Methods for Enriching and Detecting Low-Abundance Mutant DNA

Abstract

A method for enriching and detecting low-abundance mutant DNA is provided, and a wild-type amplification inhibitor is also provided. The wild-type amplification inhibitor has a Tm value (Tm1) for specifically binding to a wild-type template that is greater than a Tm value (Tm2) for specifically binding to a mutant template, and has a blocking group at the 3' end of the wild-type amplification inhibitor. A corresponding amplification inhibition mutation system and a nucleic acid detection system are also provided, which include a probe (whose Tm value for binding to a wild-type template is Tm3, where Tm1>Tm3>Tm2) that competes with the wild-type amplification inhibitor to bind to the template, and upstream and downstream primers. With the technical means provided, the system can enrich low-abundance DNA mutations and detect multiple types of base mutations, and has the advantages of high sensitivity, high accuracy, low cost, short time consumption, and simple operation.

Description

[Technical field]

[0001] The present invention relates to the technical field of biodetection, specifically to a method for enrichment and detection of low abundance mutant DNA. [Background technology]

[0002] DNA often undergoes random mutations during the replication process, and the frequency of mutations often increases significantly in harsh environments (e.g., radiation, chemical mutagens, etc.). Gene mutations are one of the important factors in biological evolution and the underlying cause of biological genetic diversity. However, gene mutations can also cause adverse effects such as the development of cancer and drug resistance in pathogenic microorganisms. These mutations are often mixed with many normal wild-type genes, and their low mutation content makes them very difficult to detect clinically. Therefore, highly sensitive mutation detection technology is of great importance in guiding drug treatment.

[0003] In recent years, techniques to enrich and / or detect low-abundance mutant DNA include sequencing, digital PCR, co-amplification at low denaturing temperature-PCR (COLD-PCR), primer amplification impaired mutation system (ARMS-PCR), wild-type amplification inhibited PCR (WTB-PCR), denaturing high performance liquid chromatography (dHPLC), etc. However, these methods have their own advantages and disadvantages in terms of sensitivity, accuracy, cost, time, and operation.

[0004] Those skilled in the art desire to develop new methods for enriching and detecting low abundance mutant DNA to overcome the deficiencies in sensitivity, accuracy, cost, time, handling, etc., present in the prior art. Summary of the Invention

[0005] The object of the present invention is to provide a method for enriching and detecting low abundance mutant DNA. The technical means provided by the present invention is optimized and improved based on wild-type amplification inhibition PCR technology. With the technical means of the present invention, the system can enrich low abundance DNA mutations and detect multiple types of base mutations, and has the advantages of high sensitivity, high accuracy, low cost, short time consumption, and simple operation.

[0006] In a first aspect, the present invention provides a wild-type amplification inhibitor, which has a Tm value of Tm1 when specifically binding to a wild-type template of a test gene and a Tm value of Tm2 when specifically binding to a mutant template of a test gene, Tm1>Tm2, and the 3'-end of the wild-type amplification inhibitor has a blocking group.

[0007] According to the technical means of the present invention, the binding site of the wild-type amplification inhibitor and the test gene covers the mutation site of the test gene, and Tm1>Tm2. Therefore, within a specific annealing temperature range, the wild-type amplification inhibitor only binds to the wild-type template, and since its 3' end is modified with a blocking group, it cannot be further extended, thereby inhibiting the amplification of the wild-type template.

[0008] Furthermore, the blocking group includes at least one selected from dideoxycytidine, reverse dT, an amino group, a phosphate group, a spacer (e.g., spacer C3, spacer C5, etc.), or other groups capable of inhibiting the wild-type amplification inhibitor from extending as a primer.

[0009] Furthermore, the difference between Tm1 and Tm2 is 5-20°C, for example, about 5°C, 6°C, 7°C, 8°C, 9°C, 10°C, 11°C, 12°C, 13°C, 14°C, 15°C, 16°C, 17°C, 18°C, 19°C, 20°C, etc.

[0010] Further, the Tm1 is 60-75°C, for example, about 60°C, 61°C, 62°C, 63°C, 64°C, 65°C, 66°C, 67°C, 68°C, 69°C, 70°C, 71°C, 72°C, 73°C, 74°C, 75°C, etc.

[0011] In some embodiments, the wild-type amplification inhibitor pairs perfectly complementary with the wild-type template of the test gene.

[0012] In some embodiments, the wild-type amplification inhibitor has at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, or at least nine bases that complementarily pair with the test gene that are RNA bases.

[0013] According to the technical means of the present invention, the difference between Tm1 and Tm2 can be further increased by substituting an RNA base for a base that complementarily pairs with the base at the mutation site in the wild-type amplification inhibitor.

[0014] Furthermore, the base in the wild-type amplification inhibitory substance that corresponds to the base at the mutation site of the test gene is located in the middle, at the 5' end or at the 3' end of the wild-type amplification inhibitory substance.

[0015] Further, the wild-type amplification inhibitor is an oligonucleotide strand having a length of 20-45 bp, for example, 20 bp, 25 bp, 30 bp, 35 bp, 40 bp, 45 bp, etc.

[0016] In a second aspect, the present invention provides an amplification inhibitor mutation system, the system comprising a forward primer, a reverse primer and at least one inhibitor-probe combination, the inhibitor-probe combination comprising a probe and a wild-type amplification inhibitor according to the present invention.

[0017] The forward primer and the reverse primer are used to amplify the test gene. The binding sites of the forward primer and the reverse primer to the test gene are located upstream and downstream of the binding site of the wild-type amplification inhibitor to the test gene, respectively.

[0018] In each set of inhibitor-probe combinations, the probe binds to the test gene in competition with the wild-type amplification inhibitor, and the binding site between the probe and the test gene covers the mutation site of the test gene.

[0019] According to the technical means of the present invention, the wild-type amplification inhibitor is located between the forward primer and the reverse primer. Compared with the prior art technical means in which the wild-type amplification inhibitor competes with the amplification primer to bind to the template, the technical means of the present invention avoids the ability of the wild-type amplification inhibitor to inhibit the amplification of the wild-type allele and the effect of said competitive binding on the binding efficiency to the mutant template by primer annealing. Thereby, the technical means of the present invention further improves the sensitivity of detection for the mutant allele.

[0020] In some embodiments, the amplification inhibition mutation system includes two or more of the inhibitor-probe combinations, for example, two, three, four, five, six, seven, eight, nine, ten, etc. The number of inhibitor-probe combinations can be determined according to the number and positions of mutation sites in the test gene, etc. This allows simultaneous detection of multiple mutation sites in the test gene in one detection system.

[0021] Furthermore, the length of the probe is 15-40 bp.

[0022] Furthermore, the probe is a fluorescent probe.

[0023] Furthermore, the 5' end of the fluorescent probe is modified with one or more fluorescent groups selected from FAM, HEX, ROX, TAMRA, Texas RED, CY5, Cy3, TET, JOE, and VIC, and the 3' end of the molecular beacon probe is provided with one or more quenching groups selected from BHQ1, BHQ2, Dabcy1, TAMRA, MGB, and ECLIPSE.

[0024] In some embodiments, the probe is a molecular beacon probe.

[0025] In some embodiments, the probe has one or two of the following modifications: peptide nucleic acid (PNA), locked nucleic acid (LNA).

[0026] Furthermore, the probe and the wild-type amplification inhibitor have overlapping bases, and the number of overlapping bases is 90%, 80%, 70%, 60%, 50%, 40%, 30%, or 20% or more of the total number of bases in the probe.

[0027] Furthermore, the probe has a Tm value at which it specifically binds to a wild-type template of a test gene, where Tm3 is greater than Tm3 and Tm2 is greater than Tm2.

[0028] In one embodiment, the difference between Tm1 and Tm3 is 5-15°C, for example, 5°C, 6°C, 7°C, 8°C, 9°C, 10°C, 11°C, 12°C, 13°C, 14°C, 15°C, etc.

[0029] Furthermore, the forward primer and the reverse primer each have a Tm value for specifically binding to a test gene of Tm 4-F and Tm 4-R and Tm1>Tm 4-F , Tm 4-R and further, 55°C < Tm 4-F , Tm 4-R ≦70°C.

[0030] In a third aspect of the present invention, there is provided a nucleic acid detection system comprising the amplification inhibition mutation system according to the second aspect of the present invention, in addition to a PCR buffer, a DNA polymerase and dNTPs.

[0031] In addition, the PCR buffer contains (NH4)2SO4, MgCl2, KCl, Tris-HCl, etc.

[0032] In some embodiments, in the PCR buffer, the concentration of (NH4)2SO4 is 5-20 mM, such as 5 mM, 10 mM, 15 mM, 20 mM, etc. The concentration of MgCl2 is 2-3 mM, such as 2 mM, 2.5 mM, 3 mM, etc. The concentration of KCl is 5-20 mM, such as 5 mM, 10 mM, 15 mM, 20 mM, etc. The concentration of Tris-HCl is 10-30 mM, such as 10 mM, 20 mM, 30 mM, etc. The pH value of the Tris-HCl is 8-8.5.

[0033] Furthermore, in the nucleic acid detection system, the final concentration of the wild-type amplification inhibitor is 0.02-0.5 μM, for example, 0.02 μM, 0.05 μM, 0.1 μM, 0.15 μM, 0.2 μM, 0.25 μM, 0.3 μM, 0.35 μM, 0.4 μM, 0.45 μM, 0.5 μM, etc.

[0034] Furthermore, in the nucleic acid detection system, the final concentration of the probe is 0.02-0.5 μM, for example, 0.02 μM, 0.05 μM, 0.1 μM, 0.15 μM, 0.2 μM, 0.25 μM, 0.3 μM, 0.35 μM, 0.4 μM, 0.45 μM, 0.5 μM, etc.

[0035] Furthermore, in the nucleic acid detection system, the final concentrations of the forward primer and reverse primer are 0.02 to 0.5 μM, for example, 0.02 μM, 0.05 μM, 0.1 μM, 0.15 μM, 0.2 μM, 0.25 μM, 0.3 μM, 0.35 μM, 0.4 μM, 0.45 μM, 0.5 μM, etc.

[0036] Furthermore, in the nucleic acid detection system, the final concentration of dNTPs is 0.2-0.5 mM, for example, 0.2 mM, 0.3 mM, 0.4 mM, 0.5 mM, etc.

[0037] Furthermore, the DNA polymerase does not have 5' to 3' exonuclease activity and 3' to 5' endonuclease activity.

[0038] In a fourth aspect of the present invention, there is provided a nucleic acid detection kit comprising the nucleic acid detection system according to the third aspect of the present invention.

[0039] In a fifth aspect of the present invention, there is provided a nucleic acid detection method comprising the steps of adding a template of a test gene to a nucleic acid detection system according to the present invention, and carrying out PCR amplification and melting curve detection.

[0040] The PCR amplification reaction process includes denaturation at 92-98°C for 10-30s, first annealing under Tm1 temperature conditions for 10-30s, and then annealing at Tm 4-F or Tm 4-R The reaction involves a second annealing at a temperature above Tm3 and below Tm1 for 10-30 s, followed by extension for 0.5-2 min at a temperature above Tm3 and below Tm1, and a total of 45-55 cycles of the above reaction.

[0041] Furthermore, the PCR amplification further includes a step of pre-denaturing at 92-98° C. for 1-10 minutes prior to the first denaturation.

[0042] In some embodiments, the PCR amplification is an asymmetric PCR amplification.

[0043] Furthermore, the reaction process of the melting curve detection includes: 92-98°C, 1 min; Tm1 temperature condition, 1 min; 45-50°C, 1 min; heating to 85°C at a heating rate of 0.01-0.05°C / s, and collecting fluorescent signals during the heating process.

[0044] In some embodiments, the fluorescent signal is collected every 1° C. during the temperature ramp.

[0045] Compared with the prior art, the technical solutions of the present invention have the following beneficial effects:

[0046] (1) The wild-type amplification inhibitor provided by the present invention can enrich low-abundance mutant DNA in large quantities without affecting the amplification efficiency of heterologous templates, thereby improving the detection specificity and sensitivity, and meeting the practical needs of clinical detection of mutant genes. (2) The probes provided by the present invention can distinguish the mutation types of the amplified low-abundance mutant DNA by melting curve detection, and can be labeled with fluorescent groups of different wavelengths to realize multi-channel, multi-target, single-tube detection. [Brief description of the drawings]

[0047] Various other benefits and advantages will become apparent to those skilled in the art upon reading the following detailed description of the preferred embodiments. The drawings are only for the purpose of illustrating the preferred embodiments and are not to be construed as limiting the invention.

[0048] [Figure 1] 1 shows the detection results of FAM channel melting curves in Example 1. [Diagram 2] 1 shows the detection results of the VIC channel melting curve in Example 1. [Diagram 3] 1 shows the detection results of the Texas RED channel melting curve in Example 1. [Figure 4] 1 shows the detection results of the CY5 channel melting curve in Example 1. [Diagram 5] 1 shows the results of FAM channel melting curve detection in Example 2, where the 95A site is mutated to 95G. [Figure 6] 1 shows the results of the detection of the Texas RED melting curve in Example 2. Here, the 1376G site is mutated to 1376T. [Figure 7] 1 shows the results of the detection of the Texas RED melting curve in Example 2. In this case, the 1388G site is mutated to 1388A. [Figure 8] 1 shows the detection results of the BRAF gene mutation melting curve in Example 3. [Figure 9] 1 shows the detection results of the CYP2C9-CC melting curve detected by the Texas channel in Example 4. [Figure 10] 1 shows the detection results of the CYP2C9-AA type melting curve detected by the Texas channel in Example 4. [Figure 11] 1 shows the detection results of a CYP2C9-CA type melting curve detected by the Texas channel in Example 4. [Figure 12] 1 shows the detection results of the VKROC1-GG melting curve detected by the CY5 channel in Example 4. [Figure 13] 1 shows the detection results of the VKROC1-AA type melting curve detected by the CY5 channel in Example 4. [Figure 14] 1 shows the detection results of the VKROC1-GA melting curve detected by the CY5 channel in Example 4. [Figure 15] 13 shows the detection results of the melting curve of HPV16 detected by the Texas channel in Example 5. [Figure 16] 13 shows the detection results of the melting curve of HPV18 detected by the Texas channel in Example 5. [Figure 17] 13 shows the detection results of the melting curve of HPV31 detected by the Texas channel in Example 5. [Figure 18] 13 shows the detection results of the melting curve of HPV33 detected by the Texas channel in Example 5. [Figure 19] 13 shows the results of melting curve detection of HPV35 using the FAM channel in Example 5. [Figure 20] 13 shows the detection results of the melting curve of HPV39 detected by the FAM channel in Example 5. [Figure 21] 13 shows the results of melting curve detection of HPV45 using the FAM channel in Example 5. [Figure 22]13 shows the results of melting curve detection of HPV51 using the FAM channel in Example 5. [Figure 23] 1 shows the results of melting curve detection of HPV52 detected by the CY5 channel in Example 5. [Figure 24] 1 shows the results of melting curve detection of HPV53 detected by the CY5 channel in Example 5. [Diagram 25] 1 shows the results of melting curve detection of HPV56 detected by the CY5 channel in Example 5. [Figure 26] 1 shows the results of melting curve detection of HPV58 detected by the CY5 channel in Example 5. [Figure 27] 13 shows the results of melting curve detection of HPV59 using the VIC channel in Example 5. [Figure 28] 13 shows the detection results of the melting curve of HPV66 detected by the VIC channel in Example 5. [Figure 29] 13 shows the detection results of the melting curve of HPV68 detected by the VIC channel in Example 5. [Diagram 30] 13 shows the results of melting curve detection of HPV73 using the VIC channel in Example 5. [Diagram 31] 13 shows the detection results of the melting curve of HPV82 detected by the VIC channel in Example 5. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0049] Hereinafter, exemplary embodiments of the present disclosure will be described in detail with reference to the drawings. Although exemplary embodiments of the present disclosure are shown in the drawings, it should be understood that the present disclosure can be realized in various forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to better understand the present disclosure and to fully convey the scope of the present disclosure to those skilled in the art.

[0050] The present invention provides a wild-type amplification inhibitor (hereinafter abbreviated as "Blocker"), an amplification inhibition mutation system, and a corresponding nucleic acid detection system and detection method.

[0051] The Blocker provided by the present invention has a Tm value (Tm1) for specific binding to a wild-type template of a test gene that is greater than the Tm value (Tm2) for specific binding to the mutant template thereof, and has a blocking group at the 3' end. In the amplification inhibition mutation system provided by the present invention, the probe binds to the template of the test gene in competition with the Blocker. The probe has a Tm value for specific binding to a wild-type template of Tm3 (Tm1>Tm3>Tm2). The forward primer and reverse primer each have a Tm value for specific binding to a test gene that is greater than Tm 4-F and Tm 4-R (Tm1>Tm 4-F , Tm 4-R ).

[0052] When only wild-type templates were present in the reaction system, the first annealing was performed under the Tm1 temperature condition. Blocker preferentially bound to the wild-type allele and showed a perfect complementarity of Tm 4-F or Tm 4-R When the second annealing was performed under temperature conditions, the amplification primer complementarily bound to the test template and extended under temperature conditions above Tm3 and below Tm1. When the amplification primer extended to the Blocker position, the polymerase used in the system did not have 5'→3' exonuclease activity, and the Blocker could not be cleaved by the enzyme, causing the extension to stop, thereby inhibiting the amplification of the wild-type template.

[0053] However, in actual detection, the blocker can usually only suppress the amplification of wild-type alleles to a certain extent, and if the initial amount of wild-type DNA is too high, false positive amplification will occur. To overcome this defect, the present invention performs subsequent melting curve detection using a probe. When performing melting curve detection, the initial temperature condition is 92-98°C, under which the PCR product is completely denatured into a single strand. When the temperature drops to Tm1, the blocker in the system competes with the probe and preferentially binds to the wild-type amplification product that can exist. As a result, under the subsequent temperature drop condition, the wild-type amplification product that can exist in the system is significantly reduced, so that the probe almost entirely binds to the mutant amplification product. This effectively reduces the interference caused by false positive amplification, and improves the accuracy of the melting curve detection result.

[0054] When only the mutant template is present in the reaction system, during extension at a temperature above Tm3 and below Tm1, neither the blocker nor the probe can bind to the template, and the amplification primer binds to the template in a perfectly complementary manner and is extended, resulting in a large amount of amplified mutant template.

[0055] When wild-type and mutant templates were simultaneously present in the system, the first annealing was performed under the Tm1 temperature condition. Blocker preferentially bound to the wild-type allele and showed a perfect complementarity with the Tm 4-F or Tm 4-R When the second annealing was performed under the temperature conditions, the amplification primers bound perfectly complementary to both the wild-type and mutant templates and extended, and terminated when the wild-type template was extended to the blocker position, whereas the mutant template was fully extended to complete the amplification, thereby enriching the low-abundance mutant DNA.

[0056] Working Example When an amino acid mutation occurs at codons 507-533 of the gene (rpoB) encoding the RNA polymerase β subunit of Mycobacterium tuberculosis, resistance to the first-line drug rifampicin (RIF) occurs. In this example, the ability of wild-type amplification inhibitors to detect low-abundance mutations was investigated using this mutation region as an example. The specific procedure is as follows.

[0057] (1) Design and synthesis of primers, probes, blockers, and wild-type and mutant plasmids

[0058] Using software such as Primer Premier 6.0, DNAMAN, and ClustalX, primers were designed based on the rifampicin resistance mutation core region in the rpoB gene of M. tuberculosis, and four probes were designed to completely cover this mutation core region with an amplified fragment length of 145 bp. Four blockers were designed to compete with the probes to inhibit the amplification of the wild-type template.

[0059] The upstream and downstream primer sequence regions were selected to synthesize the wild-type plasmid, and the 511CCG, 516GTC, 526TAC and 531TTG mutant codons were selected based on clinical high frequency mutations to synthesize the mutant plasmid.

[0060] The designed primers (F-rpoB, R-rpoB), probes (P1-rpoB, P2-rpoB, P3-rpoB, P4-rpoB) and blockers (B1-rpoB, B2-rpoB, B3-rpoB, B4-rpoB) were all compared for homology by BLAST to ensure their specificity. All primers, probes and plasmids were synthesized by a specialized company. The specific sequences are shown in Table 1.

[0061] Table 1: Primer, probe, blocker and plasmid sequences. [Table 1]

[0062] In Table 1, underlines indicate LNA modified bases and squares indicate RNA bases.

[0063] The wild-type and mutant plasmids were each 4 The concentration of the template was adjusted to copies / μl. 10% group: 10 μl of 10 4 Copies / μl of the mutant plasmid was mixed with 90 μl of the wild-type plasmid at the same concentration and shaken to mix uniformly. 5% group: 5 μl of 10 4 Copies / μl of the mutant plasmid was taken and mixed with 95 μl of the wild-type plasmid at the same concentration, and the mixture was shaken to mix uniformly. 1% group: Take 10 μl of the mixture from the 10% group and add 90 μl of the 10% group. 4 The mixture was mixed with copies / μl of the wild-type plasmid and shaken to mix uniformly. 0.5% group: Take 10 μl of the mixture from the 5% group and add 90 μl of the 10% 4 The mixture was mixed with copies / μl of the wild-type plasmid and shaken to mix uniformly. 0.1% group: Take 10 μl of the mixture from the 1% group and add 90 μl of 10 4 The mixture was mixed with copies / μl of the wild-type plasmid and shaken to mix uniformly. 0.01% group: Take 10 μl of the mixture from the 0.1% group and add 90 μl of the 10 4 The mixture was mixed with copies / μl of the wild-type plasmid and shaken to mix uniformly. Wild-type control group: 10 4 copies / μl of wild-type plasmid.

[0064] (2) Construction of real-time fluorescent PCR amplification system The reaction mixture (25 μl) contained 0.2 mM dNTPs, 10 mM (NH4)2SO4, 3 mM MgCl2, 10 mM KCl, 20 mM Tris-HCl (pH 8.3), 1 U of DNA polymerase, 0.02 μM upstream primer, 0.2 μM downstream primer, 0.1 μM each of probe and blocker, 5 μl of DNA template, and the remainder was water.

[0065] (3) PCR amplification and melting curve reaction Asymmetric PCR was performed on the DNA template in a 25 μl reaction system to amplify a large amount of single-stranded DNA, which was then subjected to melting curve analysis. The specific reaction process is as follows:

[0066] The PCR amplification process was 95°C, 10 min; (50 cycles of 95°C, 15 s; 82°C, 20 s; 61°C, 15 s; 75°C, 30 s).

[0067] The process of the melting curve reaction was as follows: 95℃, 1 min; 82℃, 1 min; 45℃, 1 min, heating to 85℃ at a rate of 0.05℃ / s, and collecting the fluorescent signal every 1℃ during the heating process.

[0068] Results and Analysis The Tm value of the FAM channel wild-type control was 74.1° C.±1° C., the Tm value of the VIC channel wild-type control was 73.5° C.±1° C., the Tm value of the Texas channel wild-type control was 74.4° C.±1° C., and the Tm value of the CY5 channel wild-type control was 71.6° C.±1° C. If the difference between the melting curve result of the test sample and the Tm value of the wild-type control was ≧2° C., it was determined to be a mutant type.

[0069] When only wild-type DNA is present in the reaction system, the Blocker binds perfectly complementary to the wild-type allele, and the amplification primer binds to the DNA template and extends to the Blocker position, but cannot continue to extend, resulting in no fluorescent signal. However, in actual detection, the Blocker can usually only suppress the amplification of the wild-type allele to a certain extent, and false-positive amplification will occur if the initial amount of wild-type DNA is too high. As a result of melting curve detection, the Tm value of each fluorescent channel was consistent with the wild-type control.

[0070] When only mutant DNA is present in the reaction system, the annealing temperature between the blocker and the mutant allele (68°C) is lower than the extension temperature between the amplification primer and DNA template (75°C), so that the amplification primer and the mutant template are extended during extension at 75°C, thereby producing a fluorescent signal.

[0071] The results, as shown in Figure 1-4, show that the wild melting peak appears in the wild control and no melting peak appears in the negative control, indicating that the detection system can effectively amplify without contamination. Only the mutant peak appears in the pure mutant type, while the wild peak and mutant peak appear simultaneously in the 5%, 1%, 0.5%, 0.1% and 0.01% groups. In Figure 1-4, 1 is the melting peak of the wild control, 2 is the melting peak of the 100% mutant sample, 3 is the melting peak of the 5% mutant content mixed sample, 4 is the melting peak of the 1% mutant content mixed sample, 5 is the melting peak of the 0.5% mutant content mixed sample, 6 is the melting peak of the 0.1% mutant content mixed sample, 7 is the melting peak of the 0.01% mutant content mixed sample, and 8 is the melting peak of the negative control.

[0072] Working Example Glucose-6-phosphate dehydrogenase (G6PD) is present in various tissues and cells of the human body and is involved in the pentose phosphate pathway. Glucose-6-phosphate dehydrogenase deficiency is a sex-linked incomplete dominant genetic disorder. G6PD deficiency significantly increases the risk of neonatal jaundice, and if left untreated or not treated in a timely manner, it may lead to kernicterus, permanent neurological damage or hemolytic anemia. Research has shown that mutations in different sites of the G6PD gene reduce G6PD enzyme activity to different degrees. Among them, 95A>G, 1376G>T and 1388G>A are the three most common types of gene mutations in our country (accounting for 70%). At present, in order to achieve the goal of giving birth to children of good quality and raising them in a good environment, G6PD testing has become one of the clinical test items, as well as premarital testing, prenatal testing and neonatal testing. Since the ratio of mutant nucleic acid to wild-type nucleic acid may differ in actual clinical samples, in order to effectively amplify low-abundance mutant nucleic acid in mixed samples, the present embodiment provides a method for detecting samples with low abundance of G6PD mutants, thereby improving the detection sensitivity.

[0073] In this example, a gene mutation in glucose-6-phosphate dehydrogenase (G6PD) was detected. The specific procedure is as follows.

[0074] (1) Design and synthesis of primers, probes and blockers Using Primer Premier 6.0, DNAMAN, ClustalX and other software, primers were designed for the mutation regions of the G6PD gene 95A>G, 1376G>T, and 1388G>A, respectively, and the lengths of the amplified fragments were 141bp and 150bp, respectively. Two probes were designed to completely cover this mutation core region. Two blockers that compete with the probes were correspondingly designed to inhibit the amplification of the wild-type template.

[0075] Table 2: Primer, probe and blocker sequences [Table 2]

[0076] In Table 2, underlines indicate LNA modified bases and squares indicate RNA bases.

[0077] (2) Synthesis of wild-type and mutant G6PD gene plasmids

[0078] Since it is difficult to obtain actual clinical samples, the plasmid was artificially synthesized and verified. The G6PD-M wild-type plasmid was synthesized by a specialized company. This plasmid contained the original sequence of 95A, 1376G, and 1388G unmutated, and pUC-GW-Amp was used as the vector. The 95A>G, 1376G>T, and 1388G>A point mutation plasmids were synthesized and named G6PD-95G-M, G6PD-1376T-M, and G6PD-1388A-M, respectively. The G6PD-M wild-type and mutant plasmid sequences are shown in Table 3.

[0079] Table 3: G6PD wild-type and mutant plasmid sequences [Table 3]

[0080] (3) Real-time fluorescent PCR amplification system The reaction mixture (25 μl) contained 0.2 mM dNTPs, 10 mM (NH4)2SO4, 3 mM MgCl2, 10 mM KCl, 20 mM Tris-HCl (pH 8.3), 2 μl DNA polymerase, 0.02 μM upstream primer, 0.2 μM downstream primer, 0.1 μM each of probe and blocker, 5 μl DNA template, and the remainder was water.

[0081] (4) PCR amplification and melting curve reaction process The PCR amplification process consisted of denaturation at 95°C for 10 min (50 cycles of 95°C, 15 s; 81°C, 20 s; 63°C, 15 s; 72°C, 30 s).

[0082] The process of the melting curve reaction was as follows: 95℃, 1 min; 81℃, 1 min; 45℃, 1 min, heating to 85℃ at a rate of 0.05℃ / s, and collecting the fluorescent signal every 1℃ during the heating process.

[0083] (5) Results and Analysis The plasmid nucleic acid in which the 95A site of the G6PD gene is mutated to 95G (G6PD-95G-M, FAM channel, FIG. 5), the plasmid nucleic acid in which the 1376G site of the G6PD gene is mutated to 1376T (G6PD-1376T-M, Texas RED channel, FIG. 6), and the plasmid nucleic acid in which the 1388G site of the G6PD gene is mutated to 1388A (G6PD-1388A-M, Texas RED channel, FIG. 7) provided in this Example (2) were detected. The results are shown in FIG. 5-7. In FIG. 5, the first line is the melting curve of the 95A wild type, with a Tm value of 77° C.; the second line is the melting curve of the mutant type in which the 95A site is mutated to 95G, with a Tm value of 62° C.; the third line is the melting curve of the detection result of a mixed sample in which the proportion of the 95G mutant type is 50%, and the fourth line is the negative control. In Figure 6, line 1 is the melting curve of the 1376G wild type, with a Tm value of 77.6 ° C, line 2 is the melting curve of the 1376T mutant type, with a Tm value of 64 ° C, line 3 is the melting curve of the mixed sample with a 50% 95G mutant type, and line 4 is the negative control. In Figure 7, line 1 is the melting curve of the 1388G wild type, with a Tm value of 77.6 ° C, line 2 is the melting curve of the 1388A mutant type, with a Tm value of 68.5 ° C, line 3 is the melting curve of the mixed sample with a 50% 95G mutant type, and line 4 is the negative control.

[0084] Working Example The BRAF gene is an oncogene located on the long arm of chromosome 7, which contains 18 exons and can encode the B-RAF protein containing 783 amino acids. It is an important transduction factor in the EGFR pathway RAS / RAF / MEK / MRK / MAPK and is involved in various physiological processes such as cell proliferation, differentiation, and apoptosis. Studies have shown that BRAF gene mutations exist in various malignant tumors, including non-small cell lung cancer and colorectal cancer, among which the V600E mutation in exon 15 is the most common, accounting for more than 90%. This mutation causes the abnormal activation of the B-RAF protein, which leads to the failure of patients to undergo treatment with EGFR-TKI drugs and EGFR monoclonal antibodies. According to the 2010 edition of the Chinese "Oncology Clinical Practice Guidelines," it is recommended that patients with non-small cell lung cancer who have normal detection results for the KRAS gene should be further tested for the mutation status of the B-RAF gene to guide targeted drug treatment plans. During the process of cancer development and progression, a small number of cancer cells will fall off from primary tumor tissue and enter into blood circulation, and when DNA is extracted from human peripheral blood, the proportion of mutant genes in the total DNA extracted will be low.Therefore, the technical means provided by the present invention can realize the enrichment of low-abundance B-RAF mutant genes and the detection of mutant bases.

[0085] In this example, human BRAF gene mutations were detected. The specific procedure is as follows.

[0086] (1) Design and synthesis of primers, probes and blockers Primers were designed for the BRAF gene 1799T>A mutation region using Primer Premier 6.0, DNAMAN, ClustalX, and other software, and the length of the amplified fragment was 200 bp. One probe was designed to completely cover this mutation core region. One blocker was designed to compete with the probe to inhibit the wild-type template.

[0087] Table 4: BRAF primers, probes and Blocker sequences [Table 4]

[0088] In Table 4, single underlines indicate LNA modified bases and underlines indicate RNA bases.

[0089] (2) Synthesis of wild-type and mutant BRAF gene plasmids

[0090] Since it is difficult to obtain actual clinical samples, the plasmids were artificially synthesized and verified. The BRAF-1799T-M wild-type plasmid and the BRAF-1799A-M mutant plasmid were synthesized by a specialized company. pUC-GW-Amp was used as the vector. The wild-type and mutant plasmid sequences are shown in Table 4.

[0091] Table 5: BRAF wild-type and mutant plasmid sequences. [Table 5]

[0092] (3) Real-time fluorescent PCR amplification system The reaction mixture (25 μl) contained 0.2 mM dNTPs, 10 mM (NH4)2SO4, 3 mM MgCl2, 10 mM KCl, 20 mM Tris-HCl (pH 8.3), 2 μl DNA polymerase, 0.02 μM upstream primer, 0.2 μM downstream primer, 0.1 μM probe and blocker, 5 μl DNA template, and the remainder was water.

[0093] (4) PCR amplification and melting curve reaction process The PCR amplification process was as follows: denaturation at 95°C for 10 min; (50 cycles of 95°C, 15 s; 80°C, 20 s; 63°C, 15 s; 70°C, 30 s).

[0094] The process of the melting curve reaction was as follows: 95℃, 1 min; 80℃, 1 min; 45℃, 1 min, heating to 85℃ at a rate of 0.05℃ / s, and collecting the fluorescent signal every 1℃ during the heating process.

[0095] (5) Results and Analysis The BRAF-1799T-M wild type plasmid, the BRAF-1799A-M mutant type plasmid, and the mixed sample with the mutant type ratio of 1‰ provided in this embodiment were detected. The results are shown in Figure 8. In Figure 8, line 1 is the melting curve of the BRAF-1799T-M wild type, with a Tm value of 70°C, line 2 is the melting curve of the BRAF-1799A-M mutant type, with a Tm value of 62°C, line 3 is the mixed sample with the BRAF-1799A-M mutant type ratio of 1‰, and line 4 is the negative control.

[0096] Working Example Warfarin is a clinical drug commonly used in the prevention and treatment of thromboembolic diseases, and is a coumarin-based anticoagulant. Overdosing on warfarin can cause bleeding, and its dosage is influenced by many factors (mainly genetics and environment), and there are large individual differences in clinical effects and side effects, making it difficult to control the dosage, so the dosage must be determined according to the individual.

[0097] A large amount of studies have shown that VKORC1 promoter polymorphism (-1639G / A) and CYP2C9 (1075A / C) mutations are important factors affecting warfarin dosing.

[0098] Therefore, in this embodiment, the mutations of CYP2C9 (rs1057910) and VKORC1 (rs9923231) genes were detected. The specific procedure is as follows:

[0099] Four primers, two probes, and two blocker sequences were designed for the region containing CYP2C9 (rs1057910) and VKORC1 (rs9923231). These primers can specifically amplify two genes, respectively. In the amplification target region, probes were designed for the mutant type, and these probes can specifically bind to the mutant target sequence, and subtypes were detected based on the difference in melting curve Tm value. In addition, to improve the detection sensitivity of the mutant type, a blocker was designed for the wild type, which can enrich the mutant sample to a certain extent. All sequences were synthesized by a specialized company.

[0100] Table 6: Primer, probe and blocker sequences [Table 6]

[0101] In Table 6, underlines indicate LNA modified bases and squares indicate RNA bases.

[0102] (2) Construction of real-time fluorescent PCR amplification system The reaction mixture (25 μl) contained 0.3 mM dNTPs, 8 mM (NH4)2SO4, 3 mM MgCl2, 10 mM KCl, 20 mM Tris-HCl (pH 8.3), 2 μl DNA polymerase, 0.02 μM upstream primer, 0.2 μM downstream primer, 0.1 μM each of probe and blocker, 5 μl DNA template, and the remainder was water.

[0103] (3) PCR amplification and melting curve reaction Asymmetric PCR was performed on the DNA template in a 25 μl reaction system to amplify a large amount of single-stranded DNA, which was then subjected to melting curve analysis. The specific reaction process is as follows:

[0104] The PCR amplification process was 95°C, 10 min; (50 cycles of 95°C, 15 s; 75°C, 20 s, 61°C, 15 s; 70°C, 30 s).

[0105] The process of the melting curve reaction was as follows: 95℃, 1 min; 75℃, 1 min; 45℃, 1 min, heating to 90℃ at a rate of 0.05℃ / s, and collecting the fluorescent signal every 1℃ during the heating process.

[0106] Results and Analysis CYP2C9 was detected by the Texas channel, and the Tm value of the CC control was 69.8°C ± 1°C (Figure 9), the Tm value of the AA control was 65.4°C ± 1°C (Figure 10), and the AC control showed a double peak with a high peak and a low peak, with the respective Tm values ​​being 65.6°C ± 1°C and 70.2°C ± 1°C (Figure 11). VKROC1 was detected by the CY5 channel, and the Tm value of the GG control was 76.4°C ± 1°C (Figure 12), the Tm value of the AA control was 69.8°C ± 1°C (Figure 13), and the GA type showed a double peak with a high peak and a low peak, with the respective Tm values ​​being 70.2°C ± 1°C and 75.6°C ± 1°C (Figure 14). If the difference between the melting curve result of the test sample and the Tm value of the control is ≦1°C, it is determined to be a subtype.

[0107] Working Example Human papilloma virus (HPV) is a double-stranded DNA virus whose genome encodes three regions: the early region (E region), the late region (L region), and the upstream regulatory region (LCR).

[0108] More than 100 HPV types have been discovered so far. Among them, the common low-risk types include HPV6, HPV11, HPV42, HPV43, and HPV44. Low-risk types generally do not cause cancer. The common high-risk types include HPV16, HPV18, HPV31, HPV33, HPV35, HPV39, HPV49, HPV51, HPV52, HPV53, HPV56, HPV58, HPV59, HPV66, HPV68, HPV73, and HPV82. According to a large amount of research, the development of female reproductive system malignancies, such as cervical cancer, is associated with repeated infection with high-risk HPV viruses.

[0109] In this example, HPV subtypes were detected. The specific procedure is as follows. Three upstream primers and one downstream primer were designed for the relatively conserved L1 gene of HPV. These primers can specifically amplify a fragment of approximately 150 bp of the HPV L1 gene. Seventeen specific probes were designed in the amplification target region. These probes can specifically bind to the HPV amplified fragment of the corresponding subtype. Subtypes were detected by the difference in the Tm value of the melting curve. In addition, five types of blockers were designed for common low-risk HPV types (6, 11, 42, 43, 44) to improve the detection sensitivity of low-abundance samples. This allows low-abundance samples to be enriched to a certain extent.

[0110] Sensitivity and specificity analyses Obtaining positive standards with different gradients: Plasmid powder synthesized by a professional company is added with water labeled by volume, and after measuring the nucleic acid concentration, the copy number of the plasmid is calculated, and a plasmid standard is constructed by gradient dilution, with a concentration of 10 7 copies / μl, 10 6 copies / μl, 10 5 copies / μl, 10 4 copies / μl, 10 3 copies / μl, 10 2Plasmid standards of different subtypes were obtained at copies / μl, 10 copies / μl and 1 copies / μl.

[0111] Negative control: genomic DNA from a normal healthy individual was diluted to 10 ng / μl in a buffer containing 0.9% NaCl, 1 mmol / L EDTA, 10 mmol / L Tris-HCl, and 0.1% Triton X-100.

[0112] Preparation of low abundance mixed sample: A plasmid mixture containing one high-risk type and five low-risk types was prepared using the dilution solution used above, with the final concentration of each high-risk type plasmid being 10 copies / μl.

[0113] (4) Detection of low-abundance mixed samples The control group was one without addition of blocker, and it was found that 10 copies / μl of plasmid could not be detected without addition of blocker, whereas it could be detected in the experimental group with addition of blocker, indicating that the method described in the present invention can improve the detection rate of low-sensitivity samples.

[0114] In this example, the subtype of high-risk HPV was identified. The specific procedure is as follows. (1) Design and synthesis of primers, probes and blockers Using software such as Primer Premier 6.0, DNAMAN, and ClustalX, we designed common primers (including three upstream primers and one downstream primer, with an amplified fragment length of approximately 145 bp) based on the relatively conserved L1 gene of HPV. We designed 17 specific probes corresponding to each subtype. We correspondingly designed five blockers that compete with the probes to inhibit the amplification of five common low-risk type templates.

[0115] Designed primers (F1-HPVL1, F2-HPVL1, F3-HPVL1, R-HPVL1) and probes (P16-HPVL1, P18-HPVL1, P31-HPVL1, P33-HPVL1, P35-HPVL1, P39-HPVL1, P45-HPVL1, P51-HPVL1, P52-HPVL1, P53-HPVL1, P56-HPVL1, P57-HPVL1, P59-HPVL1, P60-HPVL1, P61-HPVL1, P62-HPVL1, P63-HPVL1, P64-HPVL1, P65-HPVL1, P66-HPVL1, P67-HPVL1, P68-HPVL1, P69-HPVL1, P70-HPVL1, P71-HPVL1, P72-HPVL1, P73-HPVL1, P74-HPVL1, P75-HPVL1, P76-HPVL1, P77-HPVL1, P78-HPVL1, P79-HPVL1, P80-HPVL1, P81-HPVL1, P82-HPVL1, P83-HPVL1, P84-HPVL1, P85-HPVL1, P86-HPVL1, P87-HPVL1, P88-HPVL1, P89-HPVL1, P90-HPVL1, P91-HPVL1, P92-HPVL1, P93-HPVL1, P94-HPVL1, P95-HPVL1, P96-HPVL1, P97-HPVL1, P98-HPVL1, P99 1, P58-HPVL1, P59-HPVL1, P66-HPVL1, P68-HPVL1, P73-HPVL1, P82-HPVL1) and Blocker (B6-HPVL1, B11-HPVL1, B42-HPVL1, B43-HPVL1, B44-HPVL1) were compared for homology by BLAST to ensure their specificity. Since it is difficult to obtain actual clinical samples, the plasmids were artificially synthesized and verified. They were synthesized by a specialized company. The specific sequences are shown in Table 7.

[0116] Table 7: Primer, probe and blocker sequences [Table 7] TIFF2025512760000009.tif130170

[0117] In Table 7, underlines indicate LNA modified bases and squares indicate RNA bases.

[0118] (2) Construction of real-time fluorescent PCR amplification system

[0119] The reaction mixture (25 μl) contained 0.4 mM dNTPs, 10 mM (NH4)2SO4, 5 mM MgCl2, 15 mM KCl, 20 mM Tris-HCl (pH 8.3), 3.5 μl DNA polymerase, 0.02 μM upstream primer, 0.2 μM downstream primer, 0.1 μM each of probe and blocker, 5 μl DNA template, and the remainder was water.

[0120] (3) PCR amplification and melting curve reaction

[0121] Asymmetric PCR was performed using a 25 μl reaction system with a DNA template to amplify a large amount of single-stranded DNA, which was then subjected to melting curve analysis. The specific reaction process is as follows:

[0122] The PCR amplification process was 95°C, 10 min; (50 cycles of 95°C, 15 s; 75°C, 20 s, 61°C, 15 s; 70°C, 30 s).

[0123] The process of the melting curve reaction was as follows: 95℃, 1 min; 75℃, 1 min; 45℃, 1 min, heating to 85℃ at a rate of 0.05℃ / s, and collecting the fluorescent signal every 1℃ during the heating process.

[0124] Results and Analysis The Tm value of the HPV16 control detected by the Texas channel was 75.3°C ± 1°C, the Tm value of the HPV18 control was 70.5°C ± 1°C, the Tm value of the HPV31 control was 65.3°C ± 1°C, and the Tm value of the HPV33 control was 60.2°C ± 1°C. The Tm value of the HPV35 control detected by the FAM channel was 86.9°C ± 1°C, the Tm value of the HPV39 control was 79.5°C ± 1°C, the Tm value of the HPV45 control was 63.3°C ± 1°C, and the Tm value of the HPV51 control was 70.9°C ± 1°C. The Tm value of the HPV52 control detected by the CY5 channel was 60.4°C ± 1°C, the Tm value of the HPV53 control was 65.4°C ± 1°C, the Tm value of the HPV56 control was 70.1°C ± 1°C, and the Tm value of the HPV58 control was 76.2°C ± 1°C. The Tm value of the HPV59 control detected by the VIC channel was 71.7° C.±1° C., the Tm value of the HPV66 control was 66.90° C.±1° C., the Tm value of the HPV68 control was 75.6° C.±1° C., the Tm value of the HPV73 control was 82.5° C.±1° C., and the Tm value of the HPV82 control was 61.8° C.±1° C. If the difference between the melting curve result of the test sample and the Tm value of the control is ≦2° C., it is determined to be HPV of this subtype.

[0125] The above description is merely a preferred embodiment of the present invention, and does not limit the scope of protection of the present invention. Any changes or replacements that can be easily thought of by a person skilled in the art within the scope of the technology disclosed in the present invention should be included in the scope of protection of the present invention. Therefore, the scope of protection of the present invention is subject to the scope of the claims.

Claims

1. A wild-type amplification inhibitor, The wild-type amplification inhibitor has a Tm value for specifically binding to the wild-type template of the test gene of １ and the wild-type amplification inhibitor has a Tm value for specifically binding to the mutant template of the test gene of ２ and Tm １ >Tm ２ wherein the wild-type amplification inhibitor has a blocking group at its 3' end.

2. The wild-type amplification inhibitor pairs perfectly complementary with a wild-type template of a test gene, Preferably, the wild-type amplification inhibitor has at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, or at least nine bases that complementarily pair with the test gene, each of which is an RNA base; Preferably, the Tm １ and Tm ２ The difference is 5-20°C. Preferably, the Tm １ is 65-87°C, The wild-type amplification inhibitor according to claim 1, wherein the blocking group preferably contains at least one selected from the group consisting of dideoxycytidine, reverse dT, an amino group, a phosphate group, and a spacer.

3. 1. An amplification inhibition mutation system, comprising: The amplification inhibition mutation system includes a forward primer, a reverse primer, and at least one inhibitor-probe combination, the inhibitor-probe combination including a probe and the wild-type amplification inhibitor according to claim 1 or 2; the forward primer and the reverse primer are used for amplifying the test gene, and the binding positions of the forward primer and the reverse primer to the test gene are located upstream and downstream of the binding position of the wild-type amplification inhibitor to the test gene, respectively; An amplification inhibition mutation system, characterized in that in each inhibitor-probe combination, the probe binds to the test gene in competition with the wild-type amplification inhibitor, and the binding site between the probe and the test gene covers the mutation site of the test gene.

4. the probe and the wild-type amplification inhibitor have an overlap of bases, and the number of overlapping bases is 90%, 80%, 70%, 60%, 50%, 40%, 30%, or 20% or more of the total number of bases of the probe; Preferably, the probe has a Tm value for specifically binding to a wild-type template of a test gene of T ３ and Tm １ >Tm ３ >Tm ２ and Preferably, the Tm １ and Tm ３ The amplification inhibition mutation system according to claim 3, wherein the difference is 5-15°C.

5. the probe is a fluorescent probe, Preferably, the 5' end of the fluorescent probe is modified with one or more fluorescent groups selected from FAM, HEX, ROX, TAMRA, Texas RED, CY5, Cy3, TET, JOE, and VIC, and the 3' end of the molecular beacon probe is modified with one or more quenching groups selected from BHQ1, BHQ2, Dabcyl, TAMRA, MGB, and ECLIPSE, Preferably, the probe is a molecular beacon probe; The amplification inhibition mutation system according to claim 3, characterized in that the probe preferably has one or two of the following modifications: peptide nucleic acid (PNA), locked nucleic acid (LNA).

6. The forward primer and the reverse primer have Tm values ​​for specifically binding to the test gene of ４－Ｆ and Tm ４－Ｒ and Tm １ >Tm ４－Ｆ , Tm ４－Ｒ and Preferably, 55° C.≦Tm ４－Ｆ , Tm ４－Ｒ The amplification inhibition mutation system according to claim 3, characterized in that the temperature is ≦70° C.

7. A nucleic acid detection system comprising the amplification inhibition mutation system according to any one of claims 3 to 6, a PCR buffer, a DNA polymerase, and dNTPs.

8. The PCR buffer is (NH ４ ) ２ SO ４ , MgCl ２ , KCl, Tris-HCl, Preferably, in the nucleic acid detection system, the final concentration of the wild-type amplification inhibitor is 0.02-0.5 μM; Preferably, in the nucleic acid detection system, the final concentration of the probe is 0.02-0.5 μM; Preferably, in the nucleic acid detection system, the final concentrations of the forward primer and the reverse primer are 0.02-0.5 μM; Preferably, in the nucleic acid detection system, the final concentration of dNTPs is 0.2-0.5 mM; 8. The nucleic acid detection system according to claim 7, wherein the DNA polymerase has no 5' to 3' exonuclease activity and no 3' to 5' exonuclease activity.

9. A nucleic acid detection kit comprising the nucleic acid detection system according to claim 7 or 8.

10. A nucleic acid detection method comprising the steps of adding a template of a test gene to the nucleic acid detection system according to claim 7 and performing PCR amplification and melting curve detection, In the PCR amplification reaction process, denaturation is performed at 92-98° C. for 10-30 s, and Tm １ The first annealing was performed under temperature conditions for 10-30 s, and Tm ４－Ｆ Or Tm ４－Ｒ The second annealing was performed for 10-30 s under the temperature condition, and Tm ３ Exceeding Tm １ The reaction was then repeated for a total of 45-55 cycles, with extension for 0.5-2 min under a temperature condition below 25°C. Preferably, in the reaction process of the melting curve detection, 92-98°C, 1 min; Tm １ A method for detecting nucleic acid, characterized in that the temperature is increased to 85° C. at a temperature increase rate of 0.01-0.05° C. / s for 1 min under temperature conditions of 45-50° C. for 1 min, and a fluorescent signal is collected during the temperature increase process.

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