POCT detection method for V617F mutation of human JAK2 gene

Abstract

The invention relates to the field of gene mutation detection, and particularly discloses a POCT (point-of-care testing) detection method for V617F mutation of a human JAK2 gene. According to the method, an integrated detection tube is divided into a cell lysis-nucleic acid binding magnetic bead area, a nucleic acid cleaning area and a nucleic acid elution-amplification area from top to bottom in sequence through a plurality of hydrophobic layers, the cell lysis-nucleic acid binding magnetic bead area is pre-filled with a lysis solution, the nucleic acid cleaning area is pre-filled with a nucleic acid cleaning solution, and the nucleic acid elution-amplification area is pre-filled with a nucleic acid elution solution. A nucleic acid elution-amplification system is pre-installed in the nucleic acid elution-amplification area, in the detection process, only a sample needs to be added into the cell lysis-nucleic acid binding magnetic bead area, a corresponding instrument is inserted, and operation is conducted, so that nucleic acid extraction, amplification and result reporting can be completed, and additional manual processing steps are not needed. The method has the advantages of simplicity and convenience in operation, short time consumption, high sensitivity, strong specificity, low pollution risk and the like, can be directly used for whole blood sample detection, meets the rapid diagnosis requirements of clinical outpatient service, and is particularly suitable for auxiliary diagnosis of BCR-ABL negative myeloproliferative tumors.

Description

Technical Field

[0001] This invention relates to the field of gene mutation detection technology, specifically to a point-of-care testing (POCT) method for detecting human JAK2 gene V617F mutation, which is applicable to rapid molecular diagnosis of myeloproliferative neoplasms and related diseases. Background Technology

[0002] Myeloproliferative neoplasms (MPNs) are a group of malignant tumors originating from pluripotent hematopoietic stem cells. They rank among the highest in incidence and mortality, posing a serious threat to human health. Based on molecular biological characteristics, MPNs are classified into BCR-ABL-positive and BCR-ABL-negative types. Classic BCR-ABL-negative MPNs mainly include polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF). The JAK2 gene encodes a non-receptor tyrosine kinase involved in hematopoietic cytokine signaling. Under normal circumstances, activated JAK2 regulates cell proliferation, differentiation, and survival by activating the STAT, MAPK, and PI3K pathways. The V617F mutation leads to the loss of self-inhibition function in the JAK2 protein, resulting in continuous activation and stimulating cell proliferation; it is a key driver mutation in myeloproliferative neoplasms. The JAK2-V617F gene mutation has a high frequency in various MPN patient populations and plays a crucial diagnostic role in BCR-ABL-negative MPNs. In 2008, the World Health Organization (WHO) included the JAK2-V617F mutation in the main diagnostic criteria for BCR-ABL-negative MPN.

[0003] Current methods for diagnosing tumors include bone marrow biopsy, blood tests, imaging examinations, and molecular testing. Bone marrow biopsy is a common method for diagnosing myeloproliferative neoplasms (MPNs) and is considered the "gold standard," but the procedure is difficult and invasive. Blood tests are another common method for diagnosing MPNs. They examine a patient's blood sample to check for abnormal white blood cells, red blood cells, and platelets. However, this method has low sensitivity and is prone to false negatives. Imaging examinations are also used to determine MPNs. These typically use X-rays, CT scans, MRI, and PET scans to examine the patient's internal structures for abnormal masses or other abnormalities. However, these methods also have low sensitivity and rely on confirmation by other means. Molecular testing is a more sensitive and convenient technique. For BCR-ABL-negative MPN patients, the JAK2 V617F mutation is a good diagnostic target. Molecular testing includes allele-specific PCR (ARMS-PCR), NGS, high-resolution melting curve assay, and quantitative real-time PCR. Allele-specific PCR is a commonly used initial screening method for MPN patients, detecting allele mutations at the 1% level. NGS can detect a wide range of genomic mutations, but its sensitivity is low, it is time-consuming, and increasing sensitivity requires increasing sequencing depth, significantly increasing costs. High-resolution melting curve assay can detect potential mutations in the JAK2 gene without probes, but its sensitivity is low (5%-10%) and it cannot quantify or determine the specific mutation content. Quantitative real-time PCR, based on the ARMS-PCR principle, can achieve a sensitivity of up to 1%, but due to the poor sequence specificity of single-point mutations, non-specific amplification is prone to occur, leading to a high false-positive rate. Furthermore, all of the above molecular testing methods require complex pretreatment processes such as leukocyte isolation and nucleic acid extraction, which are cumbersome and time-consuming, failing to meet the demand for timely results reporting in outpatient settings. Summary of the Invention

[0004] The purpose of this invention is to address the shortcomings of existing technologies by proposing a point-of-care testing (POCT) method for detecting human JAK2 gene V617F mutations.

[0005] To achieve the above objectives, the present invention adopts the following technical solution:

[0006] A point-of-care testing (POCT) method for detecting the human JAK2 gene V617F mutation involves dividing an integrated detection tube into three parts from top to bottom: a cell lysis-nucleic acid binding magnetic bead region, a nucleic acid washing region, and a nucleic acid elution-amplification region. The cell lysis-nucleic acid binding magnetic bead region is pre-loaded with lysis buffer, the nucleic acid washing region is pre-loaded with nucleic acid washing buffer, and the nucleic acid elution-amplification region is pre-loaded with a nucleic acid elution-amplification system. The detection process only requires adding the sample to the cell lysis-nucleic acid binding magnetic bead region, inserting the corresponding instrument, and running the test to complete nucleic acid extraction, amplification, and result reporting, without any additional manual processing steps.

[0007] As a preferred embodiment of the present invention, the nucleic acid elution-nucleic acid amplification region adopts the AARMS-PCR nucleic acid amplification system, which includes an internal standard module, a detection module, and an inhibition module. The internal standard module is used to monitor the effectiveness of the entire detection process, and the inhibition module is used to specifically inhibit the amplification of wild-type alleles.

[0008] As a preferred embodiment of the present invention, the cell lysis-nucleic acid binding magnetic bead region includes a nucleic acid extraction reagent and magnetic beads. The nucleic acid extraction reagent includes isopropanol, guanidine hydrochloride and proteinase K, wherein the working concentration of isopropanol is 40%~60%, the working concentration of guanidine hydrochloride is 1~4M, and the working concentration of proteinase K is 0.2~2mg / mL.

[0009] In a preferred embodiment of the present invention, the internal standard module includes a pair of internal standard primers and an internal standard probe, the detection module includes a pair of detection primers and a detection probe, and the inhibition module includes an inhibition probe; the internal standard probe and the detection probe are modified with different fluorescence, and the 3' end of the inhibition probe is blocked and cannot be used as a primer for amplification.

[0010] In a preferred embodiment of the present invention, the internal standard primer targets a conserved sequence of the JAK2 gene, the detection primer targets the region where the V617F mutation site of the JAK2 gene is located, and the first base at the 3' end of one of the detection primers is complementary to the base at the mutation site, while the second or third base is mismatched with the target sequence; the inhibition probe has a length of 5-9 nt, is completely complementary to the wild-type allele sequence, and is mismatched with the mutant allele sequence.

[0011] As a preferred embodiment of the present invention, the nucleotide sequences of the internal standard primer, detection primer, and each probe are as follows:

[0012] Upstream internal standard primer: SEQ ID NO.1: 5'-CCTGTTTGACTGGCATTA;

[0013] Downstream internal standard primer: SEQ ID NO.2: 5'-CTGTGTAAGCCTGGAATATG;

[0014] Internal standard probe: SEQ ID NO.3: 5'-CATGATTCCTGTACCACTCTTGCTCTC;

[0015] Upstream detection primer: SEQ ID NO.4: 5'-GGACAACAGTCAAACAACA;

[0016] Downstream detection primer: SEQ ID NO.5: 5'-CTTACTCTCGTCTCCACAGTC;

[0017] Detection probe: SEQ ID NO.6: 5'-CTTGCTCATCATACTTGCTGCTTCA;

[0018] Inhibition probe: SEQ ID NO.7: 5'-AGACACA.

[0019] As a preferred embodiment of the present invention, the AARMS-PCR nucleic acid amplification system further comprises PCR buffer, dNTPs, hot-start Taq enzyme and UDG enzyme, with the following working concentrations: primers and probes 0.05~0.3μM, PCR buffer 0.5×~2×, dNTPs 450μM~900μM, hot-start Taq enzyme 5U~20U, and UDG enzyme 0.5U~2U.

[0020] As a preferred embodiment of the present invention, the specific operation steps are as follows:

[0021] 1) Add the sample to be tested into the detection tube and close the tube cap;

[0022] 2) Place the test tube into the applicable instrument. The instrument will automatically complete the lysis, nucleic acid extraction, amplification and signal detection. The lysis program is 60℃ for 10 min and 95℃ for 10 min. The amplification program is 94℃ for 5 min (1 cycle), 94℃ for 5 s and 60℃ for 30 s (50 cycles).

[0023] In a preferred embodiment of the present invention, the sample to be tested is a whole blood sample, and the sample volume is 200 μL; the mutation detection sensitivity of the detection method is not less than 0.1%.

[0024] This invention provides a point-of-care testing (POCT) method for detecting the human JAK2 gene V617F mutation, which, compared with existing technologies:

[0025] This invention designs and optimizes a method for detecting mutant alleles. This method reduces wild-type allele amplification by inhibiting probes, thereby improving the sensitivity and specificity of detecting allele mutations.

[0026] Fast and efficient: This invention takes only 1 hour from sample loading to report results.

[0027] Easy to operate: This invention only requires two steps, "sample addition and instrument loading," to complete the detection of whole blood samples and obtain the test results. The target gene can be detected in a single test tube, making the operation simple.

[0028] Low risk of contamination: The detection system adopts a fully enclosed design, which significantly reduces the risk of contamination;

[0029] Wide range of applications: It can directly test whole blood samples without separating white blood cells or purifying nucleic acids, making it highly applicable in clinical settings;

[0030] High specificity: By designing to suppress probe-primer mismatch, it reduces wild-type amplification interference and decreases false positives;

[0031] High sensitivity: The mutation detection sensitivity reaches 0.1%, which is significantly better than traditional detection methods. Attached Figure Description

[0032] Figure 1 This is a schematic diagram of the detection tube used in this invention;

[0033] Figure 2 This is a schematic diagram of the AARMS-PCR principle. Detailed Implementation

[0034] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. The specific embodiments described herein are merely used to explain the present invention and are not intended to limit the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0035] The detection tube of the present invention (hereinafter referred to as the detection tube, see below) Figure 1 From top to bottom, it includes the lysis-nucleic acid binding magnetic bead region, the magnetic bead-nucleic acid washing region, and the nucleic acid elution-nucleic acid amplification region.

[0036] The specific gene sequence of the JAK2 gene V617F mutation was detected in the nucleic acid elution-nucleic acid amplification region (hereinafter referred to as the amplification region).

[0037] This invention's internal standard system comprises a set of primers and probes targeting the conserved JAK2 sequence to monitor the effectiveness of extraction, purification, and amplification reactions. The detection tube is pre-loaded with nucleic acid extraction reagents, magnetic beads, nucleic acid purification reagents, nucleic acid elution reagents, and PCR reaction reagents. Before use, the user simply adds the sample to the detection tube. Under the control of the applicable instrument, sample lysis and nucleic acid purification are automatically completed. The purified nucleic acid and reaction reagents are mixed and heated by the applicable instrument for AARMS-PCR amplification. Simultaneously, the fluorescent probe specifically binds to the target, generating a fluorescent signal. The applicable instrument collects the fluorescence signal and automatically determines the test results by analyzing changes in the fluorescence signal.

[0038] Example 1

[0039] The optimal range and value for suppressing probe length and concentration were determined.

[0040] Table 1. Test Samples

[0041]

[0042] Table 2. Inhibition probes

[0043]

[0044] Table 3. Nucleic Acid Amplification Reagents

[0045]

[0046] Samples: The cell lines shown in Table 1 were used as the test samples; the cell concentration was 25 cells / μL, and 200 μL of sample was added.

[0047] Reagents: Nucleic acid amplification reagents: Prepare amplification reagents according to the concentrations in Table 3 (inhibition probes are prepared using the concentration gradients described in Table 4), and perform three independent replicate experiments for each condition.

[0048] Detection tube: Detection tube

[0049] Instrument: Fully automated medical PCR analysis system (Hangzhou Ustar Biotechnology Co., Ltd.)

[0050] Suppression probes: Segment sequences of different lengths from those in Table 2 were used as suppression probes.

[0051] Table 4. Suppressing probe concentration gradients

[0052]

[0053] Detection: Add the above sample (JAK2 V617F mutant or wild-type cell suspension) to the amplification reaction system of the detection tube and mix well.

[0054] The amplification conditions are shown in the table below:

[0055] Table 5. Nucleic Acid Amplification Conditions

[0056]

[0057] Experimental results: See Table 6, where UN indicates no amplification signal detected;

[0058] Experimental conclusion:

[0059] The optimal fragment size of the inhibition probe is 7 nt, and the working concentration is 0.05~0.3 μM. Preferably, the working concentration of the inhibition probe is 0.2 μM.

[0060] Table 6. Experimental Results of Example 1

[0061]

[0062] Example 2

[0063] Samples: Mixtures of JAK2 wild-type and mutant cell lines (cell concentration of 10,000 cells / μL) in different proportions as shown in Table 1. The mixing proportions are shown in Table 7.

[0064] Test tubes: Test tubes (complete system)

[0065] Instrument: Fully automated medical PCR analysis system (Hangzhou Ustar Biotechnology Co., Ltd.)

[0066] Cell lines mixed in different proportions were used as samples. 20 μL of the sample was added to the test tube and the samples were tested using a fully automated medical PCR analysis system. Each group was repeated 3 times.

[0067] Table 7. Mixed ratio of wild-type and mutant cells

[0068]

[0069] Experimental results: see Table 8;

[0070] Table 8. Experimental Results of Example 2

[0071]

[0072] Experimental conclusion: The human JAK2 gene V617F point mutation detection kit disclosed in this invention can detect mutant genes with a proportion as low as 0.1%.

[0073] In summary:

[0074] This invention designs and optimizes a method for detecting mutant alleles. This method reduces wild-type allele amplification by inhibiting probes, thereby improving the sensitivity and specificity of detecting allele mutations.

[0075] Fast and efficient: This invention takes only 1 hour from sample loading to report results.

[0076] Easy to operate: This invention only requires two steps, "sample addition and instrument loading," to complete the detection of whole blood samples and obtain the test results. The target gene can be detected in a single test tube, making the operation simple.

[0077] Low risk of contamination: The detection system adopts a fully enclosed design, which significantly reduces the risk of contamination;

[0078] Wide range of applications: It can directly test whole blood samples without separating white blood cells or purifying nucleic acids, making it highly applicable in clinical settings;

[0079] High specificity: By designing to suppress probe-primer mismatch, it reduces wild-type amplification interference and decreases false positives;

[0080] High sensitivity: The mutation detection sensitivity reaches 0.1%, which is significantly better than traditional detection methods.

[0081] It should be noted that:

[0082] In the sequence listing of this patent application, SEQ ID NO.7 is an inhibition probe with the sequence 5'-AGACACA-3' and a length of 7 bases.

[0083] Because this sequence is a short oligonucleotide, it may be automatically marked as a "skipped sequence" in some sequence listing management systems. It should be noted that this sequence is one of the key technical features of this invention, has been explicitly disclosed in the claims and description, and should be considered part of the sequence listing.

[0084] This inhibitory probe specifically blocks the amplification of the wild-type JAK2 gene in the AARMS-PCR nucleic acid amplification system, and is a key component for improving detection sensitivity and specificity.

[0085] Finally, it should be noted that the above are merely preferred embodiments of the present invention and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A point-of-care testing (POCT) method for detecting the V617F mutation in the human JAK2 gene, characterized in that: The integrated detection tube is divided into three parts from top to bottom by multiple hydrophobic layers: a cell lysis-nucleic acid binding magnetic bead area, a nucleic acid washing area, and a nucleic acid elution-amplification area. The cell lysis-nucleic acid binding magnetic bead area is pre-filled with lysis buffer, the nucleic acid washing area is pre-filled with nucleic acid washing buffer, and the nucleic acid elution-amplification area is pre-filled with a nucleic acid elution-amplification system. The detection process only requires adding the sample to the cell lysis-nucleic acid binding magnetic bead area, inserting the corresponding instrument, and running the test to complete nucleic acid extraction, amplification, and result reporting, without any additional manual processing steps.

2. The POCT detection method according to claim 1, characterized in that, The nucleic acid elution-nucleic acid amplification region uses the AARMS-PCR nucleic acid amplification system, which includes an internal standard module, a detection module, and an inhibition module. The internal standard module is used to monitor the effectiveness of the entire detection process, and the inhibition module is used to specifically inhibit the amplification of wild-type alleles.

3. The POCT detection method according to claim 2, characterized in that, The cell lysis-nucleic acid binding magnetic bead region includes a nucleic acid extraction reagent and magnetic beads. The nucleic acid extraction reagent includes isopropanol, guanidine hydrochloride, and proteinase K, wherein the working concentration of isopropanol is 40%~60%, the working concentration of guanidine hydrochloride is 1~4M, and the working concentration of proteinase K is 0.2~2mg / mL.

4. The POCT detection method according to claim 3, characterized in that, The internal standard module includes a pair of internal standard primers and an internal standard probe; the detection module includes a pair of detection primers and a detection probe; and the inhibition module includes an inhibition probe. The internal standard probe and the detection probe are modified with different fluorescence, and the 3' end of the inhibition probe is blocked, so it cannot be used as a primer for amplification.

5. The POCT detection method according to claim 4, characterized in that, The internal standard primer targets a conserved sequence of the JAK2 gene, and the detection primer targets the region where the V617F mutation site of the JAK2 gene is located. In the detection primer, the first base at the 3' end is complementary to the base at the mutation site, and the second or third base is mismatched with the target sequence. The inhibition probe is 5-9 nt in length, is completely complementary to the wild-type allele sequence, and has a mismatch with the mutant allele sequence.

6. The POCT detection method according to claim 5, characterized in that, The nucleotide sequences of the internal standard primer, detection primer, and each probe are as follows: Upstream internal standard primer: SEQ ID NO.1: 5'-CCTGTTTGACTGGCATTA; Downstream internal standard primer: SEQ ID NO.2: 5'-CTGTGTAAGCCTGGAATATG; Internal standard probe: SEQ ID NO.3: 5'-CATGATTCCTGTACCACTCTTGCTCTC; Upstream detection primer: SEQ ID NO.4: 5'-GGACAACAGTCAAACAACA; Downstream detection primer: SEQ ID NO.5: 5'-CTTACTCTCGTCTCCACAGTC; Detection probe: SEQ ID NO.6: 5'-CTTGCTCATCATACTTGCTGCTTCA; Inhibition probe: SEQ ID NO.7: 5'-AGACACA.

7. The POCT detection method according to claim 3, characterized in that, The AARMS-PCR nucleic acid amplification system also includes PCR buffer, dNTPs, hot-start Taq enzyme, and UDG enzyme. The working concentrations of each component are as follows: primers and probes 0.05~0.3μM, PCR buffer 0.5×~2×, dNTPs 450μM~900μM, hot-start Taq enzyme 5U~20U, and UDG enzyme 0.5U~2U.

8. The POCT detection method according to claim 1, characterized in that, The specific operating steps are as follows: 1) Add the sample to be tested into the detection tube and close the tube cap; 2) Place the test tube into the applicable instrument. The instrument will automatically complete the lysis, nucleic acid extraction, amplification and signal detection. The lysis program is 60℃ for 10 min and 95℃ for 10 min. The amplification program is 94℃ for 5 min (1 cycle), 94℃ for 5 s and 60℃ for 30 s (50 cycles).

9. The POCT detection method according to claim 1 or 8, characterized in that, The sample to be tested is a whole blood sample, and the sample volume is 200 μL; the mutation detection sensitivity of the detection method is not less than 0.1%.

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