Acute leukemia Pax-5a gene detection kit based on lambda exonuclease selective digestion and its use method

Abstract

The present invention belongs to the field of biological analysis and relates to a gene detection kit for the acute leukemia gene Pax‑5a based on selective digestion of lambda exonuclease. When the target gene is present, the tail sequence of HP‑HCR forms a flat end with the Pax‑5a gene. After adding lambda exonuclease, the hairpin is gradually digested from the 5' end, and the Pax‑5a sequence is released from HP‑HCR and combined with the next hairpin probe to achieve the recycling of the gene and achieve the purpose of the first step of amplification. At the same time, HP‑HCR is cleaved into a single-stranded trigger chain, triggering a hybridization chain reaction. After the H1 chain is opened by the trigger chain, it hybridizes with the H2 chain sequence. The generated hybrid chain continues to bind to new H1 and H2 chains, and the fluorescent groups and quenching groups on H1 and H2 are away from each other, and the fluorescent signal is restored to achieve the second amplification of the Pax‑5a gene. Thereby achieving the detection of the acute leukemia Pax‑5a gene.

Description

Technical Field

[0001] The present invention belongs to the field of biological analysis and relates to an acute leukemia Pax-5a gene detection kit based on lambda exonuclease selective digestion and a method for using the kit. Background Art

[0002] Acute B-cell lymphoblastic leukemia (ABCL) is a formidable cancer, especially in children. The primary characteristic of ABCL is genetic changes or chromosomal abnormalities that affect cell differentiation and proliferation. Pax-5, commonly referred to as Pax-5a, is a member of the Pax family that plays an important role in regulating the development and differentiation of B cells. Abnormal expression of the Pax-5 gene is closely associated with ABCL. Since Pax-5 is rarely positive in tumors of other lineages, it is considered a marker of the B-cell lineage. Therefore, research and detection of the Pax-5 gene is crucial for the early diagnosis and late-stage treatment of ABCL.

[0003] Lambda exonuclease is an exonuclease that digests phosphorylated double-stranded and single-stranded DNA. The reaction mechanism involves the gradual degradation of the 5' end of phosphorylated mononucleotides along a 5'-3' sequence. Lambda exonuclease has a high cleavage rate but exhibits low activity against non-phosphorylated and single-stranded DNA. This property makes lambda exonuclease useful for cleaving phosphorylated double-stranded DNA to obtain desired single-stranded DNA.

[0004] To improve the sensitivity of nucleic acid detection signals, it is often necessary to construct a signal amplification system. Researchers have proposed many technologies for amplifying biological signals, such as the CRISPR-Cas13a amplification system, helicase-linked amplification systems, polymerase chain reaction (PCR), and hybridization chain reaction (HCR). Our research group previously used electrochemical methods to amplify signals for the detection of the acute leukemia gene Pax5a. Through the synergistic interaction of DNA polymerase and restriction endonuclease, the target gene was recycled and the detection signal was amplified. The G-quadruplexes generated by enzymatic amplification can bind to hemin to form a G-quadruplex / hemin complex with horseradish peroxidase mimetic properties. The stable current signal generated by the G-quadruplex catalyzing the reduction of hydrogen peroxide enables sensitive detection of the target gene. However, electrochemical biosensors generally have certain limitations in signal generation. Firstly, when constructing electrochemical biosensors, appropriate labeling of electroactive substances is required, which may limit detection sensitivity. Secondly, typical electrochemical layer-by-layer assembly reactions continuously introduce interfering substances onto the electrode surface. Thirdly, directly incorporating appropriate signal amplification technologies into the construction of electrochemical biosensors at the electrode interface presents efficiency issues. Therefore, developing novel signal amplification strategies, anti-interference measures, and signal amplification efficiency on the electrode surface to improve analyte detection sensitivity is of great significance for expanding the application and performance of electrochemical analysis. Summary of the Invention

[0005] To solve the above problems, the present invention provides a Pax-5a gene detection kit for acute leukemia based on selective digestion with lambda exonuclease and a method for using the kit.

[0006] The technical solution of the present invention is achieved as follows:

[0007] The invention discloses an acute leukemia Pax-5a gene detection kit based on selective digestion with lambda exonuclease, comprising an HP-HCR chain, an H1 chain, an H2 chain, a buffer system and lambda exonuclease.

[0008] The HP-HCR chain sequence is 5'-ACTGAGTCTCCCCATGCCTGAGAGATGCGGTGATCCTTGAGACTTCAGATCTCAAGGATCACCGCATCTCTCA-3', the H1 chain sequence is 5'-TGAGAGATGCGGT-FAM-GATCCTTGAGACTAAGTTCTCAAGGAT-BHQ-CACCGCAT-3', and the H2 chain sequence is 5'-TCTCAAGGAT-FAM-CACCGCATCTCTCAATGCGGT-BHQ-GATCCTTGAGAACTTAG-3'.

[0009] The buffer system is 1×NEB Buffer 2 (100 mM Tris HCl, 100 mM MgCl 2 , 10 mM dithiothreitol (DTT), 500 mM NaCl, pH=7.9), and the concentration of lambda exonuclease is 500 U / mL.

[0010] The steps for using the above-mentioned acute leukemia Pax-5a gene detection kit are as follows:

[0011] (1) Prepare the HP-HCR chain solution, mix it with the standard solution of Pax-5a with gradient concentration in a reaction tube, add the buffer system, and incubate at 37°C for 1 hour;

[0012] (2) Add 1 μL of λ exonuclease to the solution treated in step (1), incubate at 37°C for 15 minutes, and then heat to inactivate the enzyme;

[0013] (3) The solution treated in step (2) was cooled to room temperature, H1 and H2 chain solutions were added, and incubated at 37°C for 75 minutes;

[0014] (4) The solution treated in step (3) is used to collect the fluorescence signal using a fluorescence spectrophotometer.

[0015] (5) Single-base mismatch sequence, three-base mismatch sequence, five-base mismatch sequence and complete mismatch sequence were used as targets to test sensitivity.

[0016] Preferably, the concentration of the HP-HCR chain in step (1) is 10 μM.

[0017] Preferably, the concentration of λ exonuclease in step (2) is 500 U / mL.

[0018] Preferably, in step (3), the concentrations of the H1 chain solution and the H2 chain solution are both 10 μM, and the amount of the H1 chain solution and the H2 chain solution added are both 1 μL.

[0019] Preferably, the excitation wavelength used when collecting the fluorescence signal in step (4) is 492 nm, the detection range of the emission wavelength is 490 nm-650 nm, and the scanning speed is 1200 nm / min.

[0020] Preferably, the linear equation in step (4) is a piecewise equation. When the fluorescence signal is 5000-7000 au, the linear equation is y=910.06lg(x)+6674.3, R 2 =0.99352; when the fluorescence signal is (1-6)×10 4au, the linear equation is y=22034.7096lg(x)+8492.9880, R 2 =0.9936.

[0021] The present invention has the following beneficial effects:

[0022] 1. The technical principle of the detection method of the present application is: when the target gene exists, the tail DNA sequence of HP-HCR is paired with the Pax-5a gene to form a blunt end. After adding lambda exonuclease, the hairpin is gradually digested from the 5' end, and the Pax-5a sequence is released from the HP-HCR and combined with the next hairpin probe to recycle Pax-5a to achieve the purpose of the first step of amplification. At the same time, the HP-HCR is cleaved to generate a single-stranded trigger chain, thereby triggering the hybridization chain reaction in the next step. After the H1 chain is opened by the trigger chain, it can hybridize with the H2 chain sequence. The generated hybrid chain continues to combine with new H1 and H2 chains to achieve the second amplification of the Pax-5a gene. At this time, the fluorescent groups on H1 and H2 are relatively far away from the quenching group, so the detection of the acute leukemia Pax-5a gene can be achieved by measuring the fluorescence intensity.

[0023] 2. The functional hairpin probe designed in this application contains a binding site for the target gene Pax-5a. After binding to the target gene, the hairpin probe can be directly cleaved by Lambda Exonuclease to produce a single-stranded DNA, triggering a hybridization chain reaction. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0025] Figure 1 This is the detection principle of this application.

[0026] Figure 2 This is the fluorescence spectrum of the Pax-5a gene detected in Example 1.

[0027] Figure 3 The results of the standard sample detection according to Example 1 are shown in Figure 1. A: Response curve of fluorescence intensity change versus target concentration in Example 1, B and C: Logarithmic-linear graphs of fluorescence intensity change versus target concentration.

[0028] Figure 4 This is the selectivity investigation result of Example 1. DETAILED DESCRIPTION

[0029] The following will clearly and completely describe the technical solutions of the present invention in conjunction with the embodiments of the present invention. Obviously, the embodiments described are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention. Example

[0030] The present invention discloses an acute leukemia Pax-5a gene detection kit based on selective digestion with lambda exonuclease, comprising an HP-HCR chain, an H1 chain, an H2 chain, a buffer system and lambda exonuclease.

[0031] The DNA sequences used are as follows:

[0032]

[0033] The buffer system is 1×NEB Buffer 2 (100 mM Tris HCl, 100 mM MgCl 2 , 10 mM dithiothreitol (DTT), 500 mM NaCl, pH=7.9), and the concentration of lambda exonuclease is 500 U / mL.

[0034] The kit of this application can be used to construct a highly sensitive biosensor:

[0035] (1) Mix 2 μL of 10 μM hairpin probe (HP-HCR) and 1 μL of Pax-5a standard aqueous solution with concentrations of 250 nM, 100 nM, 50 nM, 25 nM, 20 nM, 15 nM, 5 nM, 4 nM, 2 nM, 1 nM, 500 pM, 100 pM, 50 pM, 25 pM, and 0 pM in a reaction tube and incubate at 37°C for 1 hour.

[0036] (2) Then, 1 μL of 500 U / mL λ exonuclease was added, incubated at 37°C for 15 minutes, and then heated at 80°C for 10 minutes to inactivate the enzyme.

[0037] (3) When the solution in step (2) has cooled to room temperature, add 1 μL of 10 μM H1 and H2 chain solutions respectively and incubate at 37°C for 75 minutes.

[0038] (4) 180 μL of ultrapure water was added to the reaction mixture obtained in step (3), and then fluorescence measurement was performed; when detecting the change in the fluorescence signal, the excitation wavelength was 492 nm, the detection range of the emission wavelength was 490 nm-650 nm, and the scanning speed was 1200 nm / min.

[0039] (5) Under the same conditions, the selectivity of this method was investigated using single-base mismatch sequence (MT1), three-base mismatch sequence (MT2), five-base mismatch sequence (MT3) and complete mismatch sequence (CMT) as targets.

[0040] Figure 1 The present invention relates to a Pax-5a gene detection process and schematic diagram for acute leukemia based on selective digestion with lambda exonuclease. When the target gene is present, the tail DNA sequence of the HP-HCR pairs with the Pax-5a gene, forming blunt ends. Upon addition of lambda exonuclease, the hairpin is gradually digested starting from the 5' end, releasing the Pax-5a sequence from the HP-HCR, allowing it to bind to the next hairpin probe, enabling recycling of Pax-5a and achieving the first amplification step. Simultaneously, the HP-HCR is cleaved to generate a single-stranded trigger strand, initiating a hybridization chain reaction. After the trigger strand opens the H1 strand, it can hybridize with the H2 strand sequence. The resulting hybrid strand then continues to bind to new H1 and H2 strands, separating the fluorescent groups and quenching groups on H1 and H2, restoring the fluorescence signal and enabling a second amplification of the Pax-5a gene. However, in the absence of target DNA, the HP-HCR cannot be cleaved to generate the trigger strand, nor can it trigger the hybridization chain reaction. Because the H1 and H2 strands cannot open, the fluorescence intensity of the system is relatively weak. Therefore, high-sensitivity detection of the Pax-5a gene in acute leukemia can be achieved by measuring fluorescence intensity.

[0041] Figure 2 This is the fluorescence spectrum of the target gene Pax-5a. Figure 2 It can be seen that the fluorescence intensity is weak when there is no target gene and λ exonuclease (curve a), λ exonuclease (curve b), and target gene (curve c), respectively. Curve d shows the fluorescence intensity of a complete experimental system, which is significantly higher than the fluorescence intensity of the first three systems, which verifies the feasibility of this experimental plan.

[0042] Figure 3 A is a response curve corresponding to the change in fluorescence intensity and the concentration of the detection target substance obtained by detecting the standard sample according to Example 1. Figure 3 B and Figure 3 C reflects that the fluorescence signal of Pax-5a gene increases in two stages within the concentration range of 0 pM-250 nM. The fluorescence signal intensity of the two stages has a good linear relationship with the logarithm of the Pax-a gene concentration. When the fluorescence signal is 5000-7000 au, the linear equation is y=910.06lg(x)+6674.3, R 2 =0.99352; when the fluorescence signal is (1-6)×10 4au, the linear equation is y=22034.7096lg(x)+8492.9880, R 2 =0.9936. And the detection limit is as low as 7.6pM. ( Figure 3 B).

[0043] Figure 4 Considering the requirements of practicality, the specificity and selectivity of this method for specific targets were investigated. Single-base mismatch sequence (MT1), three-base mismatch sequence (MT2), five-base mismatch sequence (MT3), and complete mismatch sequence (CMT) were selected as interfering substances to test their effects on the sensing system. The experimental results confirmed that the sensing system had low or almost no response to different interfering components, but a significant response to specific targets, indicating that it has good selectivity and specificity. Figure 4 ).

[0044] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A Pax-5a gene detection kit for acute leukemia based on selective digestion with lambda exonuclease, characterized by: including HP-HCR chain, H1 chain, H2 chain, buffer system and lambda exonuclease; The HP-HCR chain sequence is 5'-ACTGAGTCTCCCCATGCCTGAGAGATGCGGTGATCCTTGAGACTTCAGATCTCAAGGATCACCGCATCTCTCA-3', the H1 chain sequence is 5'-TGAGAGATGCGGT-FAM-GATCCTTGAGACTAAGTTCTCAAGGAT-BHQ-CACCGCAT-3', and the H2 chain sequence is 5'-TCTCAAGGAT-FAM-CACCGCATCTCTCAATGCGGT-BHQ-GATCCTTGAGAACTTAG-3'.

2. The acute leukemia Pax-5a gene detection kit according to claim 1, characterized in that: The buffer system is 1×NEB Buffer 2 with a pH of 7.9, which contains 100 mM Tris HCl, 100 mM MgCl 2 , 10 mM dithiothreitol, and 500 mM NaCl.

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