CN113249475B - Drop-off ddPCR method and kit for quantitatively detecting NPM1 gene mutation

Info

Abstract

Description

Claims

CN113249475B

Publication number: CN113249475B
Application number: CN202110484036.1A
Authority: CN
Inventors: 金晔; 钱军; 林江; 徐子浚; 闻向梅; 马吉春
Original assignee: Zhenjiang First Peoples Hospital
Current assignee: Zhenjiang First Peoples Hospital
Priority date: 2021-04-30
Filing date: 2021-04-30
Publication date: 2022-02-11
Anticipated expiration: 2041-04-30
Also published as: CN113249475A

The invention relates to a detection method and a kit for detecting NPM1 gene mutation based on drop-offddPCR, wherein primers and probes are designed according to a DNA sequence of an NPM1 gene, two wild probes are designed aiming at a 12 th exon mutation hotspot of an NPM1 gene, one wild probe is positioned on the mutation hotspot, the other wild probe is positioned outside the mutation hotspot, and when the mutation hotspot has insertion, substitution, deletion and other mutations, the wild probe positioned at the mutation hotspot cannot be tightly combined with a template, so that the kit can detect various mutations of the 12 th exon mutation hotspot of the NPM1 gene only by using two pairs of primer probes, has high sensitivity and can be used for MRD monitoring.

Drop-off ddPCR method and kit for quantitatively detecting NPM1 gene mutation

Technical Field

The invention relates to the technical field of biology, in particular to a method and a kit for quantitatively detecting NPM1 gene mutation.

Background

The nucleophosmin 1 (NPM 1) gene is located on human chromosome 5q35, and has 12 exons in total, and the protein is composed of 294 nucleotides. The NPM1 mutation is one of the most common genetic mutations in Acute Myeloid Leukemia (AML). The detection rate of NPM1 mutation in AML cases was 25% -35%, in cytogenetically normal AML (CN-AML) was over 50%, and was also found in myeloproliferative and myelodysplastic diseases. To date, approximately 50 NPM1 exon 12 mutations have been found, with the majority of mutations being insertions between nucleotides 863 and 864. The NPM1 mutation is very stable during the course of disease and is therefore one of the important molecular markers for better diagnosis and disease monitoring in AML.

Minimal Residual Disease (MRD) refers to a microscopic lesion in a hematological tumor that achieves morphological remission, is clinically asymptomatic, but remains in vivo after treatment. MRD detection has important guiding significance for prognosis judgment and layered treatment of AML, and in recent years, the NPM1 mutation level has been used as one of important indexes for MRD monitoring of AML patients. There are studies showing that post-treatment MRD levels correlate with prognosis, and that after induction of chemotherapy in AML patients with NPM1 mutations, NPM1 mutant genes turned negative or at low levels with lower recurrence rates and longer Overall Survival (OS) than when MRD was at high levels.

At present, various methods for detecting NPM1 gene mutation are clinically used, and the main methods include Sanger sequencing, second-generation sequencing, real-time fluorescence quantitative PCR and the like. Sanger sequencing is considered as a gold standard for detecting gene mutations, but has low sensitivity and is not suitable for clinical MRD detection. The second generation sequencing has high flux and high sensitivity, but the cost is high, the operation difficulty is large, and the second generation sequencing is difficult to be applied to single-gene MRD monitoring in clinic. Real-time fluorescence quantitative PCR cannot be absolutely quantified, and is not suitable for MRD monitoring. The traditional ddPCR method is high in sensitivity and can directly reflect the NPM1 mutation load level, but the mutation type to be detected is single, only known mutation can be detected, and the detection of various mutation types on mutation hot spots is complex and easy to omit. Therefore, further optimization is needed for screening NPM1 gene mutation and MRD monitoring in the initial patients.

Disclosure of Invention

In order to overcome the defects of the prior art, the invention provides a drop-off ddPCR method and a kit for quantitatively detecting NPM1 gene mutation. The primer and the probe for detecting NPM1 gene mutation by the drop-off ddPCR technology are designed according to the DNA sequence of the NPM1 gene, two wild probes are designed aiming at the mutation hot spot of the 12 th exon of the NPM1 gene, one wild probe is positioned on the mutation hot spot, the other wild probe is positioned outside the mutation hot spot, and when the mutation hot spot has mutations such as insertion, replacement, deletion and the like, the wild probe positioned at the mutation hot spot cannot be tightly combined with a template, so that the kit can detect various mutations of the mutation hot spot of the 12 th exon of the NPM1 gene only by using two pairs of primer probes, has high sensitivity and can be used for MRD monitoring.

In order to achieve the purpose, the invention provides the following technical scheme:

the invention provides a kit for quantitatively detecting NPM1 gene mutation by using drop-off ddPCR, which comprises a specific primer pair and a probe aiming at an NPM1 gene, wherein the sequences of the primers are as follows:

NPM1 forward primer: 5'-ATGAAGTGTTGTGGTTCCTT-3'

NPM1 reverse primer: 5'-CAGAAATGAAATAAGACGGAAAA-3' are provided.

Further, the probe sequence is as follows:

NPM1 mutant site wild-type probe: 5 '-FAM-AGATCTCTGGCAGTGGAGG-BHQ 1-3'

Wild-type probe outside NPM1 mutation site: 5 '-VIC-TGTTTAAACTATTTTCTTAAAGAGACT-BHQ 1-3',

the kit comprises a mutation site probe, a fluorescence quenching group BHQ1, a fluorescence group VIC, a mutation site probe, a fluorescence quenching group BHQ1 and a mutation site, wherein the 5 'end of the mutation site probe is marked with a fluorescence group FAM, the 3' end of the probe is marked with the fluorescence quenching group BHQ1, the 5 'end of the probe outside the mutation site is marked with the fluorescence group VIC, and the 3' end of the probe is marked with the fluorescence quenching group BHQ 1.

Further, the kit also included NPM1 mutant and wild-type plasmids as positive and negative controls, respectively.

Furthermore, the gene mutation is a plurality of insertion mutations after 864 nucleotides of the 12 th exon of NPM 1.

Further, the insertion mutation is selected from the group consisting of type a, type B, type D and rare.

The invention also provides a drop-off ddPCR method for quantitatively detecting NPM1 gene mutation, which comprises the following steps:

(1) providing a sample to be detected, and obtaining DNA of the sample to be detected;

(2) taking the sample DNA as a template, mixing the sample DNA with the primer pair and the probe in the kit, and preparing a digital PCR reaction mixed solution;

(3) mixing the digital PCR mixed solution prepared in the step (2) with an oil phase, preparing PCR micro-reaction liquid drops capable of independently carrying out PCR amplification reaction by using a droplet preparation instrument, and introducing the PCR micro-reaction liquid drops into a chip;

(4) carrying out PCR amplification reaction on the PCR micro-reaction liquid drops in the chip prepared in the step (3);

(5) and (4) collecting signals of the droplets in the chip after the PCR amplification reaction in the step (4), thereby calculating the allele frequency of the NPM1 gene in the sample.

Further, the sample to be tested is peripheral blood or bone marrow tissue of a patient.

Furthermore, a FAM-marked mutation site wild-type probe and a VIC-marked mutation site outer wild-type probe are in the same PCR reaction system, according to the result of automatic analysis by a microdroplet analyzer, the copy number concentration of FAM and VIC channels in a sample is read, and the mutation load level of the NPM1 gene is calculated.

Further, NPM1 gene allele frequency (VAF) ═ 1-FAM copy number/VIC copy number.

Further, the system for preparing the mixed solution of the ddPCR reaction is as follows:

10xdPCR reaction buffer	3.5μl
		dPCR enzymes	1μl
NPM1 forward primer	10pmol
		NPM1 reverse primer	10pmol
NPM1 mutant site wild-type probe	7pmol
		NPM1 mutant site out wild type probe	7pmol
Template to be tested	1～7μl
		Supplementing 35 μ l of ribozyme-free water

。

Further, the ddPCR reaction procedure was as follows:

。

further, the blank Limit (LOB) of NPM1 gene mutation was 9.18 copies/microliter, and the lowest detection limit was 22.78 copies/microliter.

Further, the drop-off ddPCR method for quantitatively detecting a mutation in the NPM1 gene is not of diagnostic interest.

Compared with the prior art, the invention has the beneficial effects that:

(1) the primer and the probe of the detection kit for detecting the NPM1 gene mutation based on drop-off ddPCR are designed according to the DNA sequence of the NPM1 gene, and the specificity is strong.

(2) The detection method for detecting the NPM1 gene mutation based on drop-off ddPCR provided by the invention only needs one pair of universal primers, one wild-type probe positioned on the mutation hot spot and one wild-type probe positioned outside the mutation hot spot aiming at the 12 th exon mutation hot spot of the NPM1 gene, so that the mutations such as insertion, substitution, deletion and the like on the hot spot can be detected.

(3) The detection method for detecting the NPM1 gene mutation based on drop-off ddPCR provided by the invention has the lowest detection lower limit of 22.78 copies/microliter and high sensitivity, and can be used for screening the NPM1 gene mutation of a patient at initial diagnosis and monitoring MRD after treatment so as to further guide treatment.

Drawings

FIG. 1 is a graph showing the results of drop-off ddPCR detection in example 1 of the present application. The upper left quadrant represents NPM1 a-type mutant droplets, the upper right quadrant represents wild-type droplets, and the lower left quadrant represents double-negative droplets.

FIG. 2 is a graph showing the results of drop-off ddPCR assay of example 2 of the present application, the upper left quadrant representing NPM 1D-type mutant droplets, the upper right quadrant representing wild-type droplets, and the lower left quadrant representing double-negative droplets.

FIG. 3 shows a graph of drop-off ddPCR assay results of example 3 of the present application, with the upper right quadrant representing NPM1 wild-type droplets and the lower left quadrant representing double negative droplets.

FIG. 4 shows the linear analysis of the detection results of different concentrations of NPM1 gene mutant plasmid drop-off ddPCR in example 4 of the present application.

FIG. 5 shows FAM channel one-dimensional scattergrams of the results of different concentrations of NPM1 gene mutant plasmid drop-off ddPCR in example 4 of the present application.

Detailed Description

The invention will be further elucidated with reference to the specific embodiments and the accompanying drawings. It should be noted that the raw materials used in the present invention are all common commercial products, and therefore the sources thereof are not particularly limited, and the conventional conditions and methods not described in the examples are generally the ones conventionally adopted by the experimenters in the field or the procedures and conditions suggested by the manufacturers.

Example 1

The detection method is described in detail by taking a bone marrow sample of a NPM1 gene A-type mutation patient as an example.

1. Isolation of bone marrow mononuclear cells and extraction of DNA

(1) Adding 5ml erythrocyte lysate into bone marrow sample, mixing, and standing for 1 min;

(2) centrifuging at 12000 × rpm for 20sec, discarding the supernatant, and collecting the precipitate;

(3) adding 1ml of Trizol, fully blowing, beating and uniformly mixing;

(4) adding 200 μ l chloroform, oscillating with vortex oscillator for 20sec, standing for 10min, centrifuging at 12000 × rpm for 1min, and discarding the supernatant;

(5) centrifuging at 12000 × rpm again, discarding the supernatant, and collecting the white precipitate of the DNA at the bottom of the tube;

(6) adding 1ml trisodium citrate into the tube, flicking or reversing, mixing, rinsing, precipitating, standing for 20-30min, centrifuging at 12000 × rpm for 5min, and removing the supernatant;

(7) adding 1ml 70% ethanol, flicking to make it float and elute, 12000 × rpm 5min, discarding the supernatant;

(8) adding 1ml 100% ethanol, flicking to make it float and elute, 12000 × rpm 5min, discarding the supernatant;

(9) drying the precipitate, adding appropriate amount of DNA dissolving solution, and placing in 65 deg.C metal bath for 5 min;

(10) detecting the absorbance (A) value of the DNA sample by an ultraviolet spectrophotometer, adjusting the absorbance to 50 ng/mu l, and storing at 4 ℃;

2. the DNA template is used to prepare a ddPCR reaction mixed solution according to the following preparation ratio:

10xdPCR reaction buffer	3.5μl
		dPCR enzymes	1μl
NPM1 forward primer	10pmol
		NPM1 reverse primer	10pmol
NPM1 mutant site wild-type probe	7pmol
		NPM1 mutant site out wild type probe	7pmol
Template to be tested	5μl
		Supplementing 35 μ l of ribozyme-free water

And (3) uniformly mixing the reaction solution, uniformly mixing the reaction solution by vortex for 30s, and instantly centrifuging to collect the reaction solution on the bottom of the tube and then putting the reaction solution on ice for later use.

3. Preparing oil phase mixed solution according to the following proportion:

oil phase A	30μl
		Oil phase B	10μl

Mixing the reaction oil phases according to the formula shown in the table, uniformly mixing the reaction oil phases by vortex or blowing for 30s by a liquid transfer machine, removing bubbles by instantaneous centrifugation, collecting the liquid at the bottom of the tube, and then placing the tube on ice for later use.

Note: the mixed oil phase mixture can be used within 30 min.

4. Droplet preparation

(1) And opening a front panel bin gate of the instrument, connecting a back network power supply and a panel of the Biodigital Loader S100 sample processing system, starting the power supply, placing the digital PCR chip in a Loader S100 chip bin, and operating according to the prompt.

(2) And (3) sucking 25 mu L of oil phase mixed solution and 30 mu L of ddPCR reaction solution by using a pipette gun, pressing down a bin gate by a left hand, aligning the bin gate with a center hole of a chip plug through a right sample injection support, and fixing a suction head in the sample injection support.

(3) And after the sample injection is finished, taking out the chip filled with the micro-droplets.

PCR reaction

The chip is placed in a chip groove of a Cycler S100 PCR amplification instrument, and the following reactions are carried out:

6. chip reading, data analysis

After the PCR amplification reaction is finished, the chip is moved to an Imager S100 biochip reader, and the experimental result and the related data shown in FIG. 1 can be obtained after data analysis is carried out according to the reading result of the chip. From the results, it was found that 250ng of the sample DNA had a FAM concentration of 1708.35 copies/. mu.l and a VIC concentration of 2314.87 copies/. mu.l, and that the concentration of the NPM 1A type mutation template was 606.52 copies/. mu.l and the NPM1 allele frequency was 26.20% by calculation.

Example 2

Taking the NPM1 gene D-type mutation patient bone marrow sample as an example, the specific detection process is the same as that of example 1. The data analysis can obtain the experimental result and related data as shown in fig. 2. In 250ng of sample DNA, the concentration of FAM is 1660.10 copies/mu l, the concentration of VIC is 1921.90 copies/mu l, the concentration of NPM 1D type mutation template is 261.80 copies/mu l, and the allele frequency of NPM1 is 13.62%.

Example 3

Taking a bone marrow sample of a patient negative for NPM1 gene mutation as an example, the specific detection process is the same as that of example 1. The data analysis can obtain the experimental result and related data as shown in fig. 3. In 250ng of sample DNA, the concentration of FAM is 2078.37 copies/mu l, the concentration of VIC is 2078.37 copies/mu l, the concentration of NPM1 mutant template is calculated to be 0 copies/mu l and is lower than the lower detection limit (22.78 copies/mu l), so the NPM1 gene mutation of the patient is negative.

Example 4

ddPCR sensitivity assay with NPM1 Gene mutant and wild-type plasmids

According to the method of example 1, the wild type plasmid template was checked 26 times in duplicate and the LOB was calculated to be 9.18 copies/. mu.l, and the low concentration mutant template was checked 25 times in duplicate and the LOB was calculated to be 22.78 copies/. mu.l.

The NPM1 gene mutation and wild-type plasmid were quantified and diluted to 2000 copies/. mu.l according to the method of example 1, and the mutant plasmid was incorporated into the wild-type plasmid such that the NPM1 mutation template ratio was 50%, 20%, 10%, 5%, 1%, 0.5%, and the assay was repeated three times for each concentration template, yielding the following data:

as shown in FIGS. 4 and 5, the linearity between the theoretical value and the actually detected value is good, so that the lowest detection limit of the NPM1 gene mutation drop-off ddPCR method provided by the present invention is 22.78 copies/microliter, and the linearity is good.

Sequence listing

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For 45 times

1. A kit for quantitatively detecting NPM1 gene mutation by using drop-offddPCR is characterized by comprising a specific primer pair and a probe aiming at the NPM1 gene, wherein the sequence of the primer is as follows:

NPM1 forward primer: 5'-ATGAAGTGTTGTGGTTCCTT-3'

NPM1 reverse primer: 5'-CAGAAATGAAATAAGACGGAAAA-3', respectively;

the probe sequence is as follows:

NPM1 mutant site wild-type probe: 5 '-FAM-AGATCTCTGGCAGTGGAGG-BHQ 1-3'

2. The kit of claim 1, further comprising NPM1 mutant and wild-type plasmids as positive and negative controls, respectively.

3. The kit of any one of claims 1-2, wherein the genetic mutation is a plurality of insertion mutations at 863 nucleotides of exon 12 of NPM 1.

4. The kit of claim 3, wherein the insertion mutation is selected from the group consisting of type A, type B, type D, and rare.

5. The application of the kit in preparing a reagent for quantitatively detecting NPM1 gene mutation by using drop-offddPCR is characterized by comprising a specific primer pair aiming at the NPM1 gene and a probe, wherein the sequence of the primer is as follows:

NPM1 forward primer: 5'-ATGAAGTGTTGTGGTTCCTT-3'

NPM1 reverse primer: 5'-CAGAAATGAAATAAGACGGAAAA-3', respectively;

the probe sequence is as follows:

NPM1 mutant site wild-type probe: 5 '-FAM-AGATCTCTGGCAGTGGAGG-BHQ 1-3'

6. The use of claim 5, wherein the kit further comprises NPM1 mutant and wild-type plasmids as positive and negative controls, respectively.

7. The use of any one of claims 5 to 6, wherein the genetic mutation is a plurality of insertion mutations at 863 nucleotides of exon 12 of NPM 1.

8. The use according to claim 7, wherein the insertion mutation is selected from the group consisting of type A, type B, type D and rare.