Human NPM1 gene mutation digital PCR detection kit and use method thereof

Abstract

The invention discloses a digital PCR (Polymerase Chain Reaction) detection kit for human NPM1 gene mutation. The kit at least comprises ddPCR reaction liquid and ddPCR MIX3, the ddPCR reaction liquid comprises primers and a probe, the primers comprise a mutation NPM1 forward primer and a mutation NPM1 reverse primer, the mutation NPM1 reverse primer comprises seven primers of NPM1 I-NPM1 VII type, the base sequence of the mutation NPM1 forward primer is SEQ ID No.1, and the base sequences of the seven primers of NPM1 I-NPM1 VII type are SEQ ID No.2-SEQ ID No.8. The kit is characterized in that the kit comprises a kit body, the kit body and the probe, and the kit body comprises a kit body and a kit body, the kit is used for qualitatively detecting NPM1 gene I-VII type mutation of bone marrow and peripheral blood samples of clinical AML patients, and can assist clinicians to carry out prognosis risk assessment, differential diagnosis, treatment guidance and AML typing judgment on the AML patients.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biological detection, in particular to a human NPM1 gene mutation digital PCR detection kit and use method and application. BACKGROUND

[0002] Acute myeloid leukemia (AML) is a heterogeneous hematological malignancy, and insertion mutation of exon 12 of NPM1 gene causes the incidence of AML disease to be 28%-35%, and NPM1 gene is also related to the lesion of NK-AML, and the incidence is 48%-53%. Therefore, qualitative detection of NPM1 gene type I-type VII mutation in bone marrow and peripheral blood samples can assist clinicians in prognosis risk assessment, differential diagnosis, treatment guidance and AML typing judgment of AML patients, but the existing detection method is time-consuming and causes great damage to patients, so it is crucial to develop a detection method for rapid and non-invasive detection of NPM1 gene mutation in patients.

[0003] Chinese invention patent CN113249475B discloses a drop-off ddPCR method and kit for quantitative detection of NPM1 gene mutation, two wild type probes are designed for NPM1 gene exon 12 mutation hot spot, one is located in the mutation hot spot, and the other is located outside the mutation hot spot, when there are insertion, substitution, deletion and other mutations in the mutation hot spot, the wild type probe located in the mutation hot spot cannot be tightly combined with the template, and the detection sensitivity is high, but this scheme cannot classify the mutation of NPM1 gene, and cannot realize targeted treatment. Chinese invention patent CN105950775B discloses a kit for detecting NPM1 gene mutation typing, which applies one-step real-time fluorescent PCR reaction mode and LNA modification to specific probes, can sensitively and rapidly detect NPM1 gene exon 12 mutation in bone marrow and peripheral blood samples of acute myeloid leukemia patients, and can classify A type, B type and D type mutations, but the classification is less, and cannot realize comprehensive detection. SUMMARY

[0004] In order to develop a detection method which can quickly and non-invasively detect NPM1 gene mutation of a patient, a first aspect of the present application provides a human NPM1 gene mutation digital PCR detection kit, which comprises at least a ddPCR reaction solution and a ddPCR MIX3; the ddPCR reaction solution comprises primers and a probe, the primers comprise a mutant NPM1 forward primer NP(Co-F) and a mutant NPM1 reverse primer, the mutant NPM1 reverse primer comprises seven primers of NPM1 I-NPM1 VII type, the base sequence of the mutant NPM1 forward primer is SEQ ID No. 1, and the base sequences of the seven primers of NPM1 I-NPM1 VII type are SEQ ID No. 2-SEQ ID No. 8.

[0005] The seven primers of NPM1 I-NPM1 VII type comprise NP-I(A), NP-II(D), NPM1-Rtype III, NPM1-Rtype IV, NPM1-Rtype V, NPM1-Rtype VI and NPM1-Rtype VII.

[0006] As an implementation form, the probe comprises an NPM1 probe NP(Co-P) and a reference probe RP-P-VIC, the base sequence of the NPM1 probe NP(Co-P) is SEQ ID No. 9, and the base sequence of the reference probe RP-P-VIC is SEQ ID No. 12.

[0007] As an implementation form, the primers further comprise a reference RP-F forward primer and a reference RP-R reverse primer.

[0008] As an implementation form, the base sequence of the reference RP-F forward primer is SEQ ID No. 10, and the base sequence of the reference RP-R reverse primer is SEQ ID No. 11.

[0009] As an implementation form, the ddPCR reaction solution specifically comprises 50 μmol / L NP(Co-F), 0.1 μL; 50 μmol / L NP-I(A), 0.1 μL; 50 μmol / L NP-II(D), 0.1 μL; 50 μmol / L NPM1-R type III, 0.1 μL; 50 μmol / L NPM1-R type IV, 0.05 μL; 50 μmol / L NPM1-R type V, 0.05 μL; 50 μmol / L NPM1-R type VI, 0.05 μL; 50 μmol / L NPM1-R type VII, 0.05 μL; 50 μmol / L NP(Co-P), 0.05 μL; 50 μmol / L RP-F, 0.1 μL; 50 μmol / L RP-R, 0.1 μL; 50 μmol / L RP-P-VIC, 0.05 μL, and 3.1 μL of ultrapure water.

[0010] As an implementation form, the ddPCR MIX3 comprises a hot start enzyme, dNTPs, MgCl2, a buffer, and a microdroplet stabilizer.

[0011] As an implementation form, the kit further comprises a control, and the control comprises a blank control and a positive control, and the positive control comprises a plasmid DNA mixture.

[0012] As an implementation form, the plasmid DNA mixture comprises NPM1 type I to type VII mutant plasmid DNA and RPLP0 internal reference plasmid DNA.

[0013] As an implementation form, the base sequence of the NPM1 type I to type VII mutant plasmid DNA is SEQ ID No. 13-SEQ ID No. 19.

[0014] As an implementation form, the base sequence of the RPLP0 internal reference plasmid DNA is SEQ ID No. 20.

[0015] As an implementation form, the volume ratio of the NPM1 type I to type VII mutant plasmid DNA and the RPLP0 internal reference plasmid DNA is 1: (1-3).

[0016] As an implementation form, the volume ratio of the NPM1 type I to type VII mutant plasmid DNA and the RPLP0 internal reference plasmid DNA is 1:2.

[0017] The positive control stock solution is mixed with the NPM1 type I to type VII mutant plasmid DNA and the RPLP0 internal reference plasmid DNA (1:2) of a determined concentration, and is diluted by 100 times with ultrapure water to obtain the positive control.

[0018] As an implementation form, the blank control is ultrapure water.

[0019] The kit of the present application has a validity period of 12 months, and the performance is stable after repeated freezing and thawing 5 times within the validity period.

[0020] The kit of the present application detects the nucleic acid DNA extracted from the bone marrow / peripheral blood sample, extracts the sample by using a nucleic acid extraction kit, uses certain solid media to selectively adsorb nucleic acid under specific conditions, and does not adsorb protein and salt, so as to realize the separation of nucleic acid and protein and salt, and the extracted nucleic acid is used as the sample DNA to be detected.

[0021] The product uses a digital PCR detection method, and is detected by using a Bio-Rad digital PCR detection system. After the chip is loaded, the sample DNA to be detected, primers, probes and a mixture of ddPCR MIX3 (hot-start enzyme, dNTPs, MgCl2, buffer and microdroplet stabilizer) are automatically separated in uniform nanoliter microdroplets, and the PCR reaction is independently carried out in each microdroplet. After amplification, the system automatically runs to the analysis module for fluorescence reading, and a double-color fluorescence image is captured by a microscopic optical system, or the microdroplets pass through a double-color optical detection system one by one. The microdroplets with fluorescence signals are positive, and the microdroplets without fluorescence signals are negative. Then, the number of positive and negative microdroplets and the proportion of positive microdroplets are calculated, and the final analysis software can calculate the concentration or copy number of the target molecule to be detected. Therefore, the DNA extracted from the clinical bone marrow / peripheral blood sample is quantitatively detected for NPM1 gene I-VII type mutations, the method has high sensitivity and strong specificity, and thus provides a reference for evaluating the prognosis risk of patients.

[0022] The second aspect of the present application provides a use method of the human NPM1 gene mutation digital PCR detection kit, comprising the following steps: Take the DNA nucleic acid sample as a PCR reaction template; Mix the ddPCR reaction liquid and the ddPCR MIX3 to configure an amplification reagent; Mix the PCR reaction template and the amplification reagent to obtain a mixture; Prepare the mixture into microdroplets to perform a PCR amplification reaction; After the reaction, the microdroplets are read, the results are analyzed, and the detection is completed.

[0023] As an implementation form, the volume ratio of the mixture of the ddPCR reaction liquid and the ddPCR MIX3 is (1-3):5.

[0024] As an embodiment, the preparation method of the DNA nucleic acid sample is as follows: 5 mL of bone marrow or peripheral blood whole blood sample of EDTA anticoagulant blood preservation tube is selected, hemolysis is avoided, and extraction is performed within 3 days after blood extraction by using an extraction kit, so as to be used as a PCR reaction template. The extracted nucleic acid is recommended to be detected immediately, or is stored below -20±4℃.

[0025] As an embodiment, the configuration method of the amplification reagent comprises the following steps: (1) The number of tests to be prepared: the number of tests of each experiment is calculated according to the total of 1 blank control, 1 positive control and sample number N, that is, N+2 tests.

[0026] (2) Preparation of amplification reagent: the corresponding reaction solution is taken out from the kit, melted and mixed at room temperature, and then centrifuged at 2000 rpm for 10 s. The NPM1 PCR premix is prepared according to the following formula: 4 μL of ddPCR reaction solution + 10 μL of ddPCR MIX3, to obtain the amplification reagent, which is respectively dispensed into each PCR tube at an amount of 14 μL per tube.

[0027] As an embodiment, the configuration method of the mixed solution comprises the following steps: The control and the DNA nucleic acid sample in the kit are taken out, melted and mixed at room temperature, and then centrifuged at 2000 rpm for 10 s. 6 μL of the DNA nucleic acid sample is added to the PCR tube containing the amplification reagent. The total volume of each reaction system is 20 μL. The PCR tube cover is tightly covered, and then centrifuged at 2000 rpm for 10 s, and then droplet preparation is performed.

[0028] As an embodiment, the method of droplet preparation comprises the following steps: (1) Preparation of droplets 1) 8 20 μL reaction systems are added to the 8 holes in the middle row of the DG8 cartridge.

[0029] Note: a. The sample must be added to the middle row of the DG8 cartridge first (if there are less than 8 samples, add 20 μL of Bx ddPCR Buffer Control to the empty holes).

[0030] b. When adding the sample, bubbles should be avoided.

[0031] 2) 70 μl of Droplet Generation oil is added to each of the 8 holes in the bottom row of the DG8 cartridge, the gasket is covered, the DG8 cartridge is gently and stably placed in the droplet generator, and the droplet generation is started. Pay attention to the status of the indicator light on the instrument, and complete it within 2 minutes.

[0032] 3) The droplets are generated in the top row of wells in the cartridge and the generated droplets (35-45 μL) are transferred to a 96-well plate.

[0033] Note: a. Be careful when aspirating and dispensing droplets to avoid breakage.

[0034] b. Be careful to cover the Pierceable Foil Heat Seal to prevent evaporation of oil. Discard the used cartridge and gasket after each use.

[0035] (2) Sealing After the droplets are transferred to the 96-well plate, seal the plate with the PX1 Heat Sealer preheated to 180°C for 5 seconds without inverting. Seal the plate again within 30 minutes of sealing and perform PCR within 4 hours at 4°C.

[0036] (3) PCR amplification 95°C, 10 min; (94°C, 15 sec; 58°C, 60 sec) 40 cycles, ramp rate should be ≤ 2°C / s. 98°C, 10 min; 4°C, 5 min.

[0037] The reaction system is set to 40 μL.

[0038] As an embodiment, the method for reading the droplets comprises the following steps: 1) Turn on the computer and then turn on the power of the Droplet Reader. Preheat for at least 30 minutes before use. 2) Place the 96-well plate containing the PCR products into the plate holder and place it into the Droplet Reader.

[0039] 3) Open the QuantaSoft software and set the information of the samples in the 96-well plate. After completion, run the program.

[0040] Note: a. Select "ddPCR Supermix for probes" for Supermix. b. Select "FAM / VIC" for Dye Set.

[0041] As an embodiment, the method for analyzing the results comprises the following steps: After the detection is completed, click "2D Amplitude" to view the cluster plots for channel 1 and channel 2. This plot allows manual or automatic adjustment of the threshold to designate positive and negative droplets for each detection channel.

[0042] 1) Click "Auto Analyze" to reset the threshold 2) Manually specify the threshold: - Use the threshold crosshair to specify the classification region for the entire dot plot. - Use the Ellipse, Rectangle or Lasso threshold adjustment tools to classify dot plot regions: click the corresponding tool button, then click the region type in the "Working cluster selector" and use the tool to select the corresponding region.

[0043] Note: The fluorescence threshold line should be set according to the negative control and the indicator control: in 2D Amplitude, the position of the fluorescence threshold line should be such that the droplet cluster of the negative control is in the "ch1-ch2-" interval, and the four droplet clusters of the indicator control are in the four intervals, respectively.

[0044]

Reference value (reference range)

Interpretation of test results

[0045] Positive control effectiveness determination: points falling in "ch1+ch2-" region ≥4 points and mutation ratio ≥1‰.

[0046] Invalid result determination: total droplets per reaction tube should be ≥8000, if total droplets <8000, the droplet generation of this reaction well is not ideal, and droplet generation needs to be performed again.

[0047] 2. Result determination: 2.1 Qualitative determination (1) If the sample has ≥4 points falling in the "ch1+ch2-" region and the mutation ratio is ≥1‰, it is determined that there is NPM1 type I-VII mutation.

[0048] (2) If it does not meet (1), If the sample DNA is ≥50 copies If the sample has <4 points falling in the "ch1+ch2-" region or the mutation ratio is <1‰, it is determined that there is no mutation or the mutation is below the minimum detection limit.

[0049] If the sample falls in the "ch1+ch2-" area of the point is 3, and the mutation ratio is greater than or equal to 1‰, the NPM1 I ~ VII type mutation is determined to be suspected positive, and re-detection is required. The result of re-detection, if the sample falls in the "ch1+ch2-" area of the point is greater than or equal to 4 and the mutation ratio is greater than or equal to 1‰, the NPM1 I ~ VII type site mutation is determined. Otherwise, it is determined that there is no mutation or the mutation is lower than the minimum detection limit.

[0050] If the sample DNA is less than 50 copies, it indicates that the added DNA quality is poor or contains PCR inhibitors, and re-extraction of DNA or re-sampling is required. After re-detection, the sample DNA is less than 50 copies and does not meet (1), the DNA quality is determined to be unsatisfactory. Otherwise, the corresponding determination is made according to the above conditions. 2.2 Quantitative determination If the sample NPM1 I ~ VII type mutation is calculated according to the Ch1 / Ch2 formula.

[0051] The third aspect of the present application provides an application of a human NPM1 gene mutation digital PCR detection kit to the detection of nucleic acid extracted by the extraction kit.

[0052] Compared with the prior art, the present application has the following beneficial effects: (1) The human NPM1 gene mutation digital PCR detection kit of the present application contains ddPCR MIX3 for preparing water-in-oil microdroplets after microdroplet preparation, forming a heat-stable microdroplet, and ensuring that the target fragment is subjected to PCR amplification in the microdroplet, thereby improving the stability of the detection.

[0053] (2) The human NPM1 gene mutation digital PCR detection kit of the present application uses a digital PCR detection method, and is detected in combination with a Bio-Rad digital PCR detection system. After the chip is loaded into the machine, the sample DNA to be detected can be automatically separated in uniform nanoliter microdroplets, and the PCR reaction is independently carried out, thereby reducing interference.

[0054] (3) After the PCR reaction amplification of the human NPM1 gene mutation digital PCR detection kit of the present application is completed, the system automatically runs to the analysis module for fluorescence reading, and the amplified microdroplets are scanned one by one through a microscopic optical system, and a double-color fluorescence image is taken, so that the detection is convenient and fast, and the error interference of manual detection is avoided.

[0055] (4) The human NPM1 gene mutation digital PCR detection kit of the present application can calculate the concentration or copy number of the target molecule to be detected through the final analysis software, and can perform qualitative detection of NPM1 gene I ~ VII type mutation on the DNA extracted from clinical bone marrow / peripheral blood samples, so that the method has high sensitivity and strong specificity.

[0056] (5) The human NPM1 gene mutation digital PCR detection kit can qualitatively detect NPM1 gene type I to type VII mutations of bone marrow and peripheral blood samples of clinical AML patients, and can assist clinicians in prognosis risk assessment, differential diagnosis, treatment guidance and AML typing judgment of AML patients. BRIEF DESCRIPTION OF DRAWINGS

[0057] Figure 1 The detection reaction liquid of the mixed site of the mutant gene NPM1 and the reference gene RPLP0 primer probe in the kit is configured, and the detection result 2D graph of the test using the RPLP0 reference plasmid is shown in the figure; Figure 2 The detection reaction liquid of the mixed site of the mutant gene NPM1 and the reference gene RPLP0 primer probe in the kit is configured, and the detection result 2D graph of the test using the RPLP0 reference plasmid is shown in the figure; Figure 3 The detection reaction liquid of the mixed site of the mutant gene NPM1 and the reference gene RPLP0 primer probe in the kit is configured, and the detection result 2D graph of the test using the RPLP0 reference plasmid is shown in the figure; Figure 4 The detection reaction liquid of the mixed site of the mutant gene NPM1 and the reference gene RPLP0 primer probe in the kit is configured, and the detection result 2D graph of the test using the RPLP0 reference plasmid is shown in the figure; Figure 5 The detection reaction liquid of the mixed site of the mutant gene NPM1 and the reference gene RPLP0 primer probe in the kit is configured, and the detection result 2D graph of the test using the RPLP0 reference plasmid is shown in the figure; Figure 6 The detection reaction liquid of the mixed site of the mutant gene NPM1 and the reference gene RPLP0 primer probe in the kit is configured, and the detection result 2D graph of the test using the RPLP0 reference plasmid is shown in the figure; Figure 7 The detection reaction liquid of the mixed site of the mutant gene NPM1 and the reference gene RPLP0 primer probe in the kit is configured, and the detection result 2D graph of the test using the RPLP0 reference plasmid is shown in the figure; Figure 8 The detection reaction liquid of the mixed site of the mutant gene NPM1 and the reference gene RPLP0 primer probe in the kit is configured, and the detection result 2D graph of the test using the RPLP0 reference plasmid is shown in the figure. DETAILED DESCRIPTION

[0058] EMBODIMENT The human NPM1 gene mutation digital PCR detection kit comprises a ddPCR reaction solution and a ddPCR MIX3; the ddPCR reaction solution comprises primers and a probe; the primers comprise a mutant NPM1 forward primer NP(Co-F) and a mutant NPM1 reverse primer, wherein the mutant NPM1 reverse primer comprises seven primers of NPM1 I-NPM1 VII types; the base sequence of the mutant NPM1 forward primer is SEQ ID No. 1; the base sequence of the NPM1 I primer is SEQ ID No. 2; the base sequence of the NPM1 II primer is SEQ ID No. 3; the base sequence of the NPM1 III primer is SEQ ID No. 4; the base sequence of the NPM1 IV primer is SEQ ID No. 5; the base sequence of the NPM1 V primer is SEQ ID No. 6; the base sequence of the NPM1 VI primer is SEQ ID No. 7; and the base sequence of the NPM1 VII primer is SEQ ID No. 8.

[0059] The seven primers of NPM1 I-NPM1 VII types comprise NP-I(A), NP-II(D), NPM1-RtypeIII, NPM1-RtypeIV, NPM1-RtypeV, NPM1-RtypeVI, and NPM1-RtypeVII.

[0060] The probe comprises an NPM1 probe NP(Co-P) and a reference probe RP-P-VIC; the base sequence of the NPM1 probe is SEQ ID No. 9; and the base sequence of the reference probe RP-P-VIC is SEQ ID No. 12.

[0061] The primers further comprise a reference RP-F forward primer and a reference RP-R reverse primer; the base sequence of the reference RP-F forward primer is SEQ ID No. 10; and the base sequence of the reference RP-R reverse primer is SEQ ID No. 11.

[0062] The ddPCR reaction solution specifically includes 50 μmol / L NP(Co-F), 0.1 μL; 50 μmol / L NP-I(A), 0.1 μL; 50 μmol / L NP-II(D), 0.1 μL; 50 μmol / L NPM1-RtypeIII, 0.1 μL; 50 μmol / L NPM1-RtypeIV, 0.05 μL; 50 μmol / L NPM1-RtypeV, 0.05 μL; 50 μmol / L NPM1-RtypeVI, 0.05 μL; 50 μmol / L NPM1-RtypeVII, 0.05 μL; 50 μmol / L NP(Co-P), 0.05 μL; 50 μmol / L RP-F, 0.1 μL; 50 μmol / L RP-R, 0.1 μL; 50 μmol / L RP-P-VIC, 0.05 μL, and 3.1 μL of ultrapure water.

[0063] The ddPCR MIX3 includes a hot-start enzyme, dNTPs, MgCl2, a buffer, and a droplet stabilizer, which are purchased from Bio-Rad Company with a product number Cat.#1863024.

[0064] The kit further includes a control, which includes a blank control and a positive control, and the positive control includes a plasmid DNA mixture.

[0065] The plasmid DNA mixture includes NPM1 type I to type VII mutant plasmid DNA and RPLP0 internal reference plasmid DNA. The base sequence of the NPM1 type I to type VII mutant plasmid DNA is SEQ ID No. 13-SEQ ID No. 19. The base sequence of the RPLP0 internal reference plasmid DNA is SEQ ID No. 20.

[0066] The volume ratio of the NPM1 type I to type VII mutant plasmid DNA and the NPM1 plasmid DNA is 1:2.

[0067] The positive control stock solution is mixed with the NPM1 type I to type VII mutant plasmid DNA and the RPLP0 internal reference plasmid DNA (volume ratio 1:2) at a determined concentration, and is diluted with ultrapure water at a ratio of 100 times to obtain the positive control.

[0068] The blank control is ultrapure water.

[0069] A use method of a human NPM1 gene mutation digital PCR detection kit, including the following steps: Taking a DNA nucleic acid sample as a PCR reaction template; Mixing the ddPCR reaction solution and the ddPCR MIX3 to configure an amplification reagent; Mix the PCR reaction template with the amplification reagent to obtain a mixed solution; Prepare the mixed solution into droplets, and perform a PCR amplification reaction; Read the droplets after the reaction, analyze the results, and complete the detection.

[0070] The preparation method of the DNA nucleic acid sample is as follows: select 5 mL of bone marrow or peripheral blood whole blood sample of EDTA anticoagulant blood preservation tube, avoid hemolysis, extract the sample within 3 days after blood extraction using an extraction kit, and use the extracted sample as the PCR reaction template. The extracted nucleic acid is recommended to be detected immediately, otherwise it should be stored below -20±4℃.

[0071] The configuration method of the amplification reagent includes the following steps: (1) The number of tests to be prepared: the number of tests for each experiment is calculated according to the total of 1 blank control, 1 positive control and sample number N, that is, N+2 tests.

[0072] (2) Preparation of amplification reagent: take out the corresponding reaction solution from the kit, melt and mix at room temperature, centrifuge at 2000 rpm for 10 s, and prepare the NPM1 PCR premix solution as follows: 4 μL of ddPCR reaction solution + 10 μL of ddPCR MIX3, to obtain the amplification reagent, and respectively, 14 μL per tube, and distribute in each PCR tube.

[0073] The configuration method of the mixed solution includes the following steps: Take out the control and DNA nucleic acid sample in the kit, melt and mix at room temperature, centrifuge at 2000 rpm for 10 s, take 6 μL of the DNA nucleic acid sample and add it to the PCR tube containing the above amplification reagent; the total volume of each reaction system is 20 μL. Tighten the cover of the PCR tube, centrifuge at 2000 rpm for 10 s, and then prepare droplets.

[0074] The method for preparing droplets includes the following steps: (1) Preparation of droplets 1) Add 8 20 μL reaction systems to the 8 holes in the middle row of the DG8 cartridge.

[0075] Note: a. The sample must be added to the middle row of the DG8 cartridge first (if there are less than 8 samples, add 20 μL of Bx ddPCR Buffer Control to the empty holes).

[0076] b. When adding the sample, avoid generating bubbles.

[0077] 2) Add 70 μΐ of Droplet Generation oil to the bottom row of 8 wells in the DG8 cartridge, cover with gasket, and gently place the DG8 cartridge into the droplet generator. Begin droplet generation and pay attention to the status of the indicator light on the instrument. Generation should be complete within 2 minutes.

[0078] 3) Droplets are generated in the top row of wells in the cartridge. Transfer the generated droplets (35-45 μΐ) to a 96-well plate.

[0079] Note: a. Be careful when aspirating and dispensing droplets to avoid breaking the droplets.

[0080] b. Be sure to cover the cartridge with the Pierceable Foil Heat Seal to prevent oil evaporation. Discard the used cartridge and gasket after each use.

[0081] (2) Sealing After the droplets are transferred to the 96-well plate, seal the plate using the PX1 thermal cycler with the following settings: 180°C, 5s, no flip. After sealing, perform PCR within 30 minutes or store the plate in a 4°C refrigerator for up to 4 hours.

[0082] (3) PCR amplification 95°C, 10 min; (94°C, 15 sec; 58°C, 60 sec) for 40 cycles, with a ramp rate of <2°C / s. 98°C, 10 min; 4°C, 5 min.

[0083] The reaction system is set to 40 μΐ.

[0084] The method for reading the droplets includes the following steps: 1) Turn on the computer and then turn on the power of the Droplet Reader. Allow the instrument to warm up for at least 30 minutes before use. 2) Place the 96-well plate containing the PCR products into the plate holder and gently place the plate into the Droplet Reader.

[0085] 3) Open the QuantaSoft software and set the information for the samples in the 96-well plate. Once the information is set, the instrument can be run.

[0086] Note: a. Select "ddPCR Supermix for probes" for the Supermix. b. Select "FAM / VIC" for the Dye Set.

[0087] The method of analyzing the results includes the following steps: After detection, click "2D Amplitude" to view the channel 1 and channel 2 cluster diagram. This diagram allows manual or automatic adjustment of the threshold to designate positive and negative microdroplets for each detection channel.

[0088] 1) Click "Auto Analyze" to reset the threshold 2) Manually specify the threshold: - Use the threshold crosshair to specify the classification area of the entire dot diagram; - Use the ellipse, rectangle or lasso threshold adjustment tool to classify the dot diagram area: click the corresponding tool button, then click the area type in the "Working cluster selector", and use the tool to select the corresponding area.

[0089] Note: The setting of the fluorescence threshold line refers to the negative control and the indicator control: in 2D Amplitude, the position of the fluorescence threshold line should make the microdroplet cluster of the negative control in the "ch1-ch2-" interval, and the four microdroplet clusters of the indicator control in the four intervals, respectively.

[0090] The specific results are shown in Figures 1-8 .

[0091] It can be seen that: Figures 1-8 The primer probe of the NPM1 type I~VII 7 mutation sites and the primer probe of the internal reference gene RPLP0 are mixed to configure a mixed site detection reaction solution, and the positive and negative plasmids corresponding to the sites are used for testing. According to the 2D diagram of the detection results, the negative plasmid detects no mutation, and the NPM1 type I~VII 7 mutation site positive plasmid can detect mutation positive, and the 2D diagram of the detection results has good aggregation degree, which can meet the division of the cross-quadrant threshold, and meets the detection requirements of the kit.

[0092] Performance test 1. Specificity experiment: using the kit of the embodiment, the negative sample verified by NGS (second-generation sequencing) is analyzed for specificity detection experiment, see Table 1. The negative samples N1-N62 come from Wuhan Zhongnan Hospital. The blank space represents the proportion of no mutation. The kit for second-generation sequencing is a leukemia gene panoramic screening detection kit, which is purchased from Shanghai Yuanqi Biomedicine Technology Co., Ltd.

[0093] Conclusion: no mutation is detected in the negative sample, and the number of points in the FAM detection channel (Ch1+Ch2-) is ≤3, and the kit has good specificity and no non-specific amplification.

[0094] Table 1

[0095] 2. Accuracy experiment The NPM1 gene type I-VII mutation positive samples verified by NGS (next generation sequencing) were verified by the kit of the embodiment, and the results were shown in Table 2: the positive samples PM1-PM10 were patient samples from Wuhan Zhongnan Hospital. The kit for next generation sequencing was a leukemia gene panoramic screening detection kit, which was purchased from Shanghai Yuanqi Biomedicine Technology Co., Ltd.

[0096] Conclusion: The results of detecting PM1-PM10 positive samples were all NPM1 gene type I-VII mutation positive, and the kit had good accuracy.

[0097] Table 2

[0098] 3. Detection limit experiment 3.1 Determination of detection limit: The NPM1 gene type I-VII mutation positive samples verified by NGS (next generation sequencing) were detected by the kit of the embodiment, and the negative samples of the same concentration were gradient diluted, the samples of different concentrations and different mutation ratios obtained by dilution were detected, and the detection limit of the kit was determined combined with the detection results of specific samples. The results were shown in Table 3. The kit for next generation sequencing was a leukemia gene panoramic screening detection kit, which was purchased from Shanghai Yuanqi Biomedicine Technology Co., Ltd.

[0099] Table 3

[0100] 3.2 Detection limit verification According to the determined detection limit of the kit, the NPM1 gene type I-VII mutation positive DNA samples verified by NGS (next generation sequencing) were gradient diluted to 0.1% with the negative clinical samples of the same concentration, and 20 repeated detections were performed, and the results were shown in Table 4. The kit for next generation sequencing was a leukemia gene panoramic screening detection kit, which was purchased from Shanghai Yuanqi Biomedicine Technology Co., Ltd.

[0101] Table 4

[0102] Conclusion: Combined with the analysis of specificity, the establishment and verification of detection limit and the detection results, no mutation was detected in the negative samples detected by the kit, and the number of points in the FAM detection channel (Ch1+Ch2-) was ≤3. The kit detected 10 ng / μL of 0.1% NPM1 gene type I-VII mutation samples at a concentration of 0.1% and met the 95% positive detection rate. Therefore, the detection limit of the kit was determined as follows: 10 ng / L of mutation ratio of 0.1% and FAM detection channel (Ch1+Ch2-) of 4 points or more points of NPM1 gene type I-VII mutation could be detected. The results showed that the detection method established in this study had high sensitivity.

[0103] 4. Precision experiment Using the kit of the embodiment, samples verified by NGS (second-generation sequencing) as NPM1 gene type I-VII mutation ratio of 20% positive were selected as high precision (J) reference (SP1). The high precision (J) reference (SP1) was diluted by 10 times (1:9) with the same concentration of negative samples to obtain a mutation ratio of 4% as a low precision (LJ) reference (SP2). The high and low precision references were detected, and the results are shown in Table 5: Conclusion: The high and low precision references (SP1, SP2) were verified to be positive for NPM1 gene type I-VII mutation, and the CV of the results of ten repeated detections was ≤5%. The kit had good repeatability.

[0104] Table 5

Claims

1. A human NPM1 gene mutation digital PCR detection kit, characterized in that, The kit comprises at least ddPCR reaction solution and ddPCR MIX3; the ddPCR reaction solution comprises primers and probes; the primers comprise mutant NPM1 forward primers and mutant NPM1 reverse primers, wherein the mutant NPM1 reverse primers comprise seven primers of NPM1 I-NPM1 VII type; the base sequence of the mutant NPM1 forward primer is SEQ ID No. 1, and the base sequences of the seven primers of NPM1 I-NPM1 VII type are SEQ ID No. 2-SEQ ID No.

8. 2.The human NPM1 gene mutation digital PCR detection kit of claim 1, characterized in that, The probes comprise NPM1 probes and internal reference probes; the base sequence of the NPM1 probe is SEQ ID No. 9; and the base sequence of the internal reference probe RP-P-VIC is SEQ ID No.

12. 3.The human NPM1 gene mutation digital PCR detection kit of claim 1, characterized in that, The primers further comprise internal reference RP-F forward primers and internal reference RP-R reverse primers. 4.The human NPM1 gene mutation digital PCR detection kit of claim 1, characterized in that, The ddPCR MIX3 comprises hot-start enzymes, dNTPs, MgCl2, buffer and microdroplet stabilizers.

5. The human NPMl gene mutation digital PCR detection kit according to claim 1, characterized in that, The kit further comprises control samples, wherein the control samples comprise blank controls and positive controls, and the positive controls comprise plasmid DNA mixtures.

6. The human NPMl gene mutation digital PCR detection kit according to claim 5, characterized in that, The plasmid DNA mixtures comprise NPM1 I-VII type mutant plasmid DNA and RPLP0 internal reference plasmid DNA.

7. The human NPMl gene mutation digital PCR detection kit according to claim 6, characterized in that, The volume ratio of the NPM1 I-VII type mutant plasmid DNA to the RPLP0 internal reference plasmid DNA is 1: (1-3).

8. The method of using the human NPM1 gene mutation digital PCR detection kit according to any one of claims 1-7, characterized in that, The kit comprises the following steps: DNA nucleic acid samples are taken as PCR reaction templates; The ddPCR reaction solution and the ddPCR MIX3 are mixed to configure amplification reagents; The PCR reaction templates are mixed with the amplification reagents to obtain a mixture; The mixture is prepared into microdroplets for PCR amplification reaction; After the reaction, the microdroplets are read, the results are analyzed, and the detection is completed. 9.The method of using the human NPM1 gene mutation digital PCR detection kit according to claim 8, characterized in that, The volume ratio of the mixture of the ddPCR reaction solution and the ddPCR MIX3 is (1-3):

5.

10. The use of the human NPM1 gene mutation digital PCR detection kit according to any one of claims 1-7, characterized in that, The kit is applied to the detection of nucleic acids extracted by an extraction kit.

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