阴性对照(完全的野生型NPM1细胞系)的检测值明显地区分开来。实验结果如图18(A)所示，直至突变比例降到10 -4，检测结果的梯度十分好。因此，本发明提供的试剂盒的检测下限至少为10 -7
表7试剂盒灵敏度测试(对应图18(A)及18(B))
Figure PCTCN2018095601-appb-000014
Figure PCTCN2018095601-appb-000015
利用本发明提供的试剂盒和上述实验步骤，本发明对14例含有不同比例NPM1突变的患者临床样本进行了检测，并与qPCR方法检测的结果进行比对。如表8及图19所示，两种检测方法的结果具有高度的一致性。
表8
Figure PCTCN2018095601-appb-000016
Figure PCTCN2018095601-appb-000017
以上对本发明所提供的引物、引物组合、试剂盒及其应用进行了详细介绍。本文应用了具体个例对本发明的原理及实施方式进行了阐述，以上实施例的说明只是用于帮助理解本发明的方法及其核心思想。应当指出，对于本技术领域技术人员来说，在不脱离本发明原理的前提下，还可以对本发明进行若干改进和修饰，这些改进和修饰也落入本发明权利要求的保护范围内。
Figure PCTCN2018095601-appb-000018
Figure PCTCN2018095601-appb-000019
Figure PCTCN2018095601-appb-000020

Claims (10)

  1. 引物，其特征在于，具有如下所示的核苷酸序列中的任意一项：
    Ⅰ、具有SEQ ID NO:1所示的核苷酸序列；
    Ⅱ、具有SEQ ID NO:1所示的核苷酸序列经修饰、取代、缺失或添加一个或多个碱基获得的核苷酸序列；
    III、与SEQ ID NO:1所示的核苷酸序列具有至少80％同源性的序列；
    IV、如Ⅰ、Ⅱ或III所示序列的互补序列。
  2. 根据权利要求1所述的引物，其特征在于，所述引物具有SEQ ID NO:2～6任意一项所示的核苷酸序列。
  3. 引物组，其特征在于，包括如权利要求1或2任一所述引物(包括任一所述引物或其组合)、顺向通用引物和/或内对照基因的引物；所述顺向通用引物具有如下所示的核苷酸序列中的任意一项：
    V、具有SEQ ID NO:7所示的核苷酸序列；
    VI、具有SEQ ID NO:7所示的核苷酸序列经修饰、取代、缺失或添加一个或多个碱基获得的核苷酸序列；
    VII、与SEQ ID NO:7所示的核苷酸序列具有至少80％同源性的序列；
    VIII、如V、VI或VII所示序列的互补序列。
  4. 引物组和探针的组合，其特征在于，包括如权利要求3所述的引物组和探针，所述探针具有如下所示的核苷酸序列中的任意一项：
    XII、具有SEQ ID NO:10或11所示的核苷酸序列；
    XIII、具有SEQ ID NO:10或11所示的核苷酸序列经修饰、取代、缺失或添加一个或多个碱基获得的核苷酸序列；
    XIV、与SEQ ID NO:10或11所示的核苷酸序列具有至少80％同源性的序列；
    XV、如XII、XIII或XIV所示序列的互补序列。
  5. 如权利要求1或2所述的引物或如权利要求3所述的引物组或如权利要求4所述的引物组和探针的组合在扩增和/或检测NPM1基因或其突变中的应用。
  6. 如权利要求1或2所述的引物或如权利要求3所述的引物组或如权利要求4所述的引物组和探针的组合在制备检测急性髓细胞白血病、急性髓细胞白血病治疗后的微小残留病变、以及初诊患者鉴定有无NPM1突变的试剂盒中的应用。
  7. 试剂盒，其特征在于，包括如权利要求1或2所述的引物或如权利要求3所述的引物组或如权利要求4所述的引物组和探针的组合以及可接受的试剂。
  8. 根据权利要求7所述的试剂盒，其特征在于，如权利要求1或2所述的引物的浓度为4～8pmol/μl。
  9. 根据权利要求8所述的试剂盒，其特征在于，所述内对照基因的引物的浓度为4～8pmol/μl；所述顺向通用引物的浓度为20～40pmol/μl；所述的探针的浓度为4～8pmol/μl；优选的，所述试剂盒为real-time PCR试剂盒和/或ddPCR试剂盒。
  10. 一种检测NPM1突变的方法，其特征在于，包括如下步骤：
    步骤1：获得待测样本的核苷酸；
    步骤2：取所述核苷酸，利用权利要求3所述引物组进行扩增，或与如权利要求8所述的试剂盒中的试剂混合，扩增，根据扩增结果获得检测结果。
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