Primer probe combination and kit for rapidly and jointly detecting JAK2, CALR and MPL gene mutation and application of primer probe combination and kit

Abstract

The invention discloses a primer probe combination and a kit for rapidly and jointly detecting JAK2, CALR and MPL gene mutation and application of the primer probe combination and the kit. The primer probe combination and the kit provided by the invention can accurately identify and combine target mutation sites in detection of JAK2, CALR and MPL gene mutation, can significantly improve the detection sensitivity and specificity of gene mutation, and are beneficial to early diagnosis of MPN diseases. The kit provided by the invention is simple and convenient to use and operate, is more convenient and rapid compared with technologies such as next-generation sequencing (NGS) requiring tedious steps and longer time, and is suitable for clinical rapid high-throughput diagnosis.

Description

Technical Field

[0001] The present invention relates to the technical field of gene detection, and in particular to a primer-probe combination, a kit and applications thereof for rapid combined detection of JAK2, CALR and MPL gene mutations. Background Art

[0002] Myeloproliferative neoplasms (MPNs) are a group of malignant tumors that are characterized by excessive proliferation of one or more bone marrow lines and originate from clonal hematopoietic stem cells. The pathogenic mechanism is mainly due to mutations in JAK / STAT pathway-related genes (such as JAK2, CALR, and MPL), which drive excessive proliferation of myeloid cells and cause disease. Among them, BCR-ABL1-negative MPNs include polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF). Studies have shown that the mutation frequency of JAK2 gene c.1849G>T (p.V617F) is approximately 95% in PV patients, approximately 50-60% in ET patients, and approximately 50% in PMF patients; the mutation frequency of CALR gene type I mutation c.1092_1143del52 (p.L367fs*46) and CALR gene type II mutation c.1154_1155insTTGTC (p.K385fs*47) is approximately 20-25% in ET patients and approximately 25-30% in PMF patients; the mutation frequency of MPL gene c.1544G>T (p.W515L) and MPL gene c.1543_1544delinsAA (p.W515K) is approximately 1-10% in ET and PMF patients. The 2022 edition of the WHO classification and diagnosis of MPNs clearly lists JAK2, CALR, and MPL mutations as one of the molecular indicators for the diagnosis of MPNs. Both the NCCN guidelines and the Chinese expert guidelines recommend the use of this standard. Therefore, the combined detection of JAK2, CALR, and MPL genes has important clinical significance.

[0003] At present, the main methods for detecting JAK2, CALR and MPL gene mutations include high-resolution melting curve (HRM) and sequencing, but they all have many shortcomings. For example, although the HRM method can analyze different genotypes through melting curves, its sensitivity and specificity are relatively low, especially for detecting low-frequency mutations and complex mutations, which is prone to misjudgment; methods such as Sanger sequencing have low sensitivity when detecting low-frequency mutations, which may lead to low mutation detection rates; the second-generation sequencing (NGS) technology requires cumbersome steps and a long time, and the required instruments and reagents are expensive.

[0004] Therefore, there is an urgent need to develop a method with high sensitivity that can simultaneously and rapidly detect JAK2, CALR and MPL gene mutations, in order to overcome the shortcomings of traditional PCR, sequencing and other methods, such as long time consumption, cumbersome operation steps and low mutation detection sensitivity. Summary of the invention

[0005] To address the deficiencies of the prior art, the present invention provides a primer-probe combination, a kit and applications thereof for rapid combined detection of JAK2, CALR and MPL gene mutations.

[0006] Specifically, the present invention provides a combination of primers and probes for rapid detection of JAK2, CALR and MPL gene mutations, wherein the primers and the probes are selected from one or more of the following (1)-(5):

[0007] (1) The primers are specific amplification primers for detecting JAK2 gene mutations, and the nucleotide sequences of the upstream primer and the downstream primer are shown in SEQ ID NO: 1 and SEQ ID NO: 2, respectively;

[0008] The probe is a probe for detecting JAK2 gene mutation, and the nucleotide sequence of the probe is shown in SEQ ID NO: 4;

[0009] (2) The primers are specific amplification primers for detecting CALR gene mutations, and the nucleotide sequences of the upstream primer and the downstream primer are shown in SEQ ID NO: 5 and SEQ ID NO: 7, respectively;

[0010] The probe is a probe for detecting CALR gene mutation, and the nucleotide sequence of the probe is shown in SEQ ID NO: 8;

[0011] (3) The primers are specific amplification primers for detecting CALR gene mutations, and the nucleotide sequences of the upstream primer and the downstream primer are shown in SEQ ID NO: 6 and SEQ ID NO: 7, respectively;

[0012] The probe is a probe for detecting CALR gene mutation, and the nucleotide sequence of the probe is shown in SEQ ID NO: 8;

[0013] (4) The primers are specific amplification primers for detecting MPL gene mutations, and the nucleotide sequences of the upstream primer and the downstream primer are shown in SEQ ID NO: 9 and SEQ ID NO: 10, respectively;

[0014] The probe is a probe for detecting MPL gene mutation, and the nucleotide sequence of the probe is shown in SEQ ID NO: 12;

[0015] (5) The primers are specific amplification primers for detecting MPL gene mutations, and the nucleotide sequences of the upstream primer and the downstream primer are shown in SEQ ID NO: 9 and SEQ ID NO: 11, respectively;

[0016] The probe is a probe for detecting MPL gene mutation, and the nucleotide sequence of the probe is shown in SEQ ID NO:12.

[0017] In some embodiments, the primers further include a specific amplification primer for an internal reference and a probe for detecting the internal reference.

[0018] In some specific embodiments, the nucleotide sequences of the specific amplification primers of the internal reference are shown as SEQ ID NOs: 13-14, respectively.

[0019] In some specific embodiments, the nucleotide sequence of the probe for detecting the internal reference is shown in SEQ ID NO:15.

[0020] In some specific embodiments, (1) further comprises a blocking probe having a nucleotide sequence as shown in SEQ ID NO:3.

[0021] In some specific embodiments, the 3' end of the blocking probe further includes a C3 Spacer.

[0022] In some specific embodiments, the 5' end of the probe is labeled with any one of the fluorescent dyes FAM, VIC, ROX, CY5, JOE, HEX and TAMRA, and the 3' end of the probe is labeled with the quenching fluorescent dye BHQ.

[0023] In some specific embodiments, the 3' end of the probes are labeled with a quenching fluorescent dye BHQ1, BHQ2 or BHQ3.

[0024] The present invention also provides a kit for rapid detection of JAK2, CALR and MPL gene mutations, the kit comprising one or more of an MPN PCR reaction solution, an MPN enzyme mixture, an MPN primer-probe mixture A and an MPN primer-probe mixture B; wherein the MPN primer-probe mixture A comprises the primers and probes described in (1) and (2) above; and the MPN primer-probe mixture B comprises the primers and probes described in (3), (4) and (5) above.

[0025] In some embodiments, the kit further comprises one or more of MPN positive control PC, MPN negative control NC1, and MPN negative control NC2.

[0026] In some specific embodiments, the MPN-positive quality control product PC is a JAK2 / CALR / MPL gene mutant nucleic acid prepared from JAK2 gene c.1849G>T mutant cell nucleic acid, CALR gene c.1092_1143del52 mutant cell nucleic acid, CALR gene c.1154_1155insTTGTC mutant cell nucleic acid, and MPL gene c.1544G>T mutant cell nucleic acid; the MPN-negative quality control product NC1 is JAK2 / CALR / MPL gene wild-type cell nucleic acid; and the MPN-negative quality control product NC2 is normal saline.

[0027] In some embodiments, the MPN primer-probe mixture A and the MPN primer-probe mixture B further include specific amplification primers for internal references and probes for detecting the internal references.

[0028] In some specific embodiments, the nucleotide sequences of the specific amplification primers of the internal reference are shown as SEQ ID NOs: 13-14, respectively.

[0029] In some specific embodiments, the nucleotide sequence of the probe for detecting the internal reference is shown in SEQ ID NO:15.

[0030] In some specific embodiments, the MPN primer-probe mixture A further includes a blocking probe having a nucleotide sequence as shown in SEQ ID NO:3.

[0031] In some specific embodiments, the 5' end of the probe is labeled with any one of the fluorescent dyes FAM, VIC, ROX, CY5, JOE, HEX and TAMRA, and the 3' end of the probe is labeled with the quenching fluorescent dye BHQ.

[0032] In some embodiments, the concentration of the primers is 200-400 nM, and the concentration of the probes is 100-250 nM.

[0033] In some specific embodiments, the MPN primer-probe mixture A consists of: 400nM JAK2-F, 400nM JAK2-R, 150nM JAK2-B, 200nM JAK2-P, 400nM CALR-M1-F, 400nM CALR-R, 200nM CALR-P, 200nM NC-F, 200nM NC-R and 100nM NC-P.

[0034] In some specific embodiments, the MPN primer-probe mixture B consists of: 400nM CALR-M2-F, 400nM CALR-R, 200nM CALR-P, 400nM MPL-F, 400nM MPL-M1-R, 400nM MPL-M2-R, 200nM MPL-P, 200nM NC-F, 200nM NC-R and 100nM NC-P.

[0035] In some specific embodiments, the fluorescent label of JAK2-P is 5'FAM-3'BHQ1, the fluorescent label of CALR-P is 5'ROX-3'BHQ2, the fluorescent label of MPL-P is 5'FAM-3'BHQ1, and the fluorescent label of NC-P is 5'VIC-3'BHQ1.

[0036] In some specific embodiments, the MPN enzyme mixture consists of: 0.1 U / μL Hotstart TaqDNA Polymerase and 0.04 U / μL Uracil DNA Glycosylase, Heat-labile.

[0037] In some specific embodiments, the positive control product consists of: 10 6 copies / mL of genomic DNA extracted from cell cultures carrying JAK2 gene c.1849G>T, CALR gene c.1092_1143del52 / c.1154_1155insTTGTC, and MPL gene c.1544G>T / c.1543_1544delinsAA mutations.

[0038] In some specific embodiments, the negative control product is composed of: 10 6 The genomic DNA extracted from wild-type cell culture of JAK2 gene, CALR gene and MPL gene at 10 copies / mL.

[0039] In some embodiments, the MPN PCR reaction solution includes 5×Hotstart Taq PCR Buffer and dNTP.

[0040] In some embodiments, the dNTPs include 100-600 μM dATP, 100-600 μM dGTP, 100-600 μM dCTP, 50-300 μM dTTP, and 50-300 μM dUTP.

[0041] In some specific embodiments, the 5×Hotstart Taq PCR Buffer includes an enhancer.

[0042] In some specific embodiments, the enhancers are tetramethylammonium chloride and BSA.

[0043] In some specific embodiments, the dNTPs include 200 μM dATP, 200 μM dGTP, 200 μM dCTP, 200 μM dTTP and 100 μM dUTP.

[0044] In some specific embodiments, the MPN PCR reaction solution includes 5×Hotstart Taq PCR Buffer (20 mM Tris-HCl, 150 mM KCl, 150 mM (NH4)2SO4, 20 μM MgCl2, 150 mM tetramethylammonium chloride, 300 μg / mL BSA), and 20 μM dNTP.

[0045] In some embodiments, the MPN enzyme mixture includes Hotstart Taq DNA Polymerase and Uracil DNA Glycosylase, Heat-labile.

[0046] In some embodiments, the MPN enzyme mixture includes 0.005-0.5 U / μL Hotstart Taq DNA Polymerase and 0.005-0.5 U / μL Uracil DNA Glycosylase, Heat-labile.

[0047] In some specific embodiments, the MPN enzyme mixture includes 0.1 U / μL Hotstart Taq DNA Polymerase and 0.04 U / μL Uracil DNA Glycosylase, Heat-labile.

[0048] The present invention also provides a method for rapidly detecting JAK2, CALR and MPL gene mutations, the method comprising the following steps:

[0049] The combination of primers and probes described in the present disclosure or the kit described in the present disclosure is used to perform PCR detection on the nucleic acid of the sample to be tested; the method is for non-diagnostic purposes.

[0050] In some embodiments, the sample to be tested is a biological sample containing genomic DNA.

[0051] In some specific embodiments, the sample to be tested is EDTA-anticoagulated bone marrow or peripheral blood.

[0052] In some specific embodiments, the reaction conditions of the PCR detection are: 37°C UDG enzyme reaction for 2 min, 95°C pre-denaturation for 5 min; 95°C denaturation for 15 s, 60°C annealing and extension for 40 s, for a total of 45 cycles.

[0053] In some specific embodiments, the result of the method is interpreted based on the amplification of each fluorescent channel in the reaction solution of tubes A and B and the size of the amplification ct value to perform typing. Further, when an amplification curve of a corresponding color (depending on the specific fluorescent labeling group) appears and the Ct value is less than 39, it means that the test sample contains the type mutation.

[0054] The present invention also provides the use of the combination of primers and probes described in the present disclosure or the kit described in the present disclosure in detecting JAK2, CALR and MPL gene mutations; the use is for non-diagnostic purposes.

[0055] In this article, non-diagnostic application scenarios include, for example, detecting JAK2, CALR and MPL gene mutations in ex vivo samples for research and development purposes.

[0056] In some embodiments, for amplification primers, blocking probes, and detection probes for highly sensitive detection of JAK2 gene c.1849G>T mutations, since the JAK2 gene c.1849G>T site is near a high AT content, it is easy to bind to the wild-type template during PCR amplification to produce non-specific results, thereby causing false positives. In order to solve this problem, on the one hand, multiple groups of amplification primers are designed for screening, and on the other hand, the present invention introduces a blocking probe. The blocking probe can completely match the wild-type template, competitively inhibit the binding of the mutant amplification primer to the wild-type template, and thus provide specificity. At the same time, because the efficiency of the blocking probe is affected by many aspects such as sequence composition and system composition, it is difficult to carry out theoretical design and prediction. Therefore, in order to obtain higher amplification efficiency and sensitivity, it is necessary to design multiple blocking probes and screen out the optimal blocking probe.

[0057] In some embodiments, for the amplification primers and detection probes for highly sensitive detection of the CALR gene c.1092_1143del52 and c.1154_1155insTTGTC mutations, the CALR gene has multiple repeat sequences at and near the mutation site, which makes the amplification very prone to non-specificity. Only through sequence alignment and differential design of multiple primer combinations can the amplification primer combination with the best amplification efficiency and sensitivity be screened.

[0058] In some embodiments, for amplification primers and detection probes for highly sensitive detection of c.1544G>T and c.1543_1544delinsAA mutations in the MPL gene, the present invention designs multiple primer-probe combinations to screen out the amplification primer combination with the best amplification efficiency and sensitivity.

[0059] In some embodiments, the present invention provides amplification primers and detection probes for monitoring internal standards of an amplification system. By comparing the database, the internal standards of the present invention are selected as highly conserved exon regions of the JAK2 gene that are not prone to mutation.

[0060] On the basis of being in accordance with the common sense in the art, the above-mentioned preferred conditions can be arbitrarily combined to obtain the preferred embodiments of the present invention.

[0061] The reagents and raw materials used in the present invention are commercially available.

[0062] The positive and progressive effects of the present invention are:

[0063] 1. High sensitivity and specificity: The present invention uses real-time fluorescence PCR, amplification retardation mutation system (ARMS) combined with Taqman fluorescent probe technology, which can accurately identify and bind to target mutation sites, and can significantly improve the detection sensitivity and specificity of gene mutations. The detection method of the present invention can at least detect JAK2, CALR and MPL gene mutations with a mutation ratio of 1% at 5ng / μL, which is helpful for the early diagnosis of MPN diseases.

[0064] 2. Easy operation: The operation steps of multiplex fluorescence PCR are simple, and the entire detection process can be completed within 1-2 hours. Compared with technologies such as next-generation sequencing (NGS) that require cumbersome steps and a long time, multiplex fluorescence PCR is more convenient and faster, and is suitable for rapid clinical diagnosis.

[0065] 3. High-throughput detection: Multiplex fluorescence PCR can simultaneously detect multiple gene mutations in a single reaction, which significantly improves detection efficiency and throughput. BRIEF DESCRIPTION OF THE DRAWINGS

[0066] Figure 1A-1F is a schematic diagram of sample test results, where: Figure 1A This is the amplification curve of the positive sample for detecting the JAK2 gene c.1849G>T mutation; Figure 1B This is the amplification curve diagram of the sample positive for the CALR gene c.1092_1143del52 mutation; Figure 1C This is the amplification curve of the sample positive for the CALR gene c.1154_1155insTTGTC mutation; Figure 1D This is the amplification curve of the sample positive for the MPL gene c.1544G>T mutation; Figure 1EThis is the amplification curve of the sample positive for the MPL gene c.1543_1544delinsAA mutation; Figure 1F It is an amplification curve diagram for detecting the wild type of JAK2 / CALR / MPL; wherein, the ordinate of the amplification curve diagram is ΔRn, and the abscissa is the Cycle value, and the value range is 2-44.

[0067] Figure 2A-2E is a schematic diagram of the detection limit test results, where: Figure 2A This is the amplification curve diagram for detecting JAK2 gene c.1849G>T mutation-positive samples (5ng / μL, 1% mutation ratio); Figure 2B This is the amplification curve diagram for detecting the CALR gene c.1092_1143del52 mutation-positive sample (5 ng / μL, 1% mutation ratio); Figure 2C This is the amplification curve diagram for detecting the CALR gene c.1154_1155insTTGTC mutation-positive sample (5ng / μL, 1% mutation ratio); Figure 2D This is the amplification curve of the positive sample (5ng / μL, 1% mutation ratio) for detecting the c.1544G>T mutation of the MPL gene; Figure 2E This is an amplification curve diagram for detecting the MPL gene c.1543_1544delinsAA mutation-positive sample (5 ng / μL, 1% mutation ratio); wherein the ordinate of the amplification curve diagram is ΔRn, and the abscissa is the Cycle value, with a numerical range of 2-44.

[0068] Figure 3A-3E This is a schematic diagram of the sample detection results using primer probe combination 2. The left picture is reaction A, and the right picture is reaction B. Figure 3A This is the amplification curve of the positive sample for detecting the JAK2 gene c.1849G>T mutation; Figure 3B This is the amplification curve diagram of the sample positive for the CALR gene c.1092_1143del52 mutation; Figure 3C This is the amplification curve of the sample positive for the CALR gene c.1154_1155insTTGTC mutation; Figure 3D This is the amplification curve of the sample positive for the MPL gene c.1544G>T mutation; Figure 3E This is an amplification curve diagram for detecting positive samples of the MPL gene c.1543_1544delinsAA mutation; wherein the ordinate of the amplification curve diagram is ΔRn, and the abscissa is the Cycle value, with a value range of 2-44.

[0069] Figure 4A-4E This is a schematic diagram of the sample detection results using primer probe combination 3. The left picture is reaction A, and the right picture is reaction B. Figure 4AThis is the amplification curve of the positive sample for detecting the JAK2 gene c.1849G>T mutation; Figure 4B This is the amplification curve diagram of the sample positive for the CALR gene c.1092_1143del52 mutation; Figure 4C This is the amplification curve of the sample positive for the CALR gene c.1154_1155insTTGTC mutation; Figure 4D This is the amplification curve of the sample positive for the MPL gene c.1544G>T mutation; Figure 4E This is an amplification curve diagram for detecting positive samples of the MPL gene c.1543_1544delinsAA mutation; wherein the ordinate of the amplification curve diagram is ΔRn, and the abscissa is the Cycle value, with a value range of 2-44.

[0070] Figure 5A-5E This is a schematic diagram of the sample detection results using primer probe combination 4. The left picture is reaction A, and the right picture is reaction B. Figure 5A This is the amplification curve of the positive sample for detecting the JAK2 gene c.1849G>T mutation; Figure 5B This is the amplification curve diagram of the sample positive for the CALR gene c.1092_1143del52 mutation; Figure 5C This is the amplification curve of the sample positive for the CALR gene c.1154_1155insTTGTC mutation; Figure 5D This is the amplification curve of the sample positive for the MPL gene c.1544G>T mutation; Figure 5E This is an amplification curve diagram for detecting positive samples of the MPL gene c.1543_1544delinsAA mutation; wherein the ordinate of the amplification curve diagram is ΔRn, and the abscissa is the Cycle value, with a value range of 2-44.

[0071] Figure 6A-6F This is a schematic diagram of sample detection results using primers of different concentrations. The left picture is reaction A, and the right picture is reaction B. Fig. 6A This is the amplification curve of the positive sample for detecting the JAK2 gene c.1849G>T mutation; Figure 6B This is the amplification curve diagram of the sample positive for the CALR gene c.1092_1143del52 mutation; Figure 6C This is the amplification curve of the sample positive for the CALR gene c.1154_1155insTTGTC mutation; Fig.6D This is the amplification curve of the sample positive for the MPL gene c.1544G>T mutation; Fig. 6E This is the amplification curve of the sample positive for the MPL gene c.1543_1544delinsAA mutation; Fig. 6FIt is the amplification curve diagram of the negative sample; wherein, the ordinate of the amplification curve diagram is ΔRn, and the abscissa is the Cycle value, and the value range is 2-44.

[0072] Figure 7A-7F This is a schematic diagram of the sample detection results using Champagne Taq DNA Polymerase. The left picture is reaction A, and the right picture is reaction B. Fig. 7A This is the amplification curve of the positive sample for detecting the JAK2 gene c.1849G>T mutation; Figure 7B This is the amplification curve diagram of the sample positive for the CALR gene c.1092_1143del52 mutation; Figure 7C This is the amplification curve of the sample positive for the CALR gene c.1154_1155insTTGTC mutation; Fig.7D This is the amplification curve of the sample positive for the MPL gene c.1544G>T mutation; Fig. 7E This is the amplification curve of the sample positive for the MPL gene c.1543_1544delinsAA mutation; Figure 7F It is the amplification curve diagram of the negative sample; wherein, the ordinate of the amplification curve diagram is ΔRn, and the abscissa is the Cycle value, and the value range is 2-44.

[0073] Figure 8A-8F For use Schematic diagram of sample detection results using HotStart Taq DNA Polymerase. The left picture is reaction A, and the right picture is reaction B. Fig. 8A This is the amplification curve of the positive sample for detecting the JAK2 gene c.1849G>T mutation; Figure 8B This is the amplification curve diagram of the sample positive for the CALR gene c.1092_1143del52 mutation; Figure 8C This is the amplification curve of the sample positive for the CALR gene c.1154_1155insTTGTC mutation; Fig.8D This is the amplification curve of the sample positive for the MPL gene c.1544G>T mutation; Fig. 8E This is the amplification curve of the sample positive for the MPL gene c.1543_1544delinsAA mutation; Figure 8F It is the amplification curve diagram of the negative sample; wherein, the ordinate of the amplification curve diagram is ΔRn, and the abscissa is the Cycle value, and the value range is 2-44.

[0074] Figure 9A-9F This is a schematic diagram of the sample detection results using Golden taq DNA polymerase. The left picture is reaction A, and the right picture is reaction B. Fig. 9AThis is the amplification curve of the positive sample for detecting the JAK2 gene c.1849G>T mutation; Fig. 9B This is the amplification curve diagram of the sample positive for the CALR gene c.1092_1143del52 mutation; Fig. 9C This is the amplification curve of the sample positive for the CALR gene c.1154_1155insTTGTC mutation; Fig.9D This is the amplification curve of the sample positive for the MPL gene c.1544G>T mutation; Fig.9E This is the amplification curve of the sample positive for the MPL gene c.1543_1544delinsAA mutation; Fig.9F It is the amplification curve diagram of the negative sample; wherein, the ordinate of the amplification curve diagram is ΔRn, and the abscissa is the Cycle value, and the value range is 2-44.

[0075] Fig.10 Schematic diagram of the detection method principle. DETAILED DESCRIPTION

[0076] The present invention is further described below by way of examples, but the present invention is not limited to the scope of the examples. The experimental methods in the following examples without specifying specific conditions are carried out according to conventional methods and conditions, or selected according to the product specifications.

[0077] Composition of Example 1 Kit

[0078] The JAK2 / CALR / MPL gene mutation detection kit consists of MPN PCR reaction solution, MPN enzyme mixture, MPN primer probe mixture A, MPN primer probe mixture B, MPN positive quality control product PC, MPN negative quality control product NC1, and MPN negative quality control product NC2.

[0079] The MPN PCR reaction solution contained a final concentration of 5×Hotstart Taq PCR Buffer (Buffer from Suzhou Yuanqi Biotechnology Co., Ltd.), 200 μM dATP, 200 μM dGTP, 200 μM dCTP, 200 μM dTTP, and 100 μM dUTP (dNTP from Shanghai Zhaowei Technology Development Co., Ltd.).

[0080] Among them, MPN primer-probe mixture A contains primers (JAK2-F, JAK2-R, CALR-M1-F, CALR-R, NC-F, NC-R) with a final concentration of 0.2 μM and probes (JAK2-P, CALR-P, NC-P) with a final concentration of 0.1 μM.

[0081] Among them, MPN primer-probe mixture B contains primers (CALR-M2-F, CALR-R, MPL-F, MPL-M1-R, MPL-M2-R, NC-F, NC-R) with a final concentration of 0.2 μM and probes (CALR-P, MPL-P, NC-P) with a final concentration of 0.1 μM (primers and probes are from Anhui General Biotechnology Co., Ltd.).

[0082] The MPN enzyme mixture is 0.1U / μL Hotstart Taq DNA Polymerase and 0.04U / μL Racil DNA Glycosylase, Heat-labile (the enzyme raw materials are from Suzhou Yuanqi Biotechnology Co., Ltd.).

[0083] Among them, the PC of MPN positive quality control product is 10 6 copies / mL of JAK2 / CALR / MPL gene mutant nucleic acid.

[0084] Among them, the MPN negative quality control product NC1 is 10 6 copies / mL of wild-type nucleic acid of the JAK2 / CALR / MPL gene.

[0085] Among them, the MPN negative control product NC2 is normal saline.

[0086] Example 2 Establishment of detection method

[0087] (1) Sample processing

[0088] For DNA extraction from bone marrow or peripheral blood samples using EDTA anticoagulation, refer to the DNA kit extraction product manual for specific procedures.

[0089] (2) Preparation of PCR reaction system

[0090] According to the proportions in Table 1, the corresponding components in the JAK2 / CALR / MPL gene mutation detection kit in Example 1 were drawn.

[0091] Table 1 PCR reaction system preparation

[0092]

[0093] N = number of samples to be tested (n) + number of quality control products (3) + 1

[0094] (3) Sample addition

[0095] Take 4 μL of the nucleic acid extract of the sample to be tested and add it to the corresponding PCR reaction tube to perform PCR reaction.

[0096] (4) Amplification

[0097] Carry out PCR reaction according to the program in Table 2 below.

[0098] Table 2 PCR reaction cycle

[0099]

[0100] (5) Result judgment:

[0101] 1. When the Ct values ​​of the internal standard (VIC) channel of the two reactions of the sample to be tested are both ≤32, the result is valid and the result interpretation is carried out according to the following principles:

[0102] 1)JAK2 gene mutation is interpreted according to the following table:

[0103] Table 3 Interpretation criteria for JAK2 gene mutation results

[0104]

[0105] 2) MPL gene mutations are interpreted according to the following table:

[0106] Table 4 Criteria for interpreting MPL gene mutation results

[0107]

[0108] 3) CALR gene mutations are interpreted according to the following table:

[0109] Table 5 Criteria for interpretation of CALR gene mutation results

[0110]

[0111]

[0112] 2. When the Ct value of the internal standard (VIC) channel is greater than 32, it is recommended to resample or re-extract genomic DNA and retest once. (6) The established method detects JAK2 / CALR / MPL gene mutation samples and JAK2 / CALR / MPL wild-type samples.

[0113] Example 3 Primer screening

[0114] Table 6 Primer probe sequences

[0115]

[0116]

[0117]

[0118]

[0119] The results of the detection using primer-probe combination 1 are as follows Figure 1A-1F As shown; the detection results using primer probe combination 2 are as shown Figure 3A-3E As shown; the detection results using primer probe combination 3 are as follows Figure 4A-4E As shown; the detection results using primer probe combination 4 are as shown Figure 5A-5E According to the above results, primer-probe combination 1 was selected as the preferred combination.

[0120] Example 4 Primer concentration, Taq enzyme screening

[0121] The primer concentration screening is shown in Table 7.

[0122] Table 7. Primer concentration screening

[0123]

[0124]

[0125]

[0126] The results of primer concentration screening are as follows Figure 6A-6F As shown, it can be seen from the results that the concentration of combination 2 has the highest amplification efficiency, so the primer probe concentration of combination 2 is selected.

[0127] Taq enzyme screening is shown in Table 8.

[0128] Table 8. Taq enzyme screening

[0129]

[0130] Among them, the results of sample testing with Champagne Taq DNA Polymerase are as follows Figure 7A-7F Shown; Hieff The results of sample testing with HotStart Taq DNA Polymerase are as follows Figure 8A-8F As shown; Golden taq DNA polymerase sample test results are shown Figure 9A-9F As shown in the figure, the Golden taq DNA polymerase (5U / μL) from Suzhou Yuanqi Biotechnology Co., Ltd. has the best amplification efficiency and specificity.

[0131] Example 5 Sensitivity Detection

[0132] The digital PCR method was used to detect the gene copy number of the sample, and samples of various mutation types with different concentrations and mutation ratios were prepared. The optimized detection method of the present invention was used for detection to determine the lower limit of detection. The results showed that the method was able to detect nucleic acids as low as 5ng / μL and 1% mutation ratio. The detection limit evaluation test results are shown in Figure 2A-2E .

[0133] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A combination of primers and probes for rapid detection of JAK2, CALR and MPL gene mutations, characterized in that: The primer and the probe are selected from one or more of the following (1)-(5): (1) The primers are specific amplification primers for detecting JAK2 gene mutations, and the nucleotide sequences of the upstream primer and the downstream primer are shown in SEQ ID NO: 1 and SEQ ID NO: 2, respectively; The probe is a probe for detecting JAK2 gene mutation, and the nucleotide sequence of the probe is shown in SEQ ID NO: 4; (2) The primers are specific amplification primers for detecting CALR gene mutations, and the nucleotide sequences of the upstream primer and the downstream primer are shown in SEQ ID NO: 5 and SEQ ID NO: 7, respectively; The probe is a probe for detecting CALR gene mutation, and the nucleotide sequence of the probe is shown in SEQ ID NO: 8; (3) The primers are specific amplification primers for detecting CALR gene mutations, and the nucleotide sequences of the upstream primer and the downstream primer are shown in SEQ ID NO: 6 and SEQ ID NO: 7, respectively; The probe is a probe for detecting CALR gene mutation, and the nucleotide sequence of the probe is shown in SEQ ID NO: 8; (4) The primers are specific amplification primers for detecting MPL gene mutations, and the nucleotide sequences of the upstream primer and the downstream primer are shown in SEQ ID NO: 9 and SEQ ID NO: 10, respectively; The probe is a probe for detecting MPL gene mutation, and the nucleotide sequence of the probe is shown in SEQ ID NO: 12; (5) The primers are specific amplification primers for detecting MPL gene mutations, and the nucleotide sequences of the upstream primer and the downstream primer are shown in SEQ ID NO: 9 and SEQ ID NO: 11, respectively; The probe is a probe for detecting MPL gene mutation, and the nucleotide sequence of the probe is shown in SEQ ID NO:

12.

2. The combination of primers and probes according to claim 1, characterized in that The primers also include specific amplification primers for internal references and probes for detecting the internal references; preferably, the nucleotide sequences of the specific amplification primers for the internal references are shown in SEQ ID NOs: 13-14, respectively; the nucleotide sequence of the probe for detecting the internal reference is shown in SEQ ID NO: 15; and / or, (1) further comprising a blocking probe having a nucleotide sequence as shown in SEQ ID NO: 3; And / or, the 5' end of the probe is labeled with any one of the fluorescent dyes FAM, VIC, ROX, CY5, JOE, HEX and TAMRA, and the 3' end of the probe is labeled with the quenching fluorescent dye BHQ.

3. A kit for rapid detection of JAK2, CALR and MPL gene mutations, characterized in that: The kit includes one or more of MPNPCR reaction solution, MPN enzyme mixture, MPN primer probe mixture A and MPN primer probe mixture B; Wherein, the MPN primer-probe mixture A comprises the primers and probes as described in (1) and (2) of claim 1; and the MPN primer-probe mixture B comprises the primers and probes as described in (3), (4) and (5) of claim 1.

4. The kit according to claim 3, characterized in that The kit further comprises one or more of an MPN positive quality control product PC, an MPN negative quality control product NC1, and an MPN negative quality control product NC2; The MPN-positive quality control product PC is a JAK2 / CALR / MPL gene mutant nucleic acid prepared from JAK2 gene c.1849G>T mutant cell nucleic acid, CALR gene c.1092_1143del52 mutant cell nucleic acid, CALR gene c.1154_1155insTTGTC mutant cell nucleic acid, and MPL gene c.1544G>T mutant cell nucleic acid; the MPN-negative quality control product NC1 is JAK2 / CALR / MPL gene wild-type cell nucleic acid; and the MPN-negative quality control product NC2 is normal saline.

5. The kit according to claim 3 or 4, characterized in that The MPN primer-probe mixture A and the MPN primer-probe mixture B further include specific amplification primers for internal references and probes for detecting the internal references; preferably, the nucleotide sequences of the specific amplification primers for the internal references are respectively shown in SEQ ID NOs: 13-14; the nucleotide sequence of the probe for detecting the internal reference is shown in SEQ ID NO: 15; and / or, the MPN primer-probe mixture A further comprises a blocking probe having a nucleotide sequence as shown in SEQ ID NO: 3; And / or, the 5' end of the probe is labeled with any one of the fluorescent dyes FAM, VIC, ROX, CY5, JOE, HEX and TAMRA, and the 3' end of the probe is labeled with the quenching fluorescent dye BHQ.

6. The kit according to any one of claims 3 to 5, characterized in that The MPN PCR reaction solution includes 5×Hotstart Taq PCR Buffer and dNTP; the 5×Hotstart Taq PCR Buffer includes an enhancer; Preferably, the dNTPs include 100-600 μM dATP, 100-600 μM dGTP, 100-600 μM dCTP, 50-300 μM dTTP and 50-300 μM dUTP, and the enhancer is tetramethylammonium chloride and BSA; More preferably, the dNTPs include 200 μM dATP, 200 μM dGTP, 200 μM dCTP, 200 μM dTTP and 100 μM dUTP.

7. The kit according to any one of claims 3 to 6, characterized in that The MPN enzyme mixture includes Hotstart Taq DNA Polymerase and Uracil DNA Glycosylase, Heat-labile; Preferably, the MPN enzyme mixture comprises 0.005-0.5U / μL Hotstart Taq DNA Polymerase and 0.005-0.5U / μL Racil DNA Glycosylase, Heat-labile; More preferably, the MPN enzyme mixture includes 0.1U / μL Hotstart Taq DNA Polymerase and 0.04U / μL Racil DNA Glycosylase, Heat-labile.

8. A method for rapid detection of JAK2, CALR and MPL gene mutations, characterized in that: The method comprises the following steps: Using the combination of primers and probes as described in claim 1 or 2 or the kit as described in any one of claims 3 to 7 to perform PCR detection on the nucleic acid of the sample to be tested; the method is for non-diagnostic purposes; Preferably, the sample to be tested is a biological sample containing genomic DNA, preferably EDTA anticoagulated bone marrow or peripheral blood.

9. The method according to claim 8, characterized in that The reaction conditions of the PCR detection are: 37°C UDG enzyme reaction for 2 min, 95°C pre-denaturation for 5 min; 95°C denaturation for 15 s, 60°C annealing and extension for 40 s, for a total of 45 cycles.

10. Use of the combination of primers and probes according to claim 1 or 2 or the kit according to any one of claims 3 to 7 in detecting JAK2, CALR and MPL gene mutations; the use is for non-diagnostic purposes.

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