Nucleic acid testing kit and use thereof in nucleic acid testing

Abstract

Disclosed in the present invention are a nucleic acid testing kit and a use thereof in nucleic acid testing. The nucleic acid testing kit comprises a probe 1, a probe 2, an upstream primer 1, and an upstream primer 2; the probe 1 and the probe 2 are respectively complementary to DNA templates where two different bases of an allele are located, and first bases at the 5' ends of the probe 1 and the probe 2 are respectively the positions of two different bases at a certain site of the allele in the DNA templates; the upstream primer 1 and the upstream primer 2 are respectively complementary to the DNA templates where two different bases of the allele are located, and first bases at the 3' ends of the upstream primer 1 and the upstream primer 2 are respectively the positions of two different bases at a certain site of the allele in the DNA templates; and the Tm values of the probe 1 and the probe 2 are 8-15°C higher than the Tm values of the upstream primer 1 and the upstream primer 2. The design of occupancy overlapping of single bases of the upstream primers and the probes at the base position of a target site causes synchronous occupancy of the single bases of the primers and the probes, such that the specificity and the sensitivity are improved, and single-tube multiplex testing can be achieved.

Description

A nucleic acid detection kit and its application in nucleic acid detection Technical Field

[0001] The present invention belongs to the technical field of biological detection, and in particular relates to a nucleic acid detection kit and an application thereof in nucleic acid detection. Background Art

[0002] Recent research in human genetics has revealed associations between human genetic diversity and disease or other traits. Single nucleotide polymorphisms (SNPs), or differences in a single nucleotide site within an individual's genomic DNA, may influence not only the mechanisms of disease development but also individual responses to drugs.

[0003] Due to the clinical value of single-base polymorphism detection, a variety of related detection technologies have emerged. However, there are various problems to varying degrees that limit the widespread application of single-base polymorphism. (1) First-generation sequencing methods, such as Sanger sequencing and pyrophosphate sequencing, require expensive equipment and have complex operation steps. (2) Gene chip method has complex operation steps and requires professional equipment, which is expensive. (3) Second-generation sequencing has high cost, long cycle, and expensive equipment. (4) Fluorescence PCR method has low cost and good versatility and has been widely used in clinical testing. At the same time, a variety of improved methods have been derived based on this method, such as AMRS-PCR and melting curve method, but there are still various problems such as poor specificity, insufficient sensitivity, and complex operation. Therefore, it is urgent to develop a genetic detection method with good specificity and high sensitivity to meet the needs of clinical testing. Summary of the Invention

[0004] In response to the problems of poor specificity, insufficient sensitivity and complex operation in nucleic acid detection in the prior art, the present invention provides a nucleic acid detection kit and its application in nucleic acid detection. By designing an amplification blocking primer at the base position of the target site so that the single base occupancy of the probe overlaps, the single base of the primer and the probe are simultaneously occupied, introducing a competitive relationship between the single base binding sites of the primer and the probe, thereby ensuring high specificity and high sensitivity in the process of single base polymorphism detection.

[0005] The present invention is achieved through the following technical solutions:

[0006] A nucleic acid detection kit, characterized in that it comprises a probe 1, a probe 2, an upstream primer 1 and an upstream primer 2;

[0007] The probe 1 and probe 2 are complementary to the DNA template where two different bases of the allele are located, and the first base of the 5' end of probe 1 and probe 2 is the position of the two different bases of a certain site of the allele in the DNA template;

[0008] The upstream primer 1 and the upstream primer 2 are complementary to the DNA template where two different bases of the allele are located, respectively. The first base of the 3' end of the upstream primer 1 and the upstream primer 2 is the position of the two different bases of a certain site of the allele in the DNA template;

[0009] The T of the probe 1 and probe 2 sequences m The values ​​were 8~15℃ higher than the Tm values ​​of upstream primer 1 and upstream primer 2.

[0010] Furthermore, the first base at the 3' end of the upstream primer 1 is the same as the first base at the 5' end of the probe 1, and the first base at the 3' end of the upstream primer 2 is the same as the first base at the 5' end of the probe 2.

[0011] Furthermore, the length of the upstream primer 1 and the upstream primer 2 is 13 to 32 bp, T m The value is 55~65℃.

[0012] Furthermore, the length of the probe 1 and probe 2 is 10 to 26 bp, T m The value is 60~75℃.

[0013] Furthermore, the 3' ends of the probes 1 and 2 are modified with a quenching group, and the 5' ends are modified with a fluorescent group.

[0014] Furthermore, the quenching group is one of BHQ1, BHQ2, MGB-NFQ, BHQ3, etc.; the fluorescent group is one of FAM, VIC, CY5, CY3, ROX, JOE, TET, NED, TAMRA, etc.

[0015] In the present invention, the kit is used in nucleic acid detection.

[0016] Furthermore, the amplification system in the nucleic acid detection contains DNA polymerase, which has DNA polymerase activity and 5'-3' exonuclease activity, but no 3'-5' exonuclease activity.

[0017] During PCR, when a DNA template is present, probes 1 and 2 have a higher Tm than upstream primers 1 and 2, so probes 1 and 2 will preferentially bind to the DNA template first, followed by upstream primers 1 and 2. The nucleic acid detection principle diagrams of the present invention are shown in Figures 1 to 3. Figure 1 is a schematic diagram of nucleic acid detection for homozygous AA; Figure 2 is a schematic diagram of nucleic acid detection for homozygous GG; and Figure 3 is a schematic diagram of nucleic acid detection for heterozygous AA / GG. Combined with Figures 1 to 3, the specific mechanism of nucleic acid detection is as follows:

[0018] (1) When the first base at the 5' end of probe 1 bound to the DNA template is complementary to the base of the DNA template, the binding position is occupied by the first base at the 5' end of probe 1. The first base at the 3' end of upstream primer 1, which is bound to the same DNA template as probe 1, has no position to bind to this DNA template, resulting in the inability of upstream primer 1 to extend, hydrolyze probe 1, and release the fluorescent signal of probe 1.

[0019] (2) When the first base at the 5' end of probe 1 bound to the DNA template is complementary to the base of the DNA template, the binding position is occupied by the first base at the 5' end of probe 1. The first base at the 3' end of upstream primer 2 bound to the same DNA template as probe 1 is not complementary to this DNA template and has no position to bind to this DNA template. As a result, upstream primer 2 cannot extend, cannot hydrolyze probe 1, and cannot release the fluorescent signal of probe 1.

[0020] (3) When the first base at the 5' end of the probe 1 bound to the DNA template is not complementary to the base of the DNA template, the first base at the 5' end of the probe 1 does not occupy the corresponding position of the DNA template, and the first base at the 3' end of the upstream primer 1 bound to the same DNA template as the probe 1 is also not complementary to the DNA template, resulting in the inability of the upstream primer 1 to extend, the inability to hydrolyze the probe 1, and the inability to release the fluorescent signal of the probe 1.

[0021] (4) When the first base at the 5' end of probe 1 bound to the DNA template is not complementary to the base of the DNA template, the first base at the 5' end of probe 1 does not occupy the corresponding position of the DNA template. The first base at the 3' end of upstream primer 2, which is bound to the same DNA template as probe 1, is complementary to the DNA template. Upstream primer 2 extends, hydrolyzes probe 1, and releases the fluorescent signal of probe 1. That is, when upstream primer 2 extends, the fluorescent signal of probe 1 is released.

[0022] (5) When the first base at the 5' end of probe 2 bound to the DNA template is complementary to the base of the DNA template, the binding position is occupied by the first base at the 5' end of probe 2. The first base at the 3' end of upstream primer 2 bound to the same DNA template as probe 2 has no position to bind to this DNA template, resulting in the inability of upstream primer 2 to extend, the inability to hydrolyze probe 2, and the inability to release the fluorescent signal of probe 2.

[0023] (6) When the first base at the 5' end of probe 2 bound to the DNA template is complementary to the base of the DNA template, the binding position is occupied by the first base at the 5' end of probe 2. The first base at the 3' end of upstream primer 1 bound to the same DNA template as probe 2 is not complementary to this DNA template and has no position to bind to this DNA template. As a result, upstream primer 1 cannot extend, cannot hydrolyze probe 2, and cannot release the fluorescent signal of probe 2.

[0024] (7) When the first base at the 5' end of probe 2 bound to the DNA template is not complementary to the base of the DNA template, the first base at the 5' end of probe 2 does not occupy the corresponding position of the DNA template, and the first base at the 3' end of upstream primer 2 bound to the same DNA template as probe 2 is also not complementary to the DNA template, resulting in the inability of upstream primer 2 to extend, the inability to hydrolyze probe 2, and the inability to release the fluorescent signal of probe 2.

[0025] (8) When the first base at the 5' end of probe 2 bound to the DNA template is not complementary to the base of the DNA template, the first base at the 5' end of probe 2 does not occupy the corresponding position of the DNA template. The first base at the 3' end of upstream primer 1 bound to the same DNA template as probe 2 is complementary to the DNA template. Upstream primer 1 extends, hydrolyzes probe 2, and releases the fluorescent signal of probe 2. That is, when upstream primer 1 extends, the fluorescent signal of probe 2 is released. Beneficial effects

[0026] The present invention designs an amplification blocking primer (upstream primer) at the base position of the target site (at the single-base polymorphism site) to overlap with the single-base position occupied by the probe, resulting in the simultaneous single-base occupation of the primer and the probe, introducing a competitive relationship between the single-base binding sites of the primer and the probe, thereby ensuring high specificity and high sensitivity in the single-base polymorphism detection process and realizing single-tube multiple detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 is a schematic diagram of the nucleic acid detection principle of homozygous AA;

[0028] Figure 2 is a schematic diagram of the nucleic acid detection principle of homozygous GG;

[0029] Figure 3 is a schematic diagram of the nucleic acid detection principle of heterozygote AA / GG;

[0030] Figure 4 shows the amplification curve of homozygous MTHFR (677CC) at 100 ng / μL;

[0031] Figure 5 is the amplification curve of homozygous MTHFR (677CC) 10 ng / μL;

[0032] Figure 6 shows the amplification curve of homozygous MTHFR (677CC) at 0.2 ng / μL;

[0033] Figure 7 is a graph showing the amplification curve of homozygous MTHFR (677TT) at 100 ng / μL;

[0034] Figure 8 is a graph showing the amplification curve of homozygous MTHFR (677TT) at 10 ng / μL;

[0035] Figure 9 is a graph showing the amplification curve of homozygous MTHFR (677TT) at 0.2 ng / μL;

[0036] Figure 10 is a graph showing the amplification curve of heterozygous MTHFR (677CT) at 100 ng / μL;

[0037] Figure 11 is a graph showing the amplification curve of heterozygous MTHFR (677CT) at 10 ng / μL;

[0038] FIG12 is a graph showing the amplification curve of heterozygous MTHFR (677CT) at 0.2 ng / μL. Implementation Method

[0039] In order to further understand the present invention, preferred embodiments of the present invention are described below in conjunction with examples. However, it should be understood that these descriptions are only for further illustrating the features and advantages of the present invention, rather than for limiting the present invention.

[0040] The specific implementation methods of this application are now described in conjunction with the detection of human MTHFR (677C>T) gene mutation. Example

[0041] (1) Two different fluorescently modified probes, called probe 1 and probe 2, were designed for two different bases at the same allele of the human MTHFR (677C>T) gene. Two different upstream amplification blocking primers, called upstream primer 1 and upstream primer 2, were designed. The nucleic acid sequences of probe 1, probe 2, upstream primer 1, and upstream primer 2 are shown in Table 1. Each primer probe was synthesized by Shanghai Sangon Biotechnology Co., Ltd.; T of probe 1 m The value is 66.4℃, and the T of probe 2 m The value was 65.7℃, and the T of upstream primer 1 was m The T value of upstream primer 2 was 57.6℃. m The value is 56.8℃.

[0042] Table 1 Nucleic acid sequence list of probe 1, probe 2, upstream primer 1, and upstream primer 2

[0043]

[0044] (2) Each primer, probe, and blocker were diluted to a concentration of 10 μM for later use. The amplification templates were human genomic DNA, namely human genomic DNA-MTHFR (677CC), human genomic DNA-MTHFR (677TT), and human genomic DNA-MTHFR (677CT). Human genomic DNA-MTHFR (677CC), human genomic DNA-MTHFR (677TT), and human genomic DNA-MTHFR (677CT) were diluted to 100 ng / μL, 10 ng / μL, and 0.2 ng / μL, respectively. The amplification Taq mix was purchased from Shanghai Yisheng Biotechnology (with 5'-3' exonuclease activity but no 3'-5' exonuclease activity).

[0045] (3) Fluorescence PCR reaction: the amplification reaction system is shown in Table 2 below, and the amplification reaction procedure is shown in Table 3;

[0046] Table 2 Amplification reaction system

[0047]

[0048] Table 3 Amplification reaction schedule

[0049]

[0050]

[0051] (4) Analysis of test results

[0052] The amplification curves of nucleic acid detection of human genomic DNA-MTHFR (677CC), human genomic DNA-MTHFR (677TT), and human genomic DNA-MTHFR (677CT) at different concentrations (100 ng / μL) are shown in Figures 4 to 12, among which Figure 4 is the amplification curve of homozygous MTHFR (677CC) at 100 ng / μL, Figure 5 is the amplification curve of homozygous MTHFR (677CC) at 10 ng / μL, Figure 6 is the amplification curve of homozygous MTHFR (677CC) at 0.2 ng / μL, Figure 7 is the amplification curve of homozygous MTHFR (677TT) at 100 ng / μL, Figure 8 is the amplification curve of homozygous MTHFR (677TT) at 10 ng / μL, Figure 9 is the amplification curve of homozygous MTHFR (677TT) at 0.2 ng / μL, and Figure 10 is the amplification curve of heterozygous MTHFR (677CT) at 100 Figure 11 is the amplification curve of heterozygous MTHFR (677CT) 10 ng / μL, and Figure 12 is the amplification curve of heterozygous MTHFR (677CT) 0.2 ng / μL.

[0053] As shown in Figure 4, at a high concentration of 100 ng / μL human genomic DNA-MTHFR (677CC) template, an amplification curve appeared in the CY5 channel, and no amplification phenomenon occurred in the FAM channel, indicating good specificity; as shown in Figure 5, at a concentration of 10 ng / μL human genomic DNA-MTHFR (677CC) template, an amplification curve appeared in the CY5 channel, and no amplification phenomenon occurred in the FAM channel, indicating good specificity and high sensitivity; as shown in Figure 6, at a low concentration of 0.2 ng / μL human genomic DNA-MTHFR (677CC) template, an amplification curve appeared in the CY5 channel, and no amplification phenomenon occurred in the FAM channel, indicating good specificity and high sensitivity; as shown in Figure 7, at a high concentration of 100 ng / μL human genomic DNA-MTHFR (677TT) template, an amplification curve appeared in the FAM channel, and no amplification phenomenon occurred in the CY5 channel, indicating good specificity; as shown in Figure 8, at a low concentration of 0.2 ng / μL human genomic DNA-MTHFR (677TT) template, an amplification curve appeared in the FAM channel, and no amplification phenomenon occurred in the CY5 channel, indicating good specificity; ng / μL concentration of human genomic DNA-MTHFR (677TT) template, an amplification curve appeared in the FAM channel, and no amplification phenomenon appeared in the CY5 channel, with good specificity and high sensitivity; as shown in Figure 9, at a low concentration of 0.2 ng / μL human genomic DNA-MTHFR (677TT) template, an amplification curve appeared in the FAM channel, and no amplification phenomenon appeared in the CY5 channel, with good specificity and high sensitivity; as shown in Figure 10, at a high concentration of 100 ng / μL human genomic DNA-MTHFR (677CT) template, amplification curves appeared in both the FAM channel and the CY5 channel, and heterozygous MTHFR (677CT) could be correctly detected; as shown in Figure 11, at a concentration of 10 ng / μL human genomic DNA-MTHFR (677CT) template, amplification curves appeared in both the FAM channel and the CY5 channel, and heterozygous MTHFR (677CT) could be correctly detected; as shown in Figure 12, at a concentration of 0.2 Under the low concentration of human genomic DNA-MTHFR (677CT) template of ng / μL, amplification curves appeared in both the FAM channel and the CY5 channel, and heterozygous MTHFR (677CT) could still be correctly detected with high sensitivity.

Claims

1. A nucleic acid detection kit, characterized in that: comprising probe 1, probe 2, upstream primer 1 and upstream primer 2; The probe 1 and probe 2 are complementary to the DNA template where two different bases of the allele are located, and the first bases at the 5' end of probe 1 and probe 2 are the positions of the two different bases of a certain site of the allele in the DNA template; The upstream primer 1 and the upstream primer 2 are complementary to the DNA template where two different bases of the allele are located, respectively. The first base at the 3' end of the upstream primer 1 and the upstream primer 2 is the position where two different bases of a certain site of the allele are located in the DNA template; The T of the probe 1 and probe 2 sequences m The values ​​were 8~15℃ higher than the Tm values ​​of upstream primer 1 and upstream primer 2.

2. The nucleic acid detection kit according to claim 1, characterized in that The first base at the 3' end of the upstream primer 1 is the same as the first base at the 5' end of the probe 1, and the first base at the 3' end of the upstream primer 2 is the same as the first base at the 5' end of the probe 2.

3. The nucleic acid detection kit according to claim 1, characterized in that The length of the upstream primer 1 and the upstream primer 2 is 13-32 bp, T m The value is 55~65℃.

4. The nucleic acid detection kit according to claim 1, characterized in that The length of the probe 1 and probe 2 is 10-26 bp, T m The value is 60~75℃.

5. The nucleic acid detection kit according to claim 1, characterized in that The 3' ends of the probe 1 and the probe 2 are modified with a quenching group, and the 5' ends are modified with a fluorescent group.

6. The nucleic acid detection kit according to claim 5, characterized in that The quenching group is one of BHQ1, BHQ2, MGB-NFQ, BHQ3, etc.; the fluorescent group is one of FAM, VIC, CY5, CY3, ROX, JOE, TET, NED, TAMRA, etc.

7. Use of the kit according to any one of claims 1 to 6 in nucleic acid detection.

8. The use according to claim 7, characterized in that The amplification system in nucleic acid detection contains DNA polymerase, which has DNA polymerase activity and 5'-3' exonuclease activity, but no 3'-5' exonuclease activity.

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