Primer probe combination, kit and method for quantitatively detecting JAK2 V617F gene mutation rate

Abstract

The invention provides a primer and probe combination, a kit and a method for quantitatively detecting the JAK2V617F gene mutation rate, and the primer and probe combination comprises a primer and probe set targeting JAK2V617F mutation sites and a primer and probe set of reference genes GPI, according to the primer probe set targeting JAK2V617F mutation sites, the nucleotide sequence of an upstream primer of a specific mutation site is any one of SEQ ID NO.1-7, the nucleotide sequence of an upstream primer of a specific wild type site is any one of SEQ ID NO.9-14, the nucleotide sequence of a universal downstream primer is SEQ ID NO.8, and the nucleotide sequence of a probe is SEQ ID NO.15. The invention further discloses a kit for detecting the JAK2V617F mutation sites. According to the invention, the JAK2V617F mutation rate can be economically, simply, efficiently, sensitively, accurately and quantitatively detected, and the detection mutation rate is as low as 0.1%.

Description

Technical Field

[0001] The present invention relates to the technical field of quantitative detection of gene mutations, and in particular to a primer-probe combination, a kit and a method for quantitatively detecting the mutation rate of the JAK2V617F gene. Background Art

[0002] Research has discovered that a mutation (G>T) at position 1849 in exon 14 of the non-receptor tyrosine kinase (JAK2) gene results in a phenylalanine (F) substitution for valine (V) at position 617 of the JAK2 protein (JAK2V617F). This mutation enhances JAK2 protein kinase activity, leading to sustained stimulation of hematopoietic cell proliferation and differentiation, ultimately inducing myeloproliferative neoplasms (MPNs). The JAK2 V617F mutation is found in approximately 95% of patients with polycythemia vera (PV), 50%-60% of patients with essential thrombocythemia (ET), and 40%-50% of patients with primary myelofibrosis (PMF). The World Health Organization (WHO) included the JAK2 V617F mutation in the diagnostic criteria for these diseases in 2008. In addition to auxiliary diagnosis, quantitative detection of JAK2 V617F mutation rate is of great value for minimal residual disease (MRD) monitoring, efficacy evaluation and recurrence warning, and is one of the core indicators for achieving precise diagnosis and treatment.

[0003] Currently, methods used to detect JAK2 V617F gene mutations, such as the "gold standard" Sanger sequencing method, have low sensitivity (usually 15% to 20%), are cumbersome to operate, and are susceptible to contamination, making it difficult to meet the needs of low-abundance mutations or dynamic monitoring. Next-generation sequencing (NGS) technology has the advantages of high sensitivity (can detect 0.1% to 1% mutation frequency) and high throughput, but it has problems such as high equipment and reagent costs, complex data analysis, and long detection cycles. It is not cost-effective in single-gene dynamic tracking scenarios. Digital PCR (dPCR) has significant advantages in MRD monitoring due to its absolute quantitative ability without the need for a standard curve and ultra-high sensitivity (up to 0.005% mutation frequency). However, factors such as expensive equipment, high reagent and consumable costs, and complex operation have limited its clinical popularity. Allele-specific PCR (AS-PCR) is a rapid, simple, and highly sensitive method, and is currently the primary method for qualitatively detecting gene mutations. However, it has inherent technical bottlenecks: (1) Nonspecific amplification: Due to limitations in primer design, even with strict control of template DNA concentration (10-15 ng / μL), it is still difficult to completely eliminate false positives; (2) Difficulty in quantification: Mutation status must be indirectly determined by the ΔCt value between wild-type and mutant forms, which limits quantitative accuracy; (3) Limited sensitivity: Under conventional conditions, the lower limit of detection is only approximately 1%, which is difficult to meet the future needs of deep residual lesion detection. With the application of targeted drugs such as JAK2 inhibitors, the clinical demand for mutation rate detection sensitivity and quantitative accuracy will increase significantly. Combining the technical advantages of AS-PCR and addressing its shortcomings may become a key breakthrough in achieving highly sensitive quantitative detection. Summary of the Invention

[0004] The present invention proposes a primer-probe combination, a kit, and a method for quantitatively detecting the JAK2 V617F gene mutation rate. The combination can economically, simply, efficiently, highly sensitively, and accurately quantitatively detect the JAK2 V617F mutation rate, with the detection mutation rate as low as 0.1%. This has important clinical application value for disease diagnosis, disease progression, and treatment monitoring.

[0005] The technical solution of the present invention is achieved as follows: a primer-probe combination includes a primer-probe set targeting the JAK2 V617F mutation site and a primer-probe set targeting the internal reference gene GPI, wherein the primer-probe set targeting the JAK2 V617F mutation site includes an upstream primer JAK-MF for the specific mutation site, an upstream primer JAK-WF for the specific wild-type site, a universal downstream primer JAK-R, and a probe JAK-P;

[0006] The nucleotide sequence of the upstream primer JAK-MF is any one of SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6, and SEQ ID NO.7;

[0007] The nucleotide sequence of the upstream primer JAK-WF is any one of SEQ ID NO.9, SEQ ID NO.10, SEQ ID NO.11, SEQ ID NO.12, SEQ ID NO.13, and SEQ ID NO.14;

[0008] The nucleotide sequence of the universal downstream primer JAK-R is SEQ ID NO.8, and the nucleotide sequence of the probe JAK-P is SEQ ID NO.15. The 5' end of the probe JAK-P carries a fluorescent gene FAM, and the 3' end carries a quenching group BHQ1.

[0009] The upstream primers for the specific mutation site and the upstream primers for the specific wild-type site introduce a mismatched base (except SEQ ID NO. 7) at the second to seventh bases from the 3' end. By acting together with the mutation site at the 3' end, the product amplification rate of the primer in the template that is not complementary to its 3' end is significantly reduced, while the primer amplifies normally in the template that is complementary to its 3' end.

[0010] Furthermore, the primer probe set for the internal reference gene GPI includes an upstream primer GPI-F, a downstream primer GPI-R, and a probe GPI-P;

[0011] The nucleotide sequence of the upstream primer GPI-F is SEQ ID NO.16, the nucleotide sequence of the downstream primer GPI-R is SEQ ID NO.17, and the nucleotide sequence of the probe GPI-P is SEQ ID NO.18. The 5' end of the probe GPI-P carries the fluorescent gene HEX and the 3' end carries the quenching group BHQ1.

[0012] Furthermore, it also includes a hairpin structure oligonucleotide (H-oligo), and the sequence of H-oligo is SEQ ID NO.19.

[0013] Furthermore, the nucleotide sequence of the upstream primer JAK-MF is SEQ ID NO.4, and the nucleotide sequence of the upstream primer JAK-WF is SEQ ID NO.12.

[0014]

[0015]

[0016] Note: Bold bases with gray background are mutation sites, bold italic bases with underlines are artificial mismatch bases, BHQ1 is the quenching group, FAM is the fluorescent gene FAM, and HEX is the fluorescent gene HEX.

[0017] A kit for quantitatively detecting the JAK2 V617F gene mutation rate comprises the primer-probe combination.

[0018] Furthermore, the final concentration of H-oligo is 10 nM to 100 nM.

[0019] Furthermore, the final concentration of H-oligo was 25 nM.

[0020] A method for quantitatively detecting the mutation rate of the JAK2 V617F gene, using the kit, taking the GPI gene as an internal reference gene, and simultaneously detecting the wild-type and mutant JAK2 V617F genes, determining whether the sample contains the JAK2 V617F gene mutation by the Ct value of the amplification curve, and using 2 -ΔΔCt Methods The JAK2 V617F mutation rate was quantitatively calculated.

[0021] Furthermore, the JAK2 V617F mutation rate was calculated as follows:

[0022] ΔCt. JAK2-Wild =Ct. JAK2-Wild -Ct. GPI-Wild ,

[0023] ΔCt. JAK2-Mut =Ct. JAK2-Mut -Ct. GPI-Mut ,

[0024] ΔΔCt=ΔCt. JAK2-Mut -ΔCt. JAK2-Wild ,

[0025] Mutation rate = 2 -ΔΔCt / (1+2 -ΔΔCt );

[0026] Among them, Ct. JAK2-Wild is the Ct value of the FAM channel of the JAK2 V617F wild-type detection system for the JAK2 617V gene, Ct. GPI-Wild is the Ct value of the HEX channel of the JAK2 V617F wild-type detection system for the internal reference gene GPI;

[0027] Ct. JAK2-Mut is the Ct value of the FAM channel of the JAK2 V617F mutant detection system for the JAK2 617F gene, Ct. GPI-MutIt is the Ct value of the HEX channel of the JAK2 V617F mutant detection system for the internal reference gene GPI.

[0028] Beneficial effects of the present invention:

[0029] (1) The kit of the present invention has good specificity. No nonspecific amplification reaction occurs after 40 cycles of amplification at a high wild-type DNA concentration (100 ng / μL). Therefore, the result interpretation does not need to refer to the ΔCt value of the wild type and mutant types, making the result interpretation simpler and more accurate.

[0030] (2) The kit of the present invention has high detection sensitivity, with a minimum detection limit of 100 pg / μL and a minimum mutation rate of 0.1%.

[0031] (3) The kit of the present invention can quantitatively detect the JAK2 V617F gene mutation rate without establishing a standard curve.

[0032] (4) The kit of the present invention has a fast reaction time, and the amplification reaction can be completed within 45 minutes. The detection process is a closed-tube operation, which can avoid product contamination. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0034] Figure 1 This is the result of primer and probe screening for the JAK2 V617F gene mutation;

[0035] Figure 2 This is the result of primer and probe screening for the wild-type JAK2 V617F gene;

[0036] Figure 3 Optimize the specificity of JAK2 V617F gene mutation detection results;

[0037] Figure 4 This is the amplification efficiency result of the JAK2 V617F mutant detection system;

[0038] Figure 5 This is the amplification efficiency result of the JAK2 V617F wild-type detection system;

[0039] Figure 6 This is the amplification efficiency result of the internal reference gene GPI;

[0040] Figure 7 is 2-ΔΔCt Quantitative validation results of the method;

[0041] Figure 8 The sensitivity test results of the detection kit for this application for detecting JAK2 V617F gene mutation;

[0042] Figure 9 These are the clinical sample validation results of the JAK2 V617F gene mutation quantitative detection kit. DETAILED DESCRIPTION

[0043] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. All other embodiments obtained by ordinary technicians in this field based on the embodiments of the present invention without creative work are within the scope of protection of the present invention.

[0044] Unless otherwise specified, the experimental methods in the following examples are conventional methods and were performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials and reagents used in the following examples, unless otherwise specified, were all commercially available.

[0045] The 10× PCR Buffer, DNA polymerase, dNTP / dUTP Mix, and MgCl2 in the following examples are products of Nanjing Novozymes Biotechnology Co., Ltd., with the product number PM301.

[0046] The primers, probes, and hairpin oligonucleotides in the following examples were synthesized by Sangon Biotechnology (Shanghai) Co., Ltd.

[0047] The JAK2 V617F mutant plasmids in the following examples were commissioned to Jiangsu Saisuofei Biotechnology Co., Ltd. for construction.

[0048] Example 1: Design and screening of primer probes

[0049] 1. Based on the JAK2 V617F mutation gene region, upstream primers specific to the mutation site, upstream primers specific to the wild-type site, universal downstream primers, and probes were designed. Upstream primers, downstream primers, and probes were designed targeting the conserved region of the internal reference gene glucose-6-phosphate isomerase (GPI). Specific sequence information is shown in Table 1.

[0050] Table 1 Primer and probe sequences

[0051]

[0052]

[0053] Note: Bold bases with gray background are mutation sites, bold italic bases with underlines are artificial mismatch bases, BHQ1 is the quenching group, FAM is the fluorescent gene FAM, and HEX is the fluorescent gene HEX.

[0054] 2. PCR detection system (see Table 2)

[0055] Table 2 PCR detection system components

[0056]

[0057] 3. PCR Amplification Procedure

[0058] The reaction procedure of the detection system includes: pre-deformation at 95°C for 30s; deformation at 95°C for 10s, and 55°C for 30s (collecting fluorescence signals), for 40 cycles.

[0059] 4. Primer and probe screening

[0060] Use the upstream primer JAK-MF (any one of JAK-MF1 to JAK-MF7) of the specific mutation site and the universal downstream primer JAK-R and probe JAK-P to prepare the reagents according to Table 2. One JAK2 V617F mutant sample and one JAK2 V617F wild-type sample were selected to test the above primers and probes. Figure 1 Figure 2 shows the curve of the relative fluorescence intensity ΔRn of the upstream primer JAK-MF as a function of cycle number. A represents JAK-MF1, B represents JAK-MF2, C represents JAK-MF3, D represents JAK-MF4, E represents JAK-MF5, F represents JAK-MF6, and G represents JAK-MF7. Mutation represents mutant samples; Wild represents wild-type samples; and NC represents blank control. The results showed that the combination of JAK-MF4, JAK-R, and JAK-P performed best, with good sensitivity and specificity. Other combinations suffered from poor nonspecificity or low sensitivity. Therefore, the combination of JAK-MF4, JAK-R, and JAK-P was selected for the detection of the JAK2 V617F mutation.

[0061] Use the upstream primer JAK-WF (any one of JAK-WF1 to JAK-WF6) of the specific wild-type site and the universal downstream primer JAK-R and probe JAK-P to prepare the reagents according to Table 2. Select the JAK2V617F mutant plasmid and one JAK2V617F wild-type sample to test the above primers and probes. Figure 2Figure 3 is the curve of the relative fluorescence intensity ΔRn of the upstream primer JAK-WF changing with the cycle number, A is JAK-WF1, B is JAK-WF2, C is JAK-WF3, D is JAK-WF4, E is JAK-WF5, and F is JAK-WF6. Figure 2 The results showed that the JAK-WF4 + JAK-R + JAK-P combination performed best, with good sensitivity and specificity. Other combinations suffered from poor nonspecificity or low sensitivity. Therefore, the JAK-WF4 + JAK-R + JAK-P combination was selected for the detection of wild-type JAK2V617F.

[0062] Example 2: Specificity optimization

[0063] Since non-specific fluorescent signals appear when the amplification reaction is cycled to 37, in order to avoid non-specific amplification and affect the interpretation of the results, the present invention designed an oligonucleotide (H-oligo) with a hairpin structure, the sequence of which is shown in Table 1, to inhibit non-specific amplification. By setting different concentrations of H-oligo (final concentrations are 100nM, 50nM, 25nM, and 10nM, respectively), the reagents are configured according to Table 2, and the mutant detection system is tested. The results are shown in Figure 3 , showing that 25 nM concentration of H-oligo can inhibit nonspecific amplification and has no inhibitory effect on amplification efficiency. Therefore, 25 nM concentration of H-oligo was selected for subsequent experiments.

[0064] Example 3: Amplification efficiency

[0065] The JAK2 V617F mutant sample, Lw-0, was sequentially diluted to Lw-1 (1E5 pg / μL = 100 ng / μL), Lw-2 (1E4 pg / μL = 10 ng / μL), Lw-3 (1E3 pg / μL = 1 ng / μL), and Lw-4 (1E2 pg / μL = 100 pg / μL) to test the amplification efficiency of the JAK2 V617F mutant system. The JAK2 V617F wild-type sample, Lm-0, was sequentially diluted to Lm-1 (1E5 pg / μL), Lm-2 (1E4 pg / μL), Lm-3 (1E3 pg / μL), and Lm-4 (1E2 pg / μL) to test the amplification efficiency of the JAK2 617V gene and the internal reference gene GPI in the JAK2 V617F wild-type system. The test was repeated three times for each dilution concentration, and the linear correlation coefficient was calculated using the logarithm of the mean value and the Ct value. 2 ≥0.980, the amplification efficiency is in the range of 90% to 110%. Figure 4 As shown, the R of the JAK2 V617F mutant detection system 2=0.9946, the amplification efficiency is 101.34%, which meets the requirements. Figure 5 As shown, the JAK2 V617F wild-type amplified R 2 =0.9932, the amplification efficiency is 101.78%, which meets the requirements. Figure 6 As shown, the R 2 =0.9924, the amplification efficiency is 100.49%, which meets the requirements.

[0066] Example 4:2 -ΔΔCt Quantitative validation of the method

[0067] To make 2 -ΔΔCt For the calculation method to be effective, the amplification efficiencies of the target sequence and the internal reference sequence must be equal. To determine whether two reactions have the same amplification efficiency, the ΔCt values ​​of the amplified products change after serial dilution of the template concentrations. The JAK2 V617F mutant samples Lm-1 (100 ng / μL), Lm-2 (10 ng / μL), Lm-3 (1 ng / μL), and Lm-4 (100 pg / μL) were used to test whether the GPI amplification efficiencies of the JAK2 V617F mutant and the internal reference gene were consistent. The JAK2 V617F wild-type samples Lw-1 (100 ng / μL), Lw-2 (10 ng / μL), Lw-3 (1 ng / μL), and Lw-4 (100 pg / μL) were used to test whether the GPI amplification efficiencies of the JAK2 V617F wild-type and the gene were consistent. The average Ct value and ΔCt value (referred to as Ct. JAK2-Mut 、Ct. GPI-Mut , ΔCt. JAK2-Mut ), and the average Ct value and ΔCt value of JAK2 V617F wild type and the corresponding GPI (referred to as Ct. JAK2-Wild 、Ct. GPI-Wild , ΔCt. JAK2-Wild ), plot the ΔCt value against the log value of the dilution multiple. If the absolute value of the slope of the straight line is close to 0, it means that the amplification efficiency of the target gene and the internal standard gene is the same, and the target gene can be amplified by 2 -ΔΔCt The relative quantification method was used. Figure 7 As shown, 7A is the amplification product ΔCt of JAK2 V617F mutant (abbreviated as ΔCt. JAK2-Mut ), and 7B is the fitting straight line of the amplified product ΔCt of the JAK2 V617F wild type (abbreviated as ΔCt. JAK2-Wild ) of the fitting line, the slopes of the lines are -0.0917 and 0.0955 respectively, so the hypothesis is established, 2 -ΔΔCt Methods can be used to quantitatively analyze data.

[0068] Example 5: Detection limit test

[0069] 10 ng / μL of JAK2 V617F wild-type sample genomic DNA was selected as diluent, and 10 ng / μL of JAK2 V617F mutant sample genomic DNA (dPCR quantitative mutation rate was 98.37%) was diluted in order to achieve mutation rates of 20%, 2%, 0.2%, 0.1%, and 0.05%. Figure 8 As shown, 0.1% to 20% of the samples can be detected, so the lower limit of the detection mutation rate of this method can be set at 0.1%, WT is the JAK2V617F wild-type sample, and NC is the blank control.

[0070] Example 6: Clinical sample testing

[0071] In order to evaluate the accuracy of the method of the present invention in detecting clinical samples, 22 JAK2 V617F mutation-positive patient samples and 18 JAK2 V617F mutation-negative patient samples were selected and tested using the method of the present invention and the dPCR method, respectively. The results are shown in Table 3 and Figure 9 As shown in the figure, the consistency of the detection results of the two methods is 100%, and the correlation coefficient R 2 The absolute quantification result of the present invention is 0.9852, indicating that the absolute quantification result of the present invention is highly consistent with the gold standard dPCR, and can accurately and quantitatively detect the JAK2 V617F mutation rate.

[0072] Table 3 Four-cell analysis of the test results of the reagents to be tested and the control method

[0073]

[0074] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A primer-probe combination, characterized in that: The primer probe set includes a primer probe set targeting the JAK2 V617F mutation site and a primer probe set targeting the internal reference gene GPI. The primer probe set targeting the JAK2 V617F mutation site includes an upstream primer JAK-MF for the specific mutation site, an upstream primer JAK-WF for the specific wild-type site, a universal downstream primer JAK-R, and a probe JAK-P. The nucleotide sequence of the upstream primer JAK-MF is any one of SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6, and SEQ ID NO.7; The nucleotide sequence of the upstream primer JAK-WF is any one of SEQ ID NO.9, SEQ ID NO.10, SEQ ID NO.11, SEQ ID NO.12, SEQ ID NO.13, and SEQ ID NO.14; The nucleotide sequence of the universal downstream primer JAK-R is SEQ ID NO.8, and the nucleotide sequence of the probe JAK-P is SEQ ID NO.

15. The 5' end of the probe JAK-P carries a fluorescent gene FAM, and the 3' end carries a quenching group BHQ1.

2. The primer-probe combination according to claim 1, wherein: The primer-probe set for the internal reference gene GPI includes the upstream primer GPI-F, the downstream primer GPI-R, and the probe GPI-P; The nucleotide sequence of the upstream primer GPI-F is SEQ ID NO.16, the nucleotide sequence of the downstream primer GPI-R is SEQ ID NO.17, and the nucleotide sequence of the probe GPI-P is SEQ ID NO.

18. The 5' end of the probe GPI-P carries the fluorescent gene HEX and the 3' end carries the quenching group BHQ1.

3. The primer-probe combination according to claim 1 or 2, characterized in that: Also included is an oligonucleotide with a hairpin structure, and the sequence of the oligonucleotide with a hairpin structure is SEQ ID NO.

19.

4. The primer-probe combination according to claim 1 or 2, characterized in that: The nucleotide sequence of the upstream primer JAK-MF is SEQ ID NO.4, and the nucleotide sequence of the upstream primer JAK-WF is SEQ ID NO.

12.

5. A kit for quantitatively detecting the JAK2 V617F gene mutation rate, characterized in that: The method comprises the primer-probe combination according to any one of claims 1 to 4.

6. The kit according to claim 5, wherein: The final concentration of the oligonucleotide is 10 nM to 100 nM.

7. The kit according to claim 6, wherein: The final concentration of the oligonucleotide was 25 nM.

8. A method for quantitatively detecting the JAK2 V617F gene mutation rate, characterized by: Using the kit according to any one of claims 5 to 8, using 2 -ΔΔCt Methods The JAK2 V617F mutation rate was quantitatively calculated.

9. The method according to claim 8, characterized in that: The JAK2 V617F mutation rate is calculated as follows: ΔCt. JAK2-Wild =Ct. JAK2-Wild -Ct. GPI-Wild , ΔCt. JAK2-Mut =Ct. JAK2-Mut -Ct. GPI-Mut , ΔΔCt=ΔCt. JAK2-Mut -ΔCt. JAK2-Wild , Mutation rate = 2 -ΔΔCt / (1+2 -ΔΔCt ); Among them, Ct. JAK2-Wild is the Ct value of the FAM channel of the JAK2 V617F wild-type detection system for the JAK2 617V gene, Ct. GPI-Wild is the Ct value of the HEX channel of the JAK2 V617F wild-type detection system for the internal reference gene GPI; Ct. JAK2-Mut is the Ct value of the FAM channel of the JAK2 V617F mutant detection system for the JAK2 617F gene, Ct. GPI-Mut It is the Ct value of the HEX channel of the JAK2 V617F mutant detection system for the internal reference gene GPI.

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