CN104818340B - Detect primer, kit and its PCR method of JAK2 gene V617F loci polymorphisms - Google Patents

Abstract

The invention discloses it is a kind of detect JAK2 gene V617F loci polymorphisms primer, kit and its PCR method, including：The downstream primer that wild type specific forward primer, saltant type specific forward primer and wild type specific forward primer are shared with saltant type specific forward primer；And the wild type specific forward primer has SEQ No.17 sequences, the saltant type specific forward primer has SEQ No.14 sequences, and the shared downstream primer has SEQ No.16 sequences；Kit has many advantages, such as that detection is simple, quick, accurate, cheap, and strong tool is provided for scientific research and clinic JAK2 gene V617F site partings and gene mutation analysis.

Description

Primers, kit and PCR method for detecting polymorphism of JAK2 gene V617F site

technical field

The invention relates to the field of molecular biological gene detection, and in particular provides a primer for detecting JAK2 gene polymorphism, a kit and a PCR method thereof, which are used for rapid detection of the V617F polymorphism site of the JAK2 gene.

Background technique

JAK is a non-receptor tyrosine protein kinase. So far, four family members have been found, namely JAK1, JAK2, JAK3 and JAK4. STAT is the direct substrate of JAK, which can directly transmit the signal to the nucleus and regulate the expression of specific genes. The JAK-STAT signaling pathway is a signaling pathway closely related to cell growth, proliferation, and differentiation, and also participates in signal transduction in hematopoietic and immune systems, and the kinase JAK plays a key role in the activation of the entire signaling pathway. So far, many JAK gene point mutations have been found in human leukemia cells, some of which can lead to the continuous activation of STAT protein. For example, the V617F mutation of the JAK2 gene can lead to abnormal activation of the JAK-STAT signaling pathway, resulting in abnormal proliferation of bone marrow cells.

Myeloproliferative diseases (MPD) are a group of hematopoietic stem cell neoplastic proliferative diseases. On the basis of the general proliferation of bone marrow cells, one or more lines of cells are particularly prominent, which is characterized by continuous hyperproliferation and an increase in the number of mature cells in peripheral blood. The clinical manifestations are heterogeneous, but almost all subtypes are accompanied by an increase in white blood cells, platelets, and megakaryocytes. Myelofibrosis and bone marrow failure appear in the later stage, and some of them can be transformed into other diseases as the disease progresses. Its classic classification is mainly divided into chronic myelogenous leukemia (chronic myelogenous leukemia, CML), polycythemia vera (polycythemia vera, PV), essential thrombocythemia (essential Thrombocythemia, ET) and primary myelofibrosis (primary myelofibrosis) myelofibrosis, PMF), etc.

The V617F polymorphism of the JAK2 gene occurs in 65% to 97% of polycythemia vera (PV), 23% to 57% of essential thrombocythemia (ET) and 35% to 57% of primary bone marrow in patients with fibrosis (PMF). According to the close relationship between JAK2 mutation and myeloproliferative diseases, in the revised 2008 World Health Organization (WHO) classification system, the V617F polymorphism of JAK2 gene has become the main diagnostic indicator for chronic myeloproliferative diseases (MPD).

At present, the detection of gene mutation and gene polymorphism commonly includes direct sequencing method; gene chip hybridization method; PCR-RFLP method; PCR amplification product capillary electrophoresis analysis method; PCR-SSCP; PCR high-resolution melting curve analysis technology; PCR -Taqman MGB (Minor Groove Binder) probe method; AS-PCR method; Cast-PCR method, etc. These methods still have more or less shortcomings such as high instrument price, difficult operation, certain false negatives and false positives, high detection cost, low clinical popularity, and inability to simultaneously detect large-scale clinical specimens.

For example: 1) The direct sequencing method is the gold standard for mutation analysis, which can detect known and unknown mutation sites, but the sensitivity of this method to detect gene mutations is 20% (that is, the number of mutant gene DNA templates of the target gene accounts for the number of wild-type DNA templates 20% of the number). At the same time, the operation is complicated, the cycle is long, the analysis speed is slow, and it usually takes 2 days, and the method is an open-tube operation, which greatly increases the possibility of contamination. It is not suitable for the detection of large-scale samples, and it also requires expensive instruments. , there are shortcomings such as difficult implementation at the grassroots level.

2) The common PCR-RFLP method is simple and cheap, and is suitable for laboratory testing of a small number of samples. However, RFLP can only detect mutations with enzyme cleavage sites, and cannot be detected without enzyme cleavage sites. It is time-consuming and laborious, and there is PCR product contamination Risk of causing false positives, see [Mol Diagn Ther. 2010 Jun 1;14(3):163-9, United States Patent20120135406].

3) Compared with traditional instrument detection methods, chip technology has the characteristics of high throughput, miniaturization, and automation. It is suitable for whole-genome mutation scanning, but not suitable for single gene mutation site detection, and its accuracy is low and expensive.

4) PCR high-resolution melting curve analysis technology can detect gene mutations in high throughput, and the reagent cost is low, but because the fluorescent signal comes from the dye, the specificity is affected, and the detection instrument is an upgraded version of the fluorescent PCR instrument. Expensive, limited access, see [Clin Chim ACta. 2012 Nov 12;413(21-22):1781-5; United States Patent 20110045479].

5) The PCR-Taqman MGB probe method is suitable for known mutation sites, and often requires two probes, and the synthesis price of Taqman MGB probes is several times that of ordinary Taqman probes, see [J Clin Microbiol. 2010 Aug;48 (8):2909-15; United States Patent 20090311679], and cannot detect alleles or mutation sites with a small amount (≥ 1 in 1,000) in the sample.

6) Although the PCR-SSCP method is simple, it is an open detection system, which is easy to cause contamination of PCR products, and there are many operating steps, which are time-consuming and labor-intensive.

7) Allele-specific primer PCR amplification method (AS-PCR), this method is currently the simplest and fastest method for detecting gene mutations or polymorphisms (Wu DY, Ugozzoli L, Pal BK, Wallace R B., Proc NatlAcad Sci USA 1989; 86:2757-2760), its principle is the base mismatch between primer 3' terminal base and template, and the efficiency of its PCR will drop by 10 ³ -10 ^6.6 times (Chen, X., and Sullivan, PF, The Pharmacogeonomics Journal 2003, 3, 77-96). Although the principle of this method is simple, non-specific amplification will still occur with mismatched primers, and the amplification depends on the type of mutation and the base sequence around the detection site (Ayyadevara S, Thaden JJ, Shmookler Reis R J. , Anal Biochem 2000; 284:11-18), also with the influence of allelic variables present in the sample. In order to increase the specificity of AS-PCR, many scientists have made great efforts. A large number of experiments have proved that the key to this method is to design two specific primers that are complementary or mismatched to the bases of the 3' terminal mutation site. If the primer design is not good, it will lead to high background cross-amplification, which will lead to higher false positive. Although many people have worked hard to overcome this disadvantage, such as the reported TaqMAMA method, introducing a mutant base at the penultimate or third position of the 3' can indeed increase the specificity of the reaction, but it still cannot completely eliminate false positives. Moreover, there is a lack of standards in the judgment of homozygotes and heterozygotes, and confusion will occur, see [J Virol Methods. 2008 Nov;153(2):156-62;Genomics. 2004 Feb;83(2):311- 20]. For simple AS-PCR, electrophoresis is usually performed after the PCR reaction is completed, and the result is judged from the presence or absence of reaction bands. Although this method does not require expensive instruments, the electrophoresis operation increases the chance of PCR contamination and is time-consuming. strenuous. Although some people have improved this method by using fluorescent quantitative PCR technology, the results obtained are not "all or nothing", and there will always be non-specific reactions, that is, the same primers will affect both wild-type and mutant sites. Amplification, but the obtained Ct (Cycle threshold) is different, so the concept of ΔCt is introduced, that is, ΔCt=wild Ct-mutant Ct, but the calculation is complicated. Zygote, whose ΔCt value is less than the heterozygous Ct value, is judged as heterozygous. The introduction of the ΔCt value not only increases the operation steps, but also causes confusion, because the setting standard of the ΔCt value cannot be accurately located, and the operation of different personnel, different specimens and Different detection instruments will have different numbers, which brings great difficulties to clinical application, and practical application is still limited, see [Chinese patent CN101235415, CN101565742A].

8) American LIFE Company adopts a method of MGB blocking probes, called Cast-PCR (Competitiveallele specific Taqman PCR), using MGB probes to block the undetected sites, and then using allele-specific primers for fluorescent quantitative PCR method to detect the target site, although this improves the specificity of detection, but due to the addition of an MGB blocking probe, it is bound to increase the cost and interfere with the reaction efficiency to some extent. See [Exp Mol Pathol. 2012 Jun;92(3 ):275-80; United States Patent 20100221717; CN102428190A]. Some people use PCR primers of locked nucleic acid (LNA) (Plant Method 2007, 3:2) or modified bases (Anal Biochem. 2005, 340: 287-294), which can improve the detection sensitivity of AS-PCR. However, these methods increase the overall cost of the analysis and require deep optimization of the reaction.

Therefore, how to simply and accurately detect the V617F polymorphism of the JAK2 gene has become an urgent problem to be solved.

Contents of the invention

In view of this, the present invention provides a primer for detecting the V617F polymorphism site of the JAK2 gene, a kit and a PCR method thereof, to at least solve the complex interpretation of results, high price of detection instruments, difficult operation, and the existence of existing kits in the past. There must be one or more problems such as false negatives and false positives, high testing costs, low clinical popularity, and the inability to simultaneously test large-scale clinical specimens.

The solution provided by the present invention is specifically: a primer for detecting the V617F polymorphism site of the JAK2 gene, characterized in that the primers include: wild-type specific upstream primers, mutant-type specific upstream primers and wild-type specific upstream primers. A downstream primer shared by the primer with the mutant-specific upstream primer;

And the wild-type specific upstream primer has the sequence of SEQ No.17, the mutant-type specific upstream primer has the sequence of SEQ No.14, and the common downstream primer has the sequence of SEQ No.16.

On the one hand, the present invention also provides a reagent for detecting the V617F polymorphism site of JAK2 gene, characterized in that the reagent contains the above-mentioned primers.

Another aspect of the present invention also provides a kit for detecting the V617F polymorphic site of the JAK2 gene, which is characterized in that the kit also contains the above-mentioned primers.

Preferably, the kit also includes a probe used in conjunction with the primer, wherein the probe is a Taqman probe with the sequence of SEQ No.15.

Further preferably, the kit also includes PCR reaction tubes, dNTPs and Taq enzyme mixture, PCR buffer, upstream primers for detecting the housekeeping gene GAPDH, downstream primers for detecting the housekeeping gene GAPDH, probes for detecting the housekeeping gene GAPDH, positive Control product and blank control;

The upstream primer for detecting the housekeeping gene GAPDH has the sequence of SEQ No.19; the downstream primer for detecting the housekeeping gene GAPDH has the sequence of SEQ No.20; and the probe for detecting the housekeeping gene GAPDH has the sequence of SEQ No.21.

Further preferably, the mutant-specific upstream primer, the shared downstream primer, the probe, the upstream primer for detecting the housekeeping gene GAPDH, the downstream primer for detecting the housekeeping gene GAPDH, and the detection housekeeping gene GAPDH The probe is pre-coated in the T-type PCR reaction tube; and with the wild-type specific upstream primer, the shared downstream primer, the probe, the detection housekeeping gene GAPDH upstream primer, the detection The downstream primers of the housekeeping gene GAPDH and the probe for detecting the housekeeping gene GAPDH are pre-coated in the G-type PCR reaction tube.

Further preferably, the mutant-specific upstream primer in the T-type PCR reaction tube, the shared downstream primer, the probe, the upstream primer for detecting the housekeeping gene GAPDH, the downstream primer for detecting the housekeeping gene GAPDH, and the The probe concentrations for detecting the housekeeping gene GAPDH are respectively 50nM-2um, 50nM-2uM, 50nM-400nM, 1pm-20pm, 1pm-20pm, 0.05pm-5pm; and the wild-type specific upstream primers in the G-type PCR reaction tube , the shared downstream primer, the probe, the upstream primer for detecting the housekeeping gene GAPDH, the downstream primer for detecting the housekeeping gene GAPDH and the probe concentration for detecting the housekeeping gene GAPDH are respectively 50nM-2um, 50nM -2uM, 50nM-400nM, 1pm-20pm, 1pm-20pm, 0.05pm-5pm.

More preferably, the mutant-specific upstream primers in the T-shaped PCR reaction tube, the shared downstream primers, the probes, the upstream primers for detecting the housekeeping gene GAPDH, the downstream primers for detecting the housekeeping gene GAPDH, and The probe concentrations for detecting the housekeeping gene GAPDH are respectively 500nM, 500nM, 100nM, 5pm, 5pm, and 2.5pm; and the wild-type specific upstream primers in the G-type PCR reaction tube, the shared downstream primers, the probe The concentrations of the needle, the upstream primer for detecting the housekeeping gene GAPDH, the downstream primer for detecting the housekeeping gene GAPDH, and the probe for detecting the housekeeping gene GAPDH were 500nM, 500nM, 100nM, 5pm, 5pm, and 2.5pm, respectively.

The present invention also provides the PCR method of the above-mentioned primers or kits, characterized in that: the PCR reaction is carried out according to a two-step amplification cycle program, and the number of cycles of amplification in the first step is less than the number of cycles in the second step of amplification. The annealing temperature of the first step amplification is higher than the annealing temperature of the second step amplification.

Preferably, the conditions of the PCR reaction are: 37°C for 2min, 95°C for 2min for pre-denaturation; first step amplification, 95°C denaturation for 5s, 55°C~68°C annealing for 32s, cycle 10~15 times; second step, 95°C Denaturation at ℃ for 5s, annealing at 50℃~65℃, extension for 32s, cycle 30~50 times, and collect fluorescence signals; further preferably, the conditions of the PCR reaction are: 37℃ for 2 minutes, 95℃ for 2 minutes for pre-denaturation; the first step of amplification , denaturation at 95°C for 5s, annealing at 63°C for 32s, cycle 15 times; the second step, denaturation at 95°C for 5s, annealing at 60°C, extension for 32s, cycle 40 times, and collect fluorescence signals.

The primers and kits for detecting the V617F polymorphic site of the JAK2 gene provided by the present invention can greatly increase the ability of allele-specific primer PCR to distinguish different allele sites, and make non-specific cross-amplification between different sites possible. The possibility of amplification is minimized, and it can achieve true "all or nothing" amplification. It not only has good specificity, but also has very good sensitivity, and can detect the genotype distribution in 1ng of genomic DNA.

The primers and kits thereof for detecting the V617F polymorphism site of the JAK2 gene provided by the present invention have the following advantages:

1) By artificially changing the primer sequence, the test result can truly achieve an "all or nothing" judgment on the result, which greatly simplifies the difficulty of result interpretation, reduces the possibility of error, and provides unparalleled scientific research and clinical application. convenient.

2) The mixture of Taq enzyme and dNTPs ensures the stability of dNTPs, so that it can withstand the test of repeated freezing and thawing.

3) The buffer solution that can be used directly is pre-prepared in advance, which is more convenient for users.

4) The primers and probes are coated in the PCR reaction tube in advance, which avoids the possibility of site mismatch by the operator and saves a lot of operation time.

5) The primers and kits of the present invention have good specificity, high detection sensitivity and fast detection speed, and the whole process can be completed within 1 hour and 20 minutes.

6) Only one conventional Taqman probe is used in the kit, and there are no other such as blocking probes to reduce costs.

7) The kit has the advantages of simple, fast, accurate and cheap detection.

Description of drawings

Fig. 1 is the PCR amplification curve diagram when designing wild-type and mutant primers;

Fig. 2 is the PCR amplification curve chart of detection wild-type gene sequence;

Fig. 3 is the PCR amplification curve chart of detection mutant gene sequence;

Fig. 4 is the PCR amplification graph of TG site;

Figure 5 is a PCR amplification curve of the housekeeping gene GAPDH.

Detailed ways

The present invention will be further explained with specific cases below, but it is not intended to limit the protection scope of the present invention.

Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and all products used are commercially available.

The following definitions are explanations of relevant terms in the present invention:

"Allele" generally refers to a segment of DNA, a pair of genes that control relative traits at the same physical location on homologous chromosomes. In some cases, an allele may correspond to a single nucleotide substitution at a particular physical locus. In other cases, alleles may correspond to insertions or deletions of nucleotides (single or multiple).

"Allele-specific primer" means that it is complementary to the sequence of the target allele and can be extended to complete a specific PCR reaction during PCR. Allele-specific primers can be designed to detect as little as 1 nucleotide difference in the target sequence. Very specific primers can contain an allele-specific nucleotide moiety, a 3' target-specific moiety, and Tail.

"MGB group" refers to a minor groove binder. When conjugated to the 3' end of an oligonucleotide, the MGB group can function as a non-extensible blocking moiety. MGBs are molecules capable of binding in the minor groove of double-stranded DNA, usually using DPI3 (see US Pat. Nos. 6,084,102; and 6,727,356 for methods of synthesis).

"Detection probe"refers to: any of a variety of signaling molecules that indicate amplification. For example, SYBR Green and other DNA-binding dyes are detection probes. Some detection probes can be sequence-specific, such as Taqman probes (US Patent No. 5,538,848), various stem-loop molecular beacons (US Patent Nos. 6,103,476 and 5,925,517 and Tyagi and Kramer, Nature Biotechnology, 1996, 14:303- 308), stemless or linear beacons (WO99/21881), PNA Molecular Beacons™ (US Pat. Nos. 6,355,421 and 6,593,091), linear PNA beacons (Kubista et al., 2001, SPIE4264:53-58), non-FRET Probe (US Patent No. 6,150,097), Sunrise/Amplifluor probe (US Patent No. 6,548,250), stem-loop and double-stranded Scorpion TM probe (Solinas et al., 2001, Nucleic Acids Research 29: E96 and US Patent No. 6,589,743), bulged Circular probe (US Patent No. 6,590,091), pseudoknot probe (US Patent No. 6,589,250), cyclicon (US Patent No. 6,383,752), MGBEclipse TM probe (Epoch Biosciences), hairpin probe (US Patent No. 6,596,490), peptide Nucleic acid (PNA) probes. The detection probe can comprise a fluorescent reporter molecule, for example, 6-carboxyfluorescein (6-FAM) or tetrachlorofluorescein (TET), and a quencher moiety, such as tetramethylrhodamine (TAMRA), Black Hole Quenche RS (Biosearch), IowaBlack (IDT), QSY quencher (Molecular Probes) and Dabsyl and Dabcel sulfonate/carboxylate quenchers (Epoch).

"Common-specific primer" refers to a primer paired with an allele-specific primer in a PCR reaction, which can be paired with an allele-specific primer at a different locus.

"Thermostable polymerase" refers to thermostable or heat-resistant enzymes, mainly DNA polymerases, which will not be inactivated under elevated temperature conditions during PCR amplification.

The "Tm" or "melting temperature" of an oligonucleotide refers to the temperature at which 50% of the molecules in a stretch of DNA hybridize to a complementary sequence. Tm values can be calculated using well-known formulas (see, Maniatis, T. et al.: Molecular cloning, Cold Spring Harbor, N.Y.: 1982).

"Sensitivity" refers to the minimum amount (copy number) of a template that can be detected.

"Specificity" refers to the ability to distinguish a matched template from a mismatched template. Specificity is often expressed as ΔCt = Ct mismatch - Ct match.

"Selectivity" refers to the degree to which an AS-PCR assay can be used to detect the minority (usually mutant) allele in a mixture without interference from the majority (often wild-type) allele. Selectivity is often expressed as a ratio or percentage. For example, an assay capable of detecting 1 mutant template in the presence of 100 wild-type templates is said to have a selectivity of 1:100 or 1%.

The "Ct" value refers to the cycle threshold, which represents the cycle number when the PCR amplification curve changes from the baseline to the inflection point of exponential growth.

"Delta Ct" or "ΔCt" is the difference in cycle number between two different samples or reactions when the signal crosses a fixed threshold. ΔCt is the difference in cycle number between two different samples or reactions at which exponential amplification is achieved. ΔCt can be used to identify specificity between matched and mismatched primers.

The invention relates to a primer and a kit for rapidly detecting the V617F mutation site of the JAK2 gene. Specifically, the present invention is based on the Past-PCR (Perfect allele-specific Taqman PCR) method, and is successfully applied to the detection of the polymorphic site of JAK2 gene V617F. The so-called Past-PCR is a combination of the highly specific AS-PCR method for detecting gene polymorphisms or mutations and the fluorescent PCR technology of Taqman probes. It only uses one Taqman probe. The needle is a conventional Taqman probe, only in some genes due to the special sequence rich in AT bases, using Taqman-MGB or LNA probes, in addition to no additional blocking probes or reporter probes, Past -PCR method perfectly combines AS-PCR and Taqman fluorescent PCR methods, and makes the advantages of the two get extreme play, specifically in the AS-PCR method, without increasing any cost, by primer design And the unique verification method successfully eliminates non-specific amplification, making the result judgment present an "all and nothing" method, that is, if there is, there is no, so that the judgment of the result is simple and accurate, which is an improvement on the past AS-PCR The biggest improvement of the method is also an effective remedy for the criticism of the AS-PCR method, so that the advantages of the AS-PCR method can be brought into full play, which is the highest state that the method can achieve. As we all know, Taqman fluorescent PCR has airtight detection without the need for post-reaction processing, which not only saves time, but also reduces the risk of PCR product contamination. The second recognition target sequence of the probe ensures the specificity and reliability of the detection, and also It can be used for quantitative detection of genes, and has reached the advantages of clinical application standards. The Past-PCR method has the dual advantages of the AS-PCR method and the Taqman fluorescent PCR method, and is the most simplified composition that can be achieved by the detection components when the two methods are combined, so we call it a perfect equivalent. Allele-specific Taqman PCR.

Based on the Past-PCR method, a kit for detecting the V617F polymorphism site of the JAK2 gene was invented, which basically consists of: (a) highly specific allele-specific primers; (b) fluorescent detection probes; ( c) Another specific primer paired with the allele-specific primer.

Wherein, the primers for detecting the V617F polymorphic site of the JAK2 gene provided by the present invention include: a wild-type specific upstream primer, a mutant-specific upstream primer, and a downstream shared by the wild-type specific upstream primer and the mutant-specific upstream primer. Primer;

And the wild-type specific upstream primer has the sequence of SEQ No.17, the mutant-type specific upstream primer has the sequence of SEQ No.14, and the common downstream primer has the sequence of SEQ No.16.

At the same time, detection reagents and kits containing the above primers are also provided to solve the difficulties in detecting the V617F polymorphism site of the JAK2 gene in the past, certain false negatives and false positives, high detection costs, and low clinical popularity. Large-scale detection of clinical specimens and other issues has achieved the ideal requirements of simple, fast, accurate, and cheap detection, and provides a powerful tool for scientific research and clinical genotype and gene mutation analysis.

Preferably, the kit also includes a probe used in conjunction with the primer, wherein the probe is a Taqman probe with a sequence of SEQ No.15, and only one probe is used in the entire kit without other Such as blocking probes, etc., reduce the cost of the kit.

Further preferably, the kit also includes PCR reaction tubes, dNTPs and Taq enzyme mixture, PCR buffer, upstream primers for detecting the housekeeping gene GAPDH, downstream primers for detecting the housekeeping gene GAPDH, probes for detecting the housekeeping gene GAPDH, positive Control product and blank control;

The upstream primer for detecting the housekeeping gene GAPDH has the sequence of SEQ No.19; the downstream primer for detecting the housekeeping gene GAPDH has the sequence of SEQ No.20; the probe for detecting the housekeeping gene GAPDH has the sequence of SEQ No.21; wherein, Mixing dNTPs and Taq enzymes can ensure the stability of dNTPs so that they can withstand repeated freeze-thaw tests; the upstream primers, downstream primers and probes for detecting the housekeeping gene GAPDH are used as internal references to prevent False negatives occurred; the positive quality control was TG-type human genomic DNA; the blank control was sterilized ultrapure water.

Further preferably, the mutant-specific upstream primer, the shared downstream primer, the probe, the upstream primer for detecting the housekeeping gene GAPDH, the downstream primer for detecting the housekeeping gene GAPDH, and the detection housekeeping gene GAPDH The probe is pre-coated in the T-type PCR reaction tube; and with the wild-type specific upstream primer, the shared downstream primer, the probe, the detection housekeeping gene GAPDH upstream primer, the detection The downstream primers of the housekeeping gene GAPDH and the probes for detecting the housekeeping gene GAPDH are pre-coated in G-type PCR reaction tubes, and are pre-coated in the PCR reaction tubes according to the optimal reaction amount, and each reaction tube is actually coated with Six oligonucleotides, one specific upstream primer for alleles, one probe (usually a Taqman probe), one shared specific downstream primer, one upstream primer for detecting the housekeeping gene GAPDH, one detecting the housekeeping gene GAPDH Downstream primers and a probe to detect the housekeeping gene GAPDH. In this way, in order to detect different gene polymorphisms at one locus, it is often necessary to coat two or more PCR reaction tubes respectively. Among them, the probe, the common specific downstream primer, the upstream primer for detecting the housekeeping gene GAPDH, and the downstream primer It is the same as the probe, and the only difference is the specific upstream primers for identifying different types of alleles. This pre-coating avoids the possibility of site mismatch by the operator and saves a lot of operating time.

Further preferably, the mutant-specific upstream primer in the T-type PCR reaction tube, the shared downstream primer, the probe, the upstream primer for detecting the housekeeping gene GAPDH, the downstream primer for detecting the housekeeping gene GAPDH, and the The probe concentrations for detecting the housekeeping gene GAPDH are respectively 50nM-2um, 50nM-2uM, 50nM-400nM, 1pm-20pm, 1pm-20pm, 0.05pm-5pm; and the wild-type specific upstream primers in the G-type PCR reaction tube , the shared downstream primer, the probe, the upstream primer for detecting the housekeeping gene GAPDH, the downstream primer for detecting the housekeeping gene GAPDH and the probe concentration for detecting the housekeeping gene GAPDH are respectively 50nM-2um, 50nM -2uM, 50nM-400nM, 1pm-20pm, 1pm-20pm, 0.05pm-5pm. Optimally, the mutant-specific upstream primers in the T-type PCR reaction tube, the shared downstream primers, the probe, the upstream primers for detecting the housekeeping gene GAPDH, the downstream primers for detecting the housekeeping gene GAPDH, and The probe concentrations for detecting the housekeeping gene GAPDH are respectively 500nM, 500nM, 100nM, 5pm, 5pm, and 2.5pm; and the wild-type specific upstream primers in the G-type PCR reaction tube, the shared downstream primers, the probe The concentrations of the needle, the upstream primer for detecting the housekeeping gene GAPDH, the downstream primer for detecting the housekeeping gene GAPDH, and the probe for detecting the housekeeping gene GAPDH were 500nM, 500nM, 100nM, 5pm, 5pm, and 2.5pm, respectively.

Due to the introduction of mismatched bases in the allele-specific primers, the efficiency of the reaction is reduced, and different primers have different degrees of decline. Usually, it takes 10 to 20 cycles to achieve the effect without mismatches. To ensure the sensitivity of detection, the preferred PCR reaction is carried out by a two-step amplification cycle program, and the number of cycles of the first step amplification is less than the number of cycles of the second step amplification, and the annealing temperature of the first step amplification is higher than that of the second step amplification. The annealing temperature of the first step amplification, the first step amplification uses a higher annealing temperature, and the second step amplification uses a lower annealing temperature, the purpose is to increase the specificity of the primer annealing during the first step amplification, so as to ensure Detect the purpose of high specificity; specifically, the preferred conditions of the PCR reaction are: 37°C for 2 minutes, 95°C for 2 minutes for pre-denaturation; the first step of amplification, 95°C for 5 seconds, 55°C to 68°C for 32 seconds, cycle 10 ~15 times; in the second step, denature at 95°C for 5s, anneal at 50°C~65°C, extend for 32s, cycle 30~50 times, and collect fluorescence signals; the more preferred PCR reaction conditions are: 37°C for 2min, 95°C for 2min Denaturation; the first step of amplification, 95°C denaturation for 5 s, 63°C annealing for 32 s, cycle 15 times; the second step, 95°C denaturation for 5 s, 60°C annealing, extension for 32 s, cycle 40 times, and collect fluorescence signals.

The specific detection process is:

1) Put the samples to be tested into pre-coated wild-type specific primers, shared downstream primers, probes, upstream primers for detecting the housekeeping gene GAPDH, downstream primers for detecting the housekeeping gene GAPDH, and probes for detecting the housekeeping gene GAPDH And two sets of PCR reaction tubes of mutant-type specific primers, shared downstream primers, probes, upstream primers for detecting housekeeping gene GAPDH, downstream primers for detecting housekeeping gene GAPDH and probes for detecting housekeeping gene GAPDH, and add PCR buffer, dNTPs and Taq enzyme mixture, sterilized ultrapure water and genomic DNA, tightly cap the tube, react on a fluorescent quantitative PCR instrument, and collect fluorescent signals;

2) If only the mutant reaction tube in the test sample has a logarithmic growth amplification curve in the FAM channel, and the VIC channel has a logarithmic growth amplification curve, it can be determined that the sample is T type, that is, the mutant homozygote;

If only the wild-type reaction tube in the test sample has a logarithmic growth amplification curve in the FAM channel, and the VIC channel has a logarithmic growth amplification curve, it can be determined that the sample is G type, that is, wild-type homozygosity;

If the test sample mutant type and wild type reaction tubes have logarithmic growth amplification curves in the FAM channel, and the VIC channel has a logarithmic growth amplification curve, it can be determined that the sample is TG type, that is, heterozygous;

If there is no logarithmic growth amplification curve in the FAM channel of the test sample mutant and wild-type reaction tubes, there may be problems with the sample or operation, or the sample DNA concentration is too low, and the sample DNA should be re-extracted for testing.

The following details the introduction of each component in the detection process:

(1) Allele-specific primers:

The allele-specific nucleotide portion of an allele-specific primer is complementary to one allelic position of a gene, but not to the other allelic position of the gene. Typically, the allele-specific nucleotide portion of the allele-specific primer is located at the last base of the 3' end of the allele-specific primer. In some cases, the allele-specific nucleotide moiety may also be located at other base positions at the 3' end of the allele-specific primer.

In some cases, except that the 3' end of the allele-specific primer is complementary to the allelic site, we often introduce mismatched bases at other positions of the primer, such as the penultimate base at the 3' end of the primer. Introduce a base mismatch at the second or third position, or a continuous mismatch between the second and third positions; for different polymorphisms or mutation sites, introduce a base at the penultimate or third position at the 3' end of the primer On the basis of base mismatches, base mismatches can also be added at other positions of the primer to meet the requirements of reaction specificity. The farther away from the 3' end, the more bases to increase the number of mismatches. It can be reached at the 5' end. most. The length of the allele _- specific primer is mainly determined by its _Tm value, which is generally about 5-20°C lower than the annealing temperature of the PCR cycle.

The two allele-specific upstream primers for detecting corresponding gene polymorphisms have the same sequence as possible except for the base detected at the 3' end, and the T _m values of the two primers are also as consistent as possible.

Allele-specific primers (ASP) with low T _m have higher specificity. In general, allele-specific primers are short oligonucleotides that are approximately 15-30 Å in length and whose _Tm is approximately 40°C to 60°C, which is less than the annealing/extension of the PCR cycling conditions used during amplification. The temperature is about 5°C to 20°C lower.

Because the design and screening of allele-specific primers are very important, good primers need to be selected from many primers to be selected, so we have established a screening method, and the specific screening process for allele-specific primers is as follows:

(1) Design peripheral primers and PCR amplification: first, design a pair of PCR amplification primers upstream and downstream of the mutation point or polymorphic site of the gene to be detected, and then use the pair of primers to perform a conventional PCR reaction on the target gene, wherein The length of the amplified fragment can be tens to thousands of base pairs, preferably 200 to 500 base pairs;

(2) Clone the PCR product: Recombine the amplified PCR product into a common T vector plasmid by AT cloning method to obtain a recombinant plasmid, which can be sequenced and verified to ensure the correctness of the PCR product;

(3) Construction of recombinant mutants: According to the information provided by bioinformatics, molecular cloning techniques were used to construct the sequence of the mutants into the recombinant plasmids obtained above, and the sequence of the constructed recombinant mutants was sequenced to ensure that they were correct. sex;

(4) Design and screening of mutation site-specific primers: design two allele-specific primers, one primer’s 3’ end is complementary to the mutant sequence, and the other primer’s 3’ end is complementary to the normal sequence; The specific primers are paired with the corresponding conventional PCR common downstream primers, and the recombinant mutant constructed above is used as the reaction template, and the reaction system is screened using a fluorescent quantitative PCR instrument; wherein, the reaction can use fluorescent dyes, such as SYBR GREEN, Specific Taqman fluorescent probes can also be used to screen according to the standard that the added sample volume is 0.1 micrograms of whole-genome DNA, and the specific screening content is as follows:

(a) Set the PCR reaction conditions: first set the annealing temperature of the PCR reaction to be exactly the same, the specific temperature is 55 ° C ~ 68 ° C, the number of PCR cycles is 40 times, and the enzyme and buffer system of the PCR reaction are optimized and fixed. The setting of this reaction condition is to facilitate simultaneous detection of multiple gene mutations;

(b) Specific screening: Use mutant primers and wild-type recombinant plasmids as templates, and the Ct of the screened primers is greater than 40;

The reason for determining the cycle threshold is as follows. The non-specific amplification of the mutant primer in the wild-type template is 19 cycles longer than the specific amplification of the wild-type primer in the wild-type template. According to the actual clinical detection, the amount of sample added is 0.1 For the standard of micrograms of whole-genome DNA, we use the following formula, copy number = (DNA amount ng×6.022×10 ²³ )/(length bp×10 ⁹ ×660), where the length of whole-genome DNA is 3 billion base pairs, so The copy number of 0.1 μg of whole-genome DNA is: 3.09×10 ⁴ , and the Ct (quantitative cycle threshold) reflected in fluorescent quantitative PCR is about 21, so we get the second screening index, that is, using mutant primers, in the wild-type When the recombinant plasmid is used as the template, the obtained Ct should be greater than 40 to ensure that no false positives will occur in the detection. The difference in amplification efficiency between the two is 40-21=19 cycles, that is, the difference in amplification efficiency is more than 19 cycles. It will be an objective indicator for us to judge the specificity of allele-specific primers. We know that when the 3' end of the primer is mismatched with the template, the extension efficiency will decrease by 10 ³ -10 ^6.6 times, and the number of reaction cycles will decrease by about 13 to 27 cycles. This is the theoretical basis for us to screen out ideal primers. Because there is no primer screening, 19-13=6 cycles will inevitably produce false positives, and the specificity of the primer is not good, and 19-27=-8, it is impossible to produce false positives, and there are 8 cycles for the range of numbers. We screen the primers. In accordance with this requirement, we designed a series of allele-specific primers, starting from the length and artificially introducing base mismatches near the 3' end, plus their paired downstream primers, using the constructed wild-type and mutant Recombinant plasmids, carry out the cross-screening of fluorescent quantitative PCR reactions respectively, that is, when using mutant primers and wild-type templates for amplification, it takes more than 19 cycles to react with wild-type primers and wild-type templates, and vice versa Of course, such primers are highly specific.

(c) Sensitivity screening: use the screened specific primers for a sensitivity test with the recombinant mutant plasmid, and screen out specific primers with a detection sensitivity of less than 10-1000 copies, which are the required mutation-specific primers .

Through the screening of the above aspects, the obtained allele-specific primers will be ideal primers with high specificity and high sensitivity.

(2) Detection probe:

In some cases, the _Tm value of the detection probe is about 60°C to 70°C, preferably 65°C, and the length of the probe is determined according to its _Tm value. Although there are many different formats of probes available, Taqman probes (Applied Biosystems, Foster City) are often the first choice. In some special cases, such as the content of AT in the detection sequence is too high, probes such as Taqman-MGB with increased T _m value can be used.

Shared specific primers, in some cases, the T _m value of shared specific primers is about 50°C to 70°C, the best is 60°C, and its length is determined by its T _m value, generally 15-25 bases between.

(3) Other ingredients:

The DNA polymerase that the present invention uses is Taq enzyme, and mutant, derivative or fragment thereof, also can be other thermostable DNA polymerase, in some cases, also can adopt Hotstart Taq enzyme, to increase the specificity and efficient of reaction sex.

The primers and kits provided for the JAK2 gene V617F polymorphic site provided in the present invention are mainly aimed at the shortcomings of the allele-specific primer PCR amplification method. Gene wild-type and mutant-type recombinant plasmids, using the recombinant plasmid as a positive template, screen to determine two specific primers that are complementary to the 3' terminal mutation site or wild site base, once the specific primer sequence is screened , will be a key element of our detection system. The above-mentioned product of the present invention can be used in any type of fluorescent quantitative PCR instrument, and the reagent cost is equivalent to that of fluorescent quantitative PCR reagents on the market today, so it overcomes the shortcomings of other PCR amplification methods using allele-specific primers.

The advantages of the present invention are: the kit is simple to operate, low in cost, simple and easy to judge results, and high in sensitivity. The sensitivity of the kit to detect gene mutations can reach 1% (that is, the number of mutant gene DNA templates of the target gene accounts for 1% of the number of wild-type DNA templates). 1% of the number of DNA templates); while the sensitivity of detecting gene mutations in direct sequencing is 20% (that is, the number of mutant gene DNA templates of the target gene accounts for 20% of the number of wild-type DNA templates); Taqman probe technology ensures the kit’s The detection is a closed-tube reaction, which effectively avoids false positives, and its detection specificity is fully guaranteed, and the detection is fast and convenient. The entire detection process only takes 80 minutes, while direct sequencing takes 2 days, and it is an open-tube method. operation, the possibility of PCR product contamination is greatly increased.

Implementation steps of PCR (Past-PCR) in the present invention:

1. Construction of positive plasmid

Design a pair of PCR primers upstream and downstream of the V617F polymorphic site of the JAK2 gene to be detected. The length of the amplified fragment can be within the range of 500bp. Using gDNA as a template, the conventional PCR method is used to amplify this fragment , by means of AT cloning, clone the fragment into the sequenced plasmid, usually use Invitrogen’s pCR2.1 Topo T vector kit, the operation is carried out according to the instructions, T vector kits from other manufacturers can also be used, or even self-prepared T vector plasmid. The positive clone obtained was verified by sequencing to prove the correctness of the sequence, and then the Stratagene company’s Quickchange kit was used to design another genotype primer corresponding to the site, and the positive clone of the mutant type was obtained by the Quickchange method, and the operation According to the instructions, the obtained positive plasmids must be verified to be correct by sequencing before they can be used in the next step. Taqman quantifies the plasmid and is used as a template to validate the sensitivity, linear dynamic range, specificity of a given assay.

2. Design and screening of allele-specific primers (ASP)

The 3' end of the primer is complementary to the corresponding gene mutation base site, and a base mismatch is introduced at the penultimate or third position of the primer 3' end, or the second and third consecutive mismatches; for different The polymorphism or mutation site, on the basis of introducing a base mismatch at the penultimate or third position of the 3' end of the primer, base mismatches can also be added at other positions of the primer to meet the specificity of the reaction. Requirements, the farther away from the 3' end, the more bases that increase the number of mismatches, and the most accessible allele 1-specific primers at the 5' end.

ASP screening is to use the constructed wild-type and mutant recombinant plasmids to carry out cross-screening of fluorescent quantitative PCR reactions, that is, when using mutant primers and wild-type templates to amplify, the same as using wild-type primers and wild-type templates. The reaction should use more than 19 cycles, and vice versa, then such primers have a high degree of specificity. Use the screened specific primers with the corresponding recombinant plasmids for a sensitivity test. The primers with a detection sensitivity of less than 10-1000 copies meet the detection sensitivity requirements. The primers that meet the above two conditions are the ASP primers we selected. The next step of detection can be carried out.

3. Preparation of amplification reagents and addition of tested samples

(1) Take out the components from the kit, slowly melt the PCR buffer, dNTPs and Taq enzyme mixture, positive quality control, and blank control at room temperature, and shake them upside down. Each reaction tube needs to add PCR buffer, Taq enzyme (dNTPs), sterilized ultrapure water and DNA sample. Cap the tube tightly, centrifuge briefly and transfer to the PCR amplification area.

(2) It is recommended that the analysis of samples, positive quality control products, and blank control be carried out at the same time for each PCR reaction, and the sample addition method of positive quality control material and blank control is the same as the sample addition method.

4. Reaction conditions

Each reaction tube (the final volume of the reaction can be in the range of 10-50 μl, the optimal reaction volume of 20-25 μl) contains PCR buffer (10mM Tris-HCl pH8.3, 50mM KCl, 0.1%Triton X-100, 2.0 mM MgCl ₂ ), 10-100ng DNA or 1,000,000 copies of plasmid DNA, 50nM-400nM a common universal specificity Taqman probe, a 50nM-2uM common universal specificity downstream primer, 50nM-2uM different alleles Gene-specific upstream primers, 1pm-20pm detect the upstream primer of the housekeeping gene GAPDH, 1pm-20pm detect the downstream primer of the housekeeping gene GAPDH, 0.05pm-5pm detect the probe of the housekeeping gene GAPDH, plus 1.5 units of Taq polymerase, 200μM dNTPs.

1), cycle condition setting

In order to make the fluorescence curve more beautiful, it is recommended to collect the fluorescence signal from the second step.

2), instrument detection channel selection

The fluorescence signal acquisition temperature is set to 60°C, please refer to the instruction manual of the corresponding instrument for the specific setting method.

3) Detection type setting: set the blank control to "NTC", set the positive quality control and the sample to be tested to "Unknown".

5. Result Analysis

If the test sample only has a logarithmic growth amplification curve in the FAM channel of the mutant reaction tube, and at the same time, the VIC channel has a logarithmic growth amplification curve, it can be determined that the sample is T-type, that is, mutant homozygosity; If there is a wild-type reaction tube with a logarithmic growth amplification curve in the FAM channel, and a logarithmic growth amplification curve in the VIC channel, it can be determined that the sample is G type, that is, wild-type homozygosity; if the test sample mutant and wild type The reaction tubes have a logarithmic growth amplification curve in the FAM channel, and at the same time, the VIC channel has a logarithmic growth amplification curve, which can determine that the sample is a TG type, that is, a heterozygote; since the result of this method is "all or none", the result It is very easy to judge which tube has a reaction and which tube is positive.

specific embodiment

Taking the detection of the V617F polymorphic site of the JAK2 gene as an example, the present invention will be further described in detail below.

Example 1: Preparation of wild type and mutant positive plasmids for JAK2 gene V617F polymorphic site

JAK is a non-receptor tyrosine protein kinase, among which JAK2 mutation is closely related to myeloproliferative diseases. JAK2 gene V617F mutation can lead to abnormal activation of JAK-STAT signaling pathway, resulting in abnormal proliferation of bone marrow cells. In the 2008 World Health Organization (WHO) classification system, JAK2 mutation has become the main diagnostic index of chronic myeloproliferative disease (MPD).

First, we called out the gene sequence before and after the V617F polymorphic site of the JAK2 gene from the gene bank, and marked the polymorphic site with a double underline, at the appropriate position upstream and downstream of the V617F polymorphic site of the JAK2 gene ( Marked in bold and underlined), design a pair of cloning primers, the amplified fragment is 228bp, including the mutation site, the gene sequence is shown below, SEQ No.1:

catgattcctgtaccactcttgctctctctcactttgatctccatattccaggcttacacaggggtttcctcagaacgttgatggcagttgcaggtccatataaagggaccaaagcacattgtatcctcatctatagtcatgctgaaagtaggagaaagtgcatctttattatggcagagagaattttctgaactatttat ggacaacagtcaaacaacaattc tttgtacttttttttttccttagtctttctttgaagcagcaagtatgatgagcaagctttctcacaagcatttggttttaaattatggagtatgt g tctgtggagacgagagtaagtaaaactacaggctttctaatgcctttctcagagcatctgtttttgtttatatagaaaattcagttt caggatcacagctaggtgtcag tgtaaactataatttaacaggagttaagtatttttgaaactgaaaacactgtaggactattcagttatatcttgtgaaaaaggaaagcaatgaagttaaaagtagaaggttacaatgcccaaacaatagagtatt

The experimental steps are as follows:

1) Genomic DNA extraction

Genomic DNA was extracted with Genomic DNA Extraction Kit from domestic Tiangen Company or Qiagen Company, and the specific operation steps were carried out according to the instructions. The prepared DNA samples were tested for concentration by UV spectrophotometer, adjusted to 50ng/μl, stored at -20°C, or directly proceeded to the following reaction.

2) AT clones obtain positive plasmids

First, design cloning primers upstream and downstream of the mutation point, as shown in Table 1:

Table 1. Primers for wild-type cloning of JAK2 gene V617F polymorphism site

In 12.5 μl of 2×PCR Master Mix (Fermentas) PCR amplification system, add 10 μM upstream primers and 10 μM downstream primers, and 50 ng of genomic DNA, add water to a total volume of 25 μl, and set the PCR reaction conditions at 95°C for pre-denaturation for 3 minutes, 95 Denaturation at ℃ for 10 s, annealing at 60 ℃ and extension for 30 s, a total of 35 cycles. After the reaction, take 15 μl of the reaction product and perform gel electrophoresis at a concentration of 2.5%. After the electrophoresis, the band is consistent with the predicted size, indicating that the amplification is successful . Using the pCR2.0 TOPO T vector from American Life Company, according to its operating instructions, the wild-type positive plasmid of the V617F polymorphic site of the JAK2 gene was obtained by TA cloning. The specific gene sequence is as follows, where the polymorphic site is shown It is G type, indicating that it is wild type, and the sequencing proves that the obtained plasmid is correct.

SEQNo.4：ggacaacagtcaaacaacaattctttgtacttttttttttccttagtctttctttgaagcagcaagtatgatgagcaagctttctcacaagcatttggttttaaattatggagtatgt g tctgtggagacgagagtaagtaaaactacaggctttctaatgcctttctcagagcatctgtttttgtttatatagaaaattcagtttcaggatcacagctaggtgtcag

3) Rapid mutation method to obtain mutant plasmids

Then, use the method of quick mutation (Quickchange, QC) to design mutation primers. Since the JAK2-V617F site changes from g to t, the primer sequences are shown in Table 2.

Table 2. Cloning primers for the V617F polymorphism site mutation of the JAK2 gene

According to the reaction conditions and steps of the (Stratagene Company) Quickchange kit, the construction of the mutant was completed. The specific gene sequence of the obtained recombinant plasmid was as follows. It was verified by sequencing that the polymorphic site was shown as t, which was a mutant type, and then Store the mutant and wild-type plasmids separately at -20°C for future use.

SEQ No.7：ggacaacagtcaaacaacaattctttgtacttttttttttccttagtctttctttgaagcagcaagtatgatgagcaagctttctcacaagcatttggttttaaattatggagtatgt t tctgtggagacgagagtaagtaaaactacaggctttctaatgcctttctcagagcatctgtttttgtttatatagaaaattcagtttcaggatcacagctaggtgtcag

Example 2: Design and specificity screening of allele-specific primers (ASP)

For JAK2-V617F, design wild-type and a series of mutant-specific primers as follows:

JAK2-V617F-WT-R: ttacttactctcgtctccacacac (SEQ No. 8)

JAK2-V617F-mut-R: tttacttactctcgtctccacacaa (SEQ No. 9)

JAK2-V617F-mut-R1: tacttactctcgtctccacacaa (SEQ No. 10)

JAK2-V617F-mut-R2: acttactctcgtctccacacaa (SEQ No. 11)

JAK2-V617F-mut-R3: ctttacttactctcgtctccacagga (SEQ No. 12)

JAK2-V617F-mut-R4:cttacttactctcgtctccacagga (SEQ No. 13)

JAK2-V617F-mut-R5: ctacttactctcgtctccacagga (SEQ No. 14)

Simultaneously design and synthesize Taqman-specific probes:

SEQ No. 15: FAM-tgaagcagcaagtatgatgagcaagc-BHQ1.

Relevant primers and probes were synthesized at Sangon Bioengineering (Shanghai) Co., Ltd.

Then use the above 7 primers to pair with the common downstream primer JAK2-V617F-F: 5'-ggacaacagtcaaacaacaattc-3' (SEQ No.16) of the JAK2 gene, and add Taqman-specific probes to add the obtained About 30,000 copies of the wild-type recombinant plasmid were used as templates, and primer specificity screening was carried out on a fluorescent quantitative PCR instrument. The reaction conditions were set as the first step of 95°C for 2min pre-denaturation, 95°C for 5s, and 63°C for 30s for a total of 15 cycles. No fluorescence signal was collected, the second step was 95°C for 5s and 60°C for 30s for a total of 40 cycles to collect the fluorescence signal, the results are shown in Figure 1.

Among them, the curves 1~7 in Figure 1 represent JAK2-V617F-WT-R, JAK2-V617F-mut-R, JAK2-V617F-mut-R1, JAK2-V617F-mut-R2, JAK2-V617F-mut-R3 , JAK2-V617F-mut-R4, and JAK2-V617F-mut-R5 PCR amplification curves, from Figure 1 we can see that No. 1 wild-type primer generates amplification signals in 7 cycles, and the specificity of 2-6 primers Poor, non-specific amplification occurs at 7.5-9 cycles respectively, and the difference in amplification efficiency with the wild-type primer (No. 1) is less than 19 cycles. Primer No. 7 meets our requirements. At 40 cycles, it is still There is no amplification, and the difference between the amplification efficiency of the wild-type primer is more than 19 cycles. In order to have better comparability with the mutant primers, we redesigned the wild-type primers with reference to primer No. 7: ctacttactctcgtctccacaggc (SEQ No. 17).

Embodiment 3: ASP sensitivity screening

Then use the No. 7 mutant primer to pair with the common downstream primer SEQ No.16 of the JAK2 gene, use the mutant recombinant plasmid, make gradient dilutions according to 10 ⁶ , 10 ⁵ , 10 ⁴ , 10 ³ , 10 ² , 10,0, add Use Taqman specific probes, and perform sensitivity verification on a fluorescent quantitative PCR instrument. The No. 7 mutation-specific primer can detect 100 copies of the mutant, so this primer is the best primer for detecting the V617F polymorphism site of the JAK2 gene screened according to our method, as shown in Table 3.

Embodiment 4: Prefabricated PCR (Past-PCR) reaction tube of the present invention

According to the requirements of two reaction tubes for each mutation site, that is, one tube for detection of wild-type site and the other tube for detection of mutation site, pre-coat the appropriate concentration of primer probes in a 0.2ML milky white PCR reaction tube.

In order to prevent false negatives during detection, each reaction tube is pre-coated with upstream primers, downstream primers and probes for the detection of the housekeeping gene GAPDH, wherein the sequence of the GAPDH gene is as follows:

cctccgggaaactgtggcgtgatggccgcggggctctccagaacatcatccctgcctctactggcgctgccaaggctgtgggcaaggtcatccctgagctgaacgggaagctcactggcatggccttccgtgtccccactgccaacgtgtcagtggtggacctgacctgccgtctagaaaaacctgccaaatatgatgacatcaagaaggtggtgaagcaggcgtcggagggccccctcaagggcatcctgggctacactgagcaccaggtggtctcctctgacttcaacagcgacacccactcctccacctttgacgctggggctggcattgccctcaacg（SEQ No.18）

The upstream primer sequence for detecting the housekeeping gene GAPDH is: ggctgtgggcaaggtcatc (SEQ No.19)

The downstream primer sequence for detecting the housekeeping gene GAPDH is: ctccgacgcctgcttcaccac (SEQ No.20)

The probe sequence for detecting the housekeeping gene GAPDH is: ccttccgtgtccccactgccaacgt (SEQ No.21)

Among them, the 5' end of the probe for detecting the housekeeping gene GAPDH is labeled with HEX fluorescence, and the 3' end is labeled with BHQ. Since HEX and VIC belong to the same channel for detection, and many instruments often use VIC labeling, VIC is used for description in this article.

The specific details in the PCR reaction tube are shown in Table 3.

Table 3. Components and concentrations of premade JAK2 gene V617F polymorphic site Past-PCR reaction tubes

Because we prepackage the mutant and wild-type upstream primers, probes and common downstream primers, the upstream and downstream primers for detecting the housekeeping gene GAPDH, and the probe for detecting the housekeeping gene GAPDH in different numbered reaction tubes, effectively avoiding The user's operation error is reduced, and the operation efficiency is greatly improved at the same time, which greatly facilitates the actual clinical use.

Embodiment 5: verification of specimen

Tissue samples of melanoma patients were collected, and genomic DNA was extracted using the genomic DNA extraction kit of Qiagen Company, and the specific operation steps were carried out according to the instructions. The concentration of the prepared DNA sample was measured by an ultraviolet spectrophotometer, and the concentration was adjusted to 100ng/μl for later use.

We use the mutation-specific primers screened out, and use wild-type primers as controls at the same time. On the same PCR reaction plate, every two reaction tubes detect a gene locus, one of which is the mutation site and the other is the wild-type site. Each reaction tube contains 10mM Tris-HCl pH8.3, 50mM KCl, 0.1%Triton X-100, 2.0mM MgCl ₂ , 100ng melanoma patient DNA, 100nM specific Taqman probe, 500nM shared specificity downstream in 20μl reaction volume Primers, and 500nM wild-type specific primers, or mutant-specific primers, 5pm upstream primers to detect the housekeeping gene GAPDH, 5pm downstream primers to detect the housekeeping gene GAPDH, 2.5pm probes to detect the housekeeping gene GAPDH, plus 1.5 units Taq DNA polymerase, 200μM dNTPs, the rest is deionized water.

The first step PCR reaction conditions are: 37°C for 2min, 95°C for 2min pre-denaturation, then 95°C for 5s, 63°C for 30s, 15 cycles, this step does not collect fluorescence; the second step PCR reaction conditions are: 95°C for 5s, 60°C ℃ for 30s, a total of 40 cycles, this step collects fluorescence. For convenience, we identified the normal site of the V617F polymorphism of the JAK2 gene as the wild type (G site), and the site that is likely to cause abnormal activation of the JAK-STAT signaling pathway as the mutant type (T site). If the corresponding reaction tube has signal growth, it is judged as the corresponding homozygous genotype, and if both tubes grow, it is heterozygous.

1. According to the above method, detect the following mutant gene sequences:

SEQ No.22: tggagtatgtttctgtggaga

The collected FAM channel PCR amplification curves are shown in Figure 2, wherein, curve a represents the amplification curve in the detection mutant PCR reaction tube, and curve b represents the amplification curve in the detection wild-type PCR reaction tube.

2. Detect the following wild-type gene sequences according to the above method:

SEQ No.23: tggagtatgtg tctgtggaga

The collected FAM channel PCR amplification curve is shown in FIG. 3 , wherein, the c curve represents the amplification curve in the detection wild-type PCR reaction tube, and the d curve represents the amplification curve in the detection mutant PCR reaction tube.

3. Perform heterozygous (TG type) detection according to the above method, and the specific gene sequence is:

SEQ No.24: tggagtatgtt t/g tctgtggaga

The collected FAM channel PCR amplification curves are shown in Figure 4, wherein the e curve represents the amplification curve in the detection wild-type PCR reaction tube, and the f curve represents the amplification curve in the detection mutant PCR reaction tube.

At the same time, in the above three detection cases, when the VIC channel housekeeping gene GAPDH has a logarithmic growth amplification curve, as shown in Figure 5, and the FAM channel has a logarithmic growth amplification curve, the experimental results are valid.

Therefore, it can be seen from the above-mentioned detection test that the detection result of this kit is an "all or none" result, that is, if the tested sample is homozygous for the mutant allele, only the mutant PCR reaction will be detected in the two PCR reaction tubes. Both tubes have logarithmic growth amplification curves in the FAM channel and VIC channel, while the wild-type PCR reaction tube has no logarithmic growth amplification curve in the FAM channel; if the tested sample is homozygous for the wild allele, two Among the PCR reaction tubes, only the wild-type PCR reaction tube has a logarithmic growth amplification curve in the FAM channel and the VIC channel, while the mutant PCR reaction tube has no logarithmic growth amplification curve in the FAM channel; if detected The sample is heterozygous, and there are amplification curves with logarithmic growth in both the FAM channel and the VIC channel in the two PCR reaction tubes. The detection results of the kit are easy to interpret, which reduces the need for a series of complex calculations such as ΔCt in the detection results of the previous kits, reduces the misjudgment rate, and effectively avoids false negatives and false positives in the detection results of the previous kits happened. In addition, the kit provided by the present invention can be used in any type of fluorescent quantitative PCR instrument, the detection instrument is simple, and the cost is low, which provides a powerful tool for scientific research and clinical JAK2 genotyping and gene mutation analysis.

sequence listing

<110> Shenyang Youjinuo Biotechnology Co., Ltd.

<120> Primers, kit and PCR method for detecting JAK2 gene V617F polymorphism

<130> 2015

<160> 24

<170> PatentIn version 3.3

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ctacttactc tcgtctccac aggc 24

<210> 18

<211> 342

<212>DNA

<213> Synthetic

<400> 18

cctccgggaa actgtggcgt gatggccgcg gggctctcca gaacatcatc cctgcctcta 60

ctggcgctgc caaggctgtg ggcaaggtca tccctgagct gaacgggaag ctcactggca 120

tggccttccg tgtccccact gccaacgtgt cagtggtgga cctgacctgc cgtctagaaa 180

aacctgccaa atatgatgac atcaagaagg tggtgaagca ggcgtcggag ggccccctca 240

agggcatcct gggctacact gagcaccagg tggtctcctc tgacttcaac agcgacaccc 300

actcctccac ctttgacgct ggggctggca ttgccctcaa cg 342

<210> 19

<211> 19

<212>DNA

<213> Synthetic

<400> 19

ggctgtgggc aaggtcatc 19

<210> 20

<211> 21

<212>DNA

<213> Synthetic

<400> 20

ctccgacgcc tgcttcacca c 21

<210> 21

<211> 25

<212>DNA

<213> Synthetic

<400> 21

ccttccgtgt ccccactgcc aacgt 25

<210> 22

<211> 21

<212>DNA

<213> Synthetic

<400> 22

tggagtatgt ttctgtggag a 21

<210> 23

<211> 21

<212>DNA

<213> Synthetic

<400> 23

tggagtatgt gtctgtggag a 21

<210> 24

<211> 23

<212>DNA

<213> Synthetic

<400> 24

tggagtatgt ttgtctgtgg aga 23

Claims (7)

1. A primer and a probe for detecting the polymorphism of the JAK2 gene V617F site, characterized in that, the primers include: wild-type specific upstream primers, mutant-specific upstream primers and wild-type specific upstream primers and mutant Downstream primers shared by type-specific upstream primers; And the wild-type specific upstream primer sequence is shown in SEQ No.17, the mutant-type specific upstream primer sequence is shown in SEQ No.14, and the shared downstream primer sequence is shown in SEQ No.16; The probe is a Taqman probe, the sequence of which is shown in SEQ No.15. 2. A reagent for detecting the polymorphism of the V617F site of the JAK2 gene, characterized in that: the reagent contains the primer and probe according to claim 1. 3. A kit for detecting the polymorphism of the V617F site of JAK2 gene, characterized in that: said kit contains the primer and probe according to claim 1. 4. according to the test kit of the described detection JAK2 gene V617F site polymorphism of claim 3, it is characterized in that: described test kit also comprises PCR reaction tube, dNTPs and Taq enzyme mixed solution, PCR damping fluid, detection housekeeping gene GAPDH Upstream primers, downstream primers for detecting the housekeeping gene GAPDH, probes for detecting the housekeeping gene GAPDH, positive quality control substances and blank controls; The upstream primer sequence for the detection of the housekeeping gene GAPDH is shown in SEQ No.19; the downstream primer sequence for the detection of the housekeeping gene GAPDH is shown in SEQ No.20; the probe sequence for the detection of the housekeeping gene GAPDH is as shown in SEQ No. 21. 5. The kit for detecting the polymorphism of the JAK2 gene V617F site according to claim 4, characterized in that: the mutant-specific upstream primer, the shared downstream primer, the probe, the detection housekeeper The upstream primer of the gene GAPDH, the downstream primer for detecting the housekeeping gene GAPDH, and the probe for detecting the housekeeping gene GAPDH are pre-coated in a T-shaped PCR reaction tube; The downstream primer, the probe, the upstream primer for detecting the housekeeping gene GAPDH, the downstream primer for detecting the housekeeping gene GAPDH, and the probe for detecting the housekeeping gene GAPDH are pre-coated in the G-type PCR reaction tube. 6. according to the kit for detecting the polymorphism of the JAK2 gene V617F site according to claim 5, it is characterized in that: the mutant-specific upstream primer in the T-shaped PCR reaction tube, the shared downstream primer, the probe , the upstream primer for detecting the housekeeping gene GAPDH, the downstream primer for detecting the housekeeping gene GAPDH, and the probe concentration for detecting the housekeeping gene GAPDH are respectively 50nM-2μm, 50nM-2μm, 50nM-400nM, 1pm-20pm, 1pm -20pm, 0.05pm-5pm; and the wild-type specific upstream primer in the G-type PCR reaction tube, the shared downstream primer, the probe, the detection housekeeping gene GAPDH upstream primer, the detection housekeeping gene The concentrations of the downstream primers for GAPDH and the probe for detecting the housekeeping gene GAPDH are 50nM-2μm, 50nM-2μm, 50nM-400nM, 1pm-20pm, 1pm-20pm, 0.05pm-5pm, respectively. 7. According to the kit for detecting the polymorphism of the JAK2 gene V617F site according to claim 6, it is characterized in that: the mutant-specific upstream primer in the T-shaped PCR reaction tube, the shared downstream primer, the probe , the upstream primer for detecting the housekeeping gene GAPDH, the downstream primer for detecting the housekeeping gene GAPDH, and the probe concentrations for detecting the housekeeping gene GAPDH are respectively 500nM, 500nM, 100nM, 5pm, 5pm, and 2.5pm; and the G The wild-type specific upstream primer in the type PCR reaction tube, the shared downstream primer, the probe, the upstream primer for detecting the housekeeping gene GAPDH, the downstream primer for detecting the housekeeping gene GAPDH, the detection housekeeping gene GAPDH Probe concentrations were 500nM, 500nM, 100nM, 5pm, 5pm, 2.5pm, respectively.