P53 gene detection signal amplifier, kit and method for detecting p53 gene in sample

Abstract

The invention relates to the technical field of biosensors, and discloses a p53 gene detection signal amplifier, a kit and a method for detecting a p53 gene in a sample. The signal amplifier comprises a hairpin probe HP and a lock-type probe, wherein the hairpin probe HP comprises an HP-1 segment which is complementarily paired with the p53DNA segment and an HP-2 segment which is complementarily paired with the lock-type probe. The signal amplifier provided by the invention has the advantages of high specificity, high sensitivity and the like, so that the method provided by the invention can be used for rapidly, efficiently and accurately detecting the p53 gene in a sample. Moreover, the signal amplifier and the method provided by the invention do not depend on high-price instruments such as a high-quality thermal cycler, and are very suitable for popularization and application in grassroots.

Description

Technical Field

[0001] This invention relates to the field of biosensor technology, specifically to a p53 gene detection signal amplifier, a kit, and a method for detecting the p53 gene in a sample. Background Technology

[0002] The p53 gene is a tumor suppressor gene that plays a crucial role in regulating biological functions such as cell cycle, apoptosis, cell differentiation, and DNA repair. It has been reported that p53 gene mutations are present in approximately 50% of human malignant tumors, and p53 gene mutation is the most common genetic alteration in human cancer. Therefore, developing simple, rapid, highly sensitive, and highly specific methods for detecting the p53 gene is of great medical and scientific significance.

[0003] Currently, nucleic acid testing is widely used in clinical diagnosis, environmental monitoring, and the prevention and control of infectious diseases. Polymerase chain reaction (PCR) technology has advantages such as high sensitivity and is one of the most widely used DNA amplification and detection methods. However, conventional PCR technology still has many shortcomings, such as dependence on high-quality thermal cyclers, frequent false positive results due to non-specific amplification, long amplification reaction time, and difficulty in promotion and application at the grassroots level. Moreover, most importantly, simple methods such as agarose gel electrophoresis can only determine the approximate length of PCR products. Sequencing is usually required to accurately determine whether the PCR product is the target gene or whether the target gene has mutated (e.g., deletion, addition of small fragments, or nucleotide substitution mutations). This greatly limits the detection efficiency, convenience, and accuracy of PCR technology in nucleic acid testing, and the detection cost is relatively high. Therefore, to achieve simpler, faster, more sensitive, and more specific detection of the p53 gene, new nucleic acid detection tools and methods need to be developed. Summary of the Invention

[0004] The purpose of this invention is to overcome the aforementioned problems in the prior art and provide a p53 gene detection signal amplifier, a kit, and a method for detecting the p53 gene in a sample. The signal amplifier provided by this invention has advantages such as high specificity and high sensitivity, thereby enabling the method provided by this invention to detect the p53 gene in a sample rapidly, efficiently, and accurately.

[0005] To achieve the above objectives, the present invention provides a p53 gene detection signal amplifier, the signal amplifier comprising a hairpin probe HP and a lock-type probe;

[0006] The hairpin probe HP contains the HP-1 segment, which is complementary to the p53 DNA fragment, and the HP-2 segment, which is complementary to the lock probe.

[0007] A second aspect of the present invention provides a kit for detecting the p53 gene, the kit comprising the following ingredients:

[0008] (1) DNA probes: including hairpin probes (HP) and locking probes, wherein the hairpin probes (HP) contain an HP-1 segment complementary to the p53 DNA fragment and an HP-2 segment complementary to the locking probe; and,

[0009] Optional, (2) enzymes: including Nt.BstNBI nicking endonuclease and phi29 DNA polymerase.

[0010] A third aspect of the present invention provides a method for detecting the p53 gene in a sample, the method comprising: contacting and reacting the sample with a signal amplifier as described in the first aspect above, and detecting the reaction product using a detection reagent.

[0011] Through the above technical solution, the present invention can achieve the following beneficial effects:

[0012] (1) The signal amplifier provided by the present invention is based on nick endonuclease signal amplification (NESA) coupled rolling circle amplification (RCA), which effectively improves the detection sensitivity, thereby enabling trace detection of the p53 gene (experimentally verified, the detection limit is as low as 4.26pM).

[0013] (2) The signal amplifier provided by the present invention has extremely high specificity. Even if a target gene fragment with only one base mismatch is used for detection, no positive result will be produced. This improves the detection accuracy, avoids the use of subsequent detection methods such as sequencing, effectively improves detection efficiency, and reduces detection costs.

[0014] (3) The method provided by the present invention is simple to operate during the detection process, does not require high-quality thermal cyclers or other instruments, and can be detected in a visual way, making it very suitable for large-scale promotion and application at the grassroots level. Attached Figure Description

[0015] Figure 1 This is a schematic diagram illustrating the detection process and principle of the p53 gene detection method provided by this invention.

[0016] Figure 2 This is a standard curve plotted in Example 1 showing the fluorescence concentration versus the p53 DNA fragment concentration. Detailed Implementation

[0017] The endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0018] Isothermal amplification of nucleic acids is a method of nucleic acid amplification that does not require multiple cycles of denaturation, annealing, and extension, and has a shorter amplification time. Compared with traditional PCR, isothermal amplification of nucleic acids does not rely on special instruments such as high-quality thermal cyclers, and it is also faster and easier to operate. Currently, commonly used high-efficiency isothermal amplification techniques include loop-mediated isothermal amplification (LAMP), rolling circle amplification (RCA), strand displacement amplification (SDA), nick endonuclease signal amplification (NESA), and exonuclease signal amplification.

[0019] The inventors of this invention ingeniously discovered that by coupling existing nucleic acid isothermal amplification techniques when detecting the p53 gene, a signal amplifier with extremely high specificity and sensitivity can be constructed, thereby achieving sensitive detection of trace amounts of the p53 gene in samples. By selecting specific probe sequences, when using this signal amplifier in conjunction with specific detection methods to detect the p53 gene, even mutations with only one base mismatch will not produce false positive results, greatly improving the specificity of the detection.

[0020] Based on the above findings, the present invention provides a p53 gene detection signal amplifier, the signal amplifier comprising a hairpin probe HP and a lock probe;

[0021] The hairpin probe HP contains the HP-1 segment, which is complementary to the p53 DNA fragment, and the HP-2 segment, which is complementary to the lock probe.

[0022] For ease of operation, according to a preferred embodiment of the present invention, the hairpin probe HP is fixed on a hairpin probe carrier. Considering the convenience of separation and washing procedures during detection, the hairpin probe carrier is preferably a micron-sized magnetic bead. For example, it can be a magnetite micron-sized magnetic bead, a silicon oxide micron-sized magnetic bead, etc.

[0023] According to a preferred embodiment of the present invention, the loading amount of the hairpin probe HP on the hairpin probe carrier is not less than 20 pmol / mg. Preferably, it is 20-50 pmol / mg. For example, it can be 20 pmol / mg, 25 pmol / mg, 30 pmol / mg, 35 pmol / mg, 40 pmol / mg, 45 pmol / mg, 50 pmol / mg, or any range of any two of the above values, or any intermediate value within that range.

[0024] According to a preferred embodiment of the present invention, the 5' end of the hairpin probe HP has a modifying group for attachment to a hairpin probe carrier. Preferably, the modifying group is biotin.

[0025] According to a preferred embodiment of the present invention, the 5' end of the lock probe is phosphorylated. The purpose of the phosphorylation is to enable the lock probe to form a "U-shaped" structure, thereby complementarily pairing with the HP-2 segment of the hairpin probe HP and initiating a rolling circle amplification reaction.

[0026] Since hairpin probes are typically synthesized as single-stranded probes, they require subsequent heating and annealing processes to form a hairpin structure. Furthermore, even if a hairpin probe is directly fabricated, improper storage can cause the stem portion to unwind and become a single strand. To avoid false positives caused by combining the single-stranded hairpin probe HP with the lock probe, it is preferable that the hairpin probe HP and the lock probe are stored independently.

[0027] In this invention, the HP-1 segment of the hairpin probe HP is located in the circular region of the hairpin probe. When it comes into contact with the p53 DNA fragment, base complementarity pairing occurs, causing the hairpin probe HP to open and exposing the HP-2 segment located in the stem region. In subsequent detection, the HP-2 segment complementarizes with the lock probe, resulting in rolling circle amplification (RoBAM) and forming a long repeating sequence detectable by specific reagents, thus showing a positive result. If the sample does not contain the p53 gene, or if the p53 gene is mutated and cannot complementarize with the HP-1 segment of the hairpin probe HP, the hairpin probe HP will not open, and the lock probe will also be unable to complementarize with the double-stranded HP-2 segment. The RoBAM reaction will not occur, resulting in a negative result.

[0028] In this invention, there are no particular restrictions on the specific probe sequence used in the provided signal amplifier. The design can be carried out according to the above design principles based on the selected p53 DNA fragment.

[0029] According to a preferred embodiment of the present invention, the nucleotide sequence of the p53 DNA fragment is as shown in SEQ ID NO:1.

[0030] TCA TCA CAC TGG AAG ACT C(SEQ ID NO:1)

[0031] When the p53 DNA fragment is the sequence of SEQ ID NO:1, preferably, the nucleotide sequence of the hairpin probe HP is as shown in SEQ ID NO:2.

[0032] TTT TTT TCC CGA TCC ATG AGT CTT CCA GTG TGA TGA GGA TCG GGA(SEQ IDNO:2)

[0033] Preferably, the nucleotide sequence of the lock probe is shown in SEQ ID NO:3.

[0034] TGG ATC GGG ATA TCC TTT GGT TGA AAC TTC TTC CTT TCT TGG AAG ACT CA(SEQ ID NO:3)

[0035] A second aspect of the present invention provides a kit for detecting the p53 gene, the kit comprising the following ingredients:

[0036] (1) DNA probes: including hairpin probes (HP) and locking probes, wherein the hairpin probes (HP) contain an HP-1 segment complementary to the p53 DNA fragment and an HP-2 segment complementary to the locking probe; and,

[0037] Optional, (2) Enzymes: including endonucleases and DNA polymerases.

[0038] In this invention, "optional" means that (2) the enzyme is not a necessary component of the kit provided by this invention, but considering the convenience of the experiment, the kit may be equipped with the corresponding reagent, or those skilled in the art may also purchase the optional reagent themselves with reference to the content of this invention and related prior art, and use it in conjunction with the kit provided by this invention.

[0039] To facilitate experimental operations, the DNA probes (e.g., hairpin probes HP) provided by this invention can be attached to a suitable carrier before use. Based on this, according to a preferred embodiment of the invention, the kit may further include: (3) a hairpin probe carrier. Preferably, the hairpin probe carrier is a micron-sized magnetic bead. For example, it can be a magnetite micron-sized magnetic bead, a silica micron-sized magnetic bead, etc.

[0040] The DNA probe in the kit provided by this invention is the same probe included in the signal amplifier in the first aspect mentioned above, and its features are as described above, and will not be repeated here.

[0041] According to a preferred embodiment of the present invention, the endonuclease is selected from Nt.BstNBI nicking endonuclease; and the DNA polymerase is selected from phi29 DNA polymerase.

[0042] Preferably, the enzyme (2) in the kit also includes DNA ligase.

[0043] According to a preferred embodiment of the present invention, the kit further comprises (4) buffer solutions: including buffer A and buffer B; wherein buffer A comprises 0.05-0.15M NaCl and 0.05-0.15M PBS buffer, with the remainder being water, and the pH is 7.2-7.5; buffer B comprises 0.05-0.15M NaCl, 0.05-0.15M PBS buffer and 0.01-0.1 vol% Tween 20, with the remainder being water, and the pH is 7.2-7.5.

[0044] Preferably, the buffer further includes at least one of Nt.BstNBI reaction buffer, DNA ligase reaction buffer, and phi29 DNA polymerase reaction buffer.

[0045] More preferably, the Nt.BstNBI reaction buffer comprises: 80-120 mM NaCl, 30-80 mM Tris-HCl, 5-15 mM MgCl2, 80-120 μg / mL BSA, and pH 7.6-8.3.

[0046] More preferably, the DNA ligase reaction buffer (10×) comprises: 60-70 mM Tris-HCl, 5-15 mM MgCl2, 0.5-1.5 mM dithiothreitol (DTT), 0.5-1.5 mM ATP, 3-8 wt% polyethylene glycol (e.g., PEG 6000), and pH 7.4-7.8.

[0047] More preferably, the phi29 DNA polymerase reaction buffer comprises: 330 mM tris(hydroxymethyl)aminomethane acetate (pH 7.6-8.2 at 37°C), 80-120 mM magnesium acetate, 650-700 mM potassium acetate, 0.5-1.5 vol% Tween 20, and 8-12 mM DTT.

[0048] According to a preferred embodiment of the present invention, the kit further includes (5) a detection reagent. The detection reagent is used to detect the macromolecular product (a long-chain product formed by a DNA repeat sequence) obtained by rolling circle amplification after the HP-2 segment of the hairpin probe HP is complementaryly paired with the lock-lock probe. Any reagent capable of detecting this macromolecular product is suitable for the kit provided by the present invention. Preferably, the detection reagent is selected from molecular beacons.

[0049] According to a preferred embodiment of the present invention, the nucleotide sequence of the molecular beacon is as shown in SEQ ID NO:4. Preferably, the 5' end of the molecular beacon is modified with a fluorescent group (e.g., FAM fluorescent group), and the 3' end is modified with a quenching group corresponding to the fluorescent group (e.g., BHQ1 quenching group). The molecular beacon sequence can undergo base pairing with the RCA product on the surface of the magnetic beads, thereby releasing a fluorescent signal. The intensity of the fluorescent signal can be used to determine the content of the p53 gene in the sample.

[0050] TTC TTC CTT TCT TGG AAG ACT(SEQ ID NO:4)

[0051] According to a preferred embodiment of the present invention, the kit further includes (6) adenosine triphosphate and / or dNTPs.

[0052] According to a preferred embodiment of the present invention, the kit comprises the following:

[0053] (1) DNA probe;

[0054] Optional, (2) enzyme;

[0055] Optionally, (3) hairpin probe carrier (e.g., micron magnetic beads);

[0056] Optional, (4) buffer solution;

[0057] Optional, (5) molecular beacons;

[0058] Optional, (6) adenosine triphosphate and / or dNTPs

[0059] The specific characteristics of the contents of the above reagent kits have been described above and will not be repeated here. Among them, the contents marked "optional" are non-essential components of the reagent kit, and their inclusion in the kit can be selected based on actual production and experimental conditions.

[0060] A third aspect of the present invention provides a method for detecting the p53 gene in a sample, the method comprising: contacting and reacting the sample with the signal amplifier described in the first aspect above, and detecting the reaction product using a detection reagent;

[0061] Alternatively, the sample can be tested using the kit described in the second aspect.

[0062] refer to Figure 1 According to a preferred embodiment of the present invention, the method includes:

[0063] (i) The hairpin probe vector immobilized with hairpin probe HP is first contacted with the sample. The p53 gene fragment in the sample causes the hairpin structure of the hairpin probe HP to open. The HP-1 segment in the opened hairpin probe HP is complementary to the p53 gene fragment to form a double-stranded structure. Then, the first contact product is second contacted with a nuclease (preferably Nt.BstNBI cleavage endonuclease) to cleave the double-stranded structure, release the p53 gene fragment, and leave the hairpin probe HP fragment containing the HP-2 segment on the hairpin probe vector.

[0064] (ii) In the presence of adenosine triphosphate, the second contact product, the lock probe and the DNA ligase are made into a third contact, such that the lock probe is complementary to the HP-2 segment in the residual hairpin probe HP fragment on the hairpin probe carrier. Then, the third contact product is made into a fourth contact with DNA polymerase (preferably phi DNA polymerase) in the presence of dNTPs, so that it undergoes a rolling circle amplification reaction.

[0065] (iii) The fourth contact product was detected using a detection reagent.

[0066] In the above method, step (i) refers to the recognition of the p53 gene fragment in the sample by the hairpin probe HP, and the recycling process of the p53 gene fragment (i.e., the NESA process). The first contact is the process in which the HP hairpin probe contacts and opens the p53 gene fragment in the sample, and the two form a double-stranded structure. The second contact is the process in which the formed double-stranded structure contacts the endonuclease and is cleaved. After the double-stranded structure is cleaved, the released p53 gene fragment can bind to a new hairpin probe HP and repeat the process of "opening the HP hairpin structure → forming a double strand → double-strand cleavage → releasing the p53 gene fragment", thereby providing enough hairpin probe residual fragments containing the HP-2 segment for pairing with the lock probe added in the subsequent step (ii) for rolling circle amplification. Here, "first contact" and "second contact" are only used for descriptive purposes to facilitate the distinction of different reaction processes in this step.

[0067] According to a preferred embodiment of the present invention, in step (i), the conditions for the first contact include a temperature of 35-40°C and a time of 1-5 hours.

[0068] According to a preferred embodiment of the present invention, in step (i), the conditions for the second contact include a temperature of 53-58°C and a time of 40-80 min.

[0069] Preferably, in step (i), the content of the p53 gene in the sample is such that the concentration of the p53 gene fragment in the first contact system is not less than 5 pM. More preferably, it is 10 pM-10 nM. For example, it can be 5 pM, 10 pM, 20 pM, 30 pM, 40 pM, 50 pM, 80 pM, 100 pM, 150 pM, 200 pM, 300 pM, 400 pM, 500 pM, 800 pM, 1 nM, 2 nM, 3 nM, 4 nM, 5 nM, 6 nM, 7 nM, 8 nM, 9 nM, 10 nM, or a range consisting of any two of the above values, or any intermediate value within that range.

[0070] Preferably, in step (i), the amount of hairpin probe HP used is 50-100 nM relative to 1 unit of nicking endonuclease (such as Nt.BstNBI). For example, it can be 50 nM, 55 nM, 60 nM, 65 nM, 70 nM, 75 nM, 80 nM, 85 nM, 90 nM, 95 nM, 100 nM, or any range consisting of any two of the above values, or any intermediate value within that range.

[0071] In the above method, step (ii) is the process by which the lock probe undergoes a rolling circle amplification reaction with the hairpin probe HP fragment (containing the HP-2 segment) remaining on the hairpin probe carrier, generating a macromolecular product formed from a DNA repetitive sequence (i.e., the RCA process). The third contact connects the lock probe with the HP-2 segment in the remaining fragment of the hairpin probe HP, and then in the fourth contact, a rolling circle amplification reaction is carried out under the action of a specific DNA polymerase.

[0072] According to a preferred embodiment of the present invention, in step (ii), the conditions for the third contact include a temperature of 35-40°C and a time of 40-80 min.

[0073] Preferably, step (ii) further includes, prior to the third contact, heating the second contact product and the lock probe at 90-100°C for 1-5 min in the presence of adenosine triphosphate, then cooling to 20-30°C at a cooling rate not exceeding 3°C / min, and holding at this temperature for at least 2 h, to allow the lock probe to fully hybridize with the second contact product. The cooling rate can be 1-3°C / min, for example, 1°C / min, 1.5°C / min, 2°C / min, 2.5°C / min, 3°C / min, or any range of any two of the above values, or any intermediate value within that range.

[0074] Preferably, in step (ii), the amount of adenosine triphosphate added is such that its concentration in the third contact system is 0.5-5 μM, more preferably 0.5-2.5 μM. For example, it can be 0.5 μM, 0.8 μM, 1 μM, 1.2 μM, 1.5 μM, 1.6 μM, 1.7 μM, 1.8 μM, 1.9 μM, 2 μM, 2.1 μM, 2.2 μM, 2.5 μM, or any range consisting of any two of the above values, or any intermediate value within that range.

[0075] Preferably, in step (ii), the amount of DNA ligase used is 1 × 10⁻⁶. 3 -5×10 3 Units / mL, preferably 1.5 × 10⁻⁶ 3 -2.5×10 3 Units: per mL. For example, 1.5 × 10⁻⁶. 3 Units / mL, 1.6 × 10 3 Units / mL, 1.7 × 10 3 Units / mL, 1.8 × 10 3 Units / mL, 1.9 × 10 3 Units / mL, 2×10 3 Units / mL, 2.2 × 10 3 Units / mL, 2.5 × 10 3 The unit is / mL, or it can be a range consisting of any two of the above values, or any intermediate value within that range.

[0076] According to a preferred embodiment of the present invention, in step (ii), the conditions for the fourth contact include a temperature of 35-40°C and a time of 1-5 hours. For example, the time of the fourth contact can be 1 hour, 1.5 hours, 2 hours, 2.5 hours, 3 hours, 3.5 hours, 4 hours, 4.5 hours, or 5 hours, or a range consisting of any two of the above values, or any intermediate value within that range. Preferably, the time of the fourth contact is 1-3 hours.

[0077] Preferably, in step (ii), the amount of dNTPs added is such that its concentration in the fourth contact system is 50-150 μM, more preferably 50-100 μM. For example, it can be 50 μM, 55 μM, 60 μM, 65 μM, 70 μM, 75 μM, 80 μM, 85 μM, 90 μM, 95 μM, or 100 μM, or it can be a range consisting of any two of the above values, or any intermediate value within that range.

[0078] Preferably, in step (ii), the amount of phi29 DNA polymerase used is 1-30 units / mL, more preferably 5-20 units / mL. For example, it can be 5 units / mL, 6 units / mL, 7 units / mL, 8 units / mL, 9 units / mL, 10 units / mL, 11 units / mL, 12 units / mL, 13 units / mL, 14 units / mL, 15 units / mL, 16 units / mL, 18 units / mL, 20 units / mL, or it can be any range of any two of the above values, or any intermediate value within that range.

[0079] More preferably, step (ii) further includes terminating the rolling circle amplification reaction after the fourth contact. A preferred method for terminating the rolling circle amplification reaction includes raising the temperature of the fourth contact system to 60-70°C and holding it therefore for 5-15 minutes.

[0080] In the above method, step (iii) is the process of detecting the rolling circle amplification product. Any detection reagent that can be used to detect rolling circle amplification products is applicable to this invention. According to a preferred embodiment of the present invention, in step (iii), the detection reagent is selected from molecular beacons.

[0081] According to a preferred embodiment of the present invention (see reference), Figure 1 In step (iii), the fourth contact product is detected by adding a molecular beacon to the fourth contact product and reacting it at 20-30°C for 30-90 min. The reaction product is then subjected to fluorescence detection to quantitatively detect the p53 gene in the sample. Preferred fluorescence detection conditions include: excitation wavelength 486 nm and emission wavelength 500-600 nm.

[0082] The characteristics of the reagents used in the above methods (such as HP hairpin probes, lock probes, enzymes, molecular beacons, etc.) are as described above and will not be repeated here.

[0083] The present invention will be described in detail below through embodiments. It should be understood that the following embodiments are only used to further explain and illustrate the content of the present invention by way of example, and are not intended to limit the present invention.

[0084] In the following examples, the Nt.BstNBI nicking endonuclease used was purchased from New England Biolabs (brand name R0607S), the T4 ligase was purchased from New England Biolabs (brand name M0202V), and the phi29 DNA polymerase was purchased from Thermo Fisher Scientific (brand name EP0091). The deoxynucleotide mixture (dNTPs) was purchased from New England Biolabs, containing deoxyguanosine, deoxyadenosine, deoxycytidine, and deoxythymidine in a molar ratio of 1:1:1:1. Unless otherwise specified, all reagents and materials used were commercially available from reputable chemical or biological reagent / material suppliers, and all reagents were of analytical grade.

[0085] Unless otherwise specified, the operating temperature in the following embodiments is room temperature (25±5℃).

[0086] Preparation Example 1

[0087] (I) Synthesis of DNA probes, molecular beacons and target nucleic acid fragments

[0088] Referring to Table 1, Sangon Biotech (Shanghai) Co., Ltd. was commissioned to synthesize DNA probes, molecular beacons, and DNA sequence fragments for simulating target genes and mutated target genes in the samples.

[0089] Table 1

[0090]

[0091] *The synthesized hairpin probe HP is in a single-stranded state and requires annealing to form a stem-loop structure before use. The HP-1 sequence is GAGTCTTCCAGTGTGATGA (5'→3'), and the HP-2 sequence is TCCCGATCCAT. GAGT C TTCC(5'→3').

[0092] (II) Preparation of buffer solution

[0093] PBS buffer: 8mM Na2HPO4, 136mM NaCl, 2mM KH2PO4, 2.6mM KCl, pH = 7.2-7.4.

[0094] Buffer A: 0.1M NaCl, 0.1M PBS buffer, pH=7.4.

[0095] Buffer B: 0.1M NaCl, 0.1M PBS buffer, 0.05% Tween-20, pH=7.4.

[0096] NEBuffer 3.1: 100mM NaCl, 50mM Tris-HCl, 10mM MgCl2, 100μg / mL BSA, pH=7.9.

[0097] Nt.BstNBI reaction buffer: 100mM NaCl, 50mM Tris-HCl, 10mM MgCl2, 100μg / ml BSA, pH=7.9@25℃;

[0098] 10× ligase reaction buffer: 66mM Tris-HCl, 10mM MgCl2, 1mM DTT, 1mM ATP, 6% wt% polyethylene glycol (PEG 6000), pH=7.6@25℃;

[0099] Phi29 DNA polymerase reaction buffer: 330 mM tris(hydroxymethyl)aminomethane acetate (pH = 7.9 @ ​​37℃), 100 mM magnesium acetate, 660 mM potassium acetate, 1 v / v Tween 20, 10 mM DTT.

[0100] Example 1

[0101] This embodiment illustrates the use of the signal amplifier provided by the present invention for the detection of the p53 gene.

[0102] (1) Fix the hairpin probe HP onto the hairpin probe carrier.

[0103] The hairpin probe HP synthesized in Preparation Example 1 was placed in PBS buffer, heated to 95°C for 5 min, and then slowly cooled (about 1°C / min) to room temperature. It was then kept at room temperature for 2 h to allow the hairpin probe HP to form a stem-loop structure.

[0104] Take 200 μL of streptavidin-modified magnetic beads (purchased from New England Biolabs, average diameter 1 μm, 4 mg / mL), wash 3 times with buffer B, and then disperse in 0.5 mL of buffer A to obtain a magnetic bead dispersion.

[0105] The hairpin probe HP, which forms a stem-loop structure, was added to the magnetic bead dispersion at a final concentration of 1 μM. The mixture was then vortexed at room temperature for 1 h to allow the biotin modified at the 5' end of the hairpin probe HP to fully interact with the streptavidin on the surface of the magnetic beads and bind together.

[0106] After the reaction, the centrifuge tube was placed on a magnetic rack for 30 seconds to allow the magnetic beads to be attracted to the bottom. The supernatant was then aspirated to remove any unbound hairpin probes (HP) from the magnetic beads. The tube was then washed three times with buffer B to remove any non-specifically adsorbed HP from the magnetic bead surface, yielding hairpin probe-magnetic bead conjugates (HP-MBs). These conjugates were dispersed in 0.5 mL of buffer A to obtain an HP-MBs dispersion. The HP loading on the magnetic beads was approximately 25 pmol / mg.

[0107] (2) NESA reaction

[0108] 15 μL of HP-MBs dispersion was mixed with 25 μL of p53 DNA fragment solutions of different concentration gradients. The mixture was incubated at 37°C for 2 h to allow the p53 DNA fragment to recognize and open the hairpin structure of the hairpin probe HP, and then pair complementaryly with the HP-1 segment on the hairpin probe HP to form a double-stranded structure. Then, 10 μL of Nt.BstNBI reaction buffer (0.5 units / μL) containing Nt.BstNBI nicking endonuclease was added to the reaction system, and the mixture was incubated at 55°C for 60 min to cleave the double-stranded structure formed by the p53 DNA fragment and the HP-1 segment. The released p53 DNA fragment can then react with the remaining unopened hairpin probe HP.

[0109] After the reaction was completed, the magnetic beads were washed with buffer B in the same way as in step (1) for a total of 3 times. Then, they were redispersed in 0.5 mL of buffer A to obtain HP residual fragment-MBs dispersion, wherein the HP residual fragment contains HP-2 segment that can complementarily pair with the lock probe.

[0110] (3) RCA reaction

[0111] The HP residual fragment-MBs dispersion, 4 μL of lock probe solution (100 nM, prepared with PBS buffer), 4 μL of 10× ligase reaction buffer, 28.5 μL of ddH2O, and 1 μL of 1 mM adenosine triphosphate (ATP) were mixed and heated at 95 °C for 3 min, then slowly cooled (approximately 1 °C / min) to room temperature and incubated at room temperature for 2 h to allow the lock probe to form a "U"-shaped structure. Then, 2.5 μL of T4 DNA ligase (400,000 units / mL) was added to the reaction system, and the mixture was incubated at 37 °C for 1 h to allow the "U"-shaped lock probe to bind to the HP-2 segment of the HP residual fragment, obtaining the ligation product dispersion.

[0112] The RCA reaction system solution was prepared according to the following ratio: 5.5 μL 10×phi29 DNA polymerase reaction buffer, 0.5 μL phi29 DNA polymerase (10000 units / mL), and 4 μL 10 mM dNTPs. The concentration of dNTPs refers to the total concentration of the four nucleotides. The ligation product dispersion was then mixed with 10 μL of the RCA reaction system solution and reacted at 37 °C for 2 h, followed by incubation at 65 °C for 10 min to terminate the RCA reaction. The resulting RCA reaction macromolecule product-MBs dispersion was obtained, wherein the RCA reaction macromolecule product is a long-chain DNA molecule composed of DNA repetitive sequences amplified from the lock probe and HP residual fragments.

[0113] (4) Detection of RCA reaction products

[0114] Quantitative detection using molecular beacons is performed as follows:

[0115] Mix 10 μL of molecular beacon solution (MB solution, 100 μM) with the RCA reaction macromolecular product-MBs dispersion and react with shaking at room temperature for 1 h (approximately 300 rpm). After the reaction, place the centrifuge tube on a magnetic rack for 30 s, remove the supernatant, and wash 5 times with buffer B. Then redisperse the washed RCA reaction macromolecular product-magnetic bead conjugate with buffer to obtain a pure RCA reaction macromolecular product-MBs dispersion.

[0116] Fluorescence detection of the pure RCA reaction macromolecular product-MBs dispersion was performed using a fluorescence spectrometer (Shimadzu RF-6000 model, Japan). The excitation wavelength was set to 486 nm, the emission wavelength to 500-600 nm, and both the excitation and emission slits were set to 5 nm.

[0117] A standard curve was plotted based on the detection results, comparing fluorescence concentration with p53 DNA fragment concentration. See details below. Figure 2 .

[0118] Example 2

[0119] This embodiment is used to illustrate the sensitivity and specificity of the signal amplifier provided by the present invention.

[0120] (a) Sensitivity

[0121] p53 DNA fragment solutions with concentration gradients of 0.005 nM, 0.01 nM, 0.02 nM, 0.05 nM, 0.1 nM, 0.2 nM, 0.5 nM, 1 nM, 2 nM, 5 nM, and 10 nM were prepared and detected using the method described in Example 1 to investigate the sensitivity of the signal amplifier provided by the present invention.

[0122] The results showed that the detection limit was 4.26 pM when using molecular beacons for detection. The detection limit refers to the lowest concentration of p53 DNA fragment in solution that can be detected. The method for calculating the detection limit is as follows:

[0123] By detecting p53 DNA fragment solutions of different concentrations, a standard curve was plotted between fluorescence concentration and p53 DNA fragment solution concentration. The detection limit of the signal amplifier was calculated by dividing the slope by three times the standard deviation.

[0124] (II) Specificity

[0125] The single-base mismatched DNA fragment T1, double-base mismatched DNA fragment T2, triple-base mismatched DNA fragment T3, and completely mismatched DNA fragment Tn in Table 1 were detected according to the method in Example 1.

[0126] The results showed that when T1, T2, T3, and Tn participated in the reaction, only very weak fluorescence signals were observed, which were essentially the same as the signal intensity in the blank control experiment. This demonstrates that the signal amplifier provided by this invention has extremely high specificity.

[0127] Example 3

[0128] This embodiment is used to illustrate the condition optimization in the p53 detection method provided by the present invention.

[0129] (a) Concentration of lock probe

[0130] The p53 DNA fragment solution with a concentration of 5 nM was detected according to the method in Example 1. In step (3), different concentrations of lock probe solutions were used to investigate the effect of lock probe concentration on the detection results. The concentration gradient of the lock probe solution was set as follows: 10 nM, 20 nM, 50 nM, 100 nM, 150 nM, and 200 nM. The results are shown in Table 2 below.

[0131] Table 2

[0132] Lock-on probe concentration / nM Fluorescence value (au) 10 1314.3 20 6133.4 50 10076.3 100 12485.8 150 11390.6 200 11609.7

[0133] (ii) Reaction concentration of phi29 DNA polymerase

[0134] The p53 DNA fragment solution with a concentration of 5 nM was detected according to the method in Example 1. In step (3), different concentrations of phi29 DNA polymerase were used to investigate the effect of phi29 DNA polymerase concentration on the detection results. The amounts of phi29 DNA polymerase added to the reaction system were 0.5 U, 1.5 U, 3 U, 5 U, 7 U, and 10 U, respectively. The results are shown in Table 3 below.

[0135] Table 3

[0136] phi29 DNA polymerase addition amount / U Fluorescence value (au) 0.5 1904.0 1.5 6854.4 3 9900.8 5 11062.4 7 10616.3 10 10281.6

[0137] (III) Reaction concentration of dNTPs

[0138] The p53 DNA fragment solution with a concentration of 5 nM was detected according to the method in Example 1. In step (3), different concentrations of dNTPs were used to investigate the effect of dNTP concentration on the detection results. The concentration gradient of dNTPs was set as follows: 1 mM, 2 mM, 5 mM, 10 mM, 15 mM, and 20 mM. The results are shown in Table 4 below.

[0139] Table 4

[0140] dNTPs concentration / mM Fluorescence value (au) 1 1801.4 2 5854.7 5 10329.2 10 12610.8 15 12259.5 20 11709.3

[0141] (iv) RCA reaction time

[0142] The p53 DNA fragment solution with a concentration of 5 nM was detected according to the method in Example 1. In step (3), different RCA reaction times were used to investigate the effect of RCA reaction time on the detection results. The RCA reaction times were set to: 0.5 h, 1 h, 1.5 h, 2 h, 3 h, and 4.5 h. The results are shown in Table 5 below.

[0143] Table 5

[0144] RCA reaction time / h Fluorescence value (au) 0.5 1246.1 1 8307.2 1.5 10384.5 2 11630.2 3 10795.4 4.5 10951.3

[0145] Example 4

[0146] This embodiment is used to illustrate the effect of using the signal amplifier provided by the present invention for actual sample detection.

[0147] p53 DNA was added to human serum samples diluted 10-fold with NEBuffer 3.1 to form human serum p53 DNA solutions with concentrations of 20 pM, 50 pM, and 80 pM, and then detected according to the method in Example 1.

[0148] The results showed that the spiked recovery rate ranged from 95.0% to 103.0%, and the RSD ranged from 3.5% to 5.8% (n=5). This demonstrates that the signal amplifier provided by this invention exhibits extremely high specificity for actual sample detection and can be widely applied in practical scenarios such as clinical testing.

[0149] The preferred embodiments of the present invention have been described in detail above; however, the present invention is not limited thereto. Within the scope of the inventive concept, various simple modifications can be made to the technical solutions of the present invention, including combinations of various technical features in any other suitable manner. These simple modifications and combinations should also be considered as the content disclosed in the present invention and are all within the protection scope of the present invention.

Claims

1. A p53 gene detection signal amplifier, characterized in that, The signal amplifier includes a hairpin probe HP and a lock probe; The hairpin probe HP contains the HP-1 segment, which is complementary to the p53 DNA fragment, and the HP-2 segment, which is complementary to the lock probe.

2. The signal amplifier according to claim 1, wherein, The hairpin probe HP is fixed on the hairpin probe carrier. Preferably, the hairpin probe carrier is a micron magnetic bead. Preferably, the loading amount of the hairpin probe HP on the hairpin probe carrier is not less than 20 pmol / mg, and more preferably 20-50 pmol / mg. And / or, the 5' end of the hairpin probe HP has a modifying group for connection with the hairpin probe carrier, preferably the modifying group is biotin; And / or, the 5' end of the locking probe is phosphorylated; Preferably, the hairpin probe HP and the lock probe are stored independently.

3. The signal amplifier according to claim 1 or 2, wherein, The nucleotide sequence of the p53 DNA fragment is shown in SEQ ID NO:1; Preferably, the nucleotide sequence of the hairpin probe HP is as shown in SEQ ID NO:2; Preferably, the nucleotide sequence of the lock probe is shown in SEQ ID NO:

3.

4. A kit for detecting the p53 gene, characterized in that, The kit contains the following: (1) DNA probes: including hairpin probes (HP) and locking probes, wherein the hairpin probes (HP) contain an HP-1 segment complementary to the p53 DNA fragment and an HP-2 segment complementary to the locking probe; and, Optional, (2) enzymes: including endonucleases and DNA polymerases.

5. The kit according to claim 4, wherein, The kit also includes: (3) a hairpin probe carrier, preferably a micron magnetic bead; And / or, the 5' end of the hairpin probe HP has a modifying group for connection with the hairpin probe carrier, preferably the modifying group is biotin; And / or, the 5' end of the locking probe is phosphorylated; Preferably, the nucleotide sequence of the p53 DNA fragment is shown in SEQ ID NO:1; More preferably, the nucleotide sequence of the hairpin probe HP is shown in SEQ ID NO:2; More preferably, the nucleotide sequence of the lock probe is shown in SEQ ID NO:

3.

6. The kit according to claim 4, wherein, The endonuclease is selected from Nt.BstNBI nicking endonuclease; the DNA polymerase is selected from phi29 DNA polymerase, and preferably the enzymes in the kit also include DNA ligase; And / or, the kit further comprises (4) buffers: including buffer A and buffer B, wherein buffer A comprises 0.05-0.15M NaCl and 0.05-0.15M PBS buffer, the balance being water, and the pH is 7.2-7.5; buffer B comprises 0.05-0.15M NaCl, 0.05-0.15M PBS buffer and 0.01-0.1 vol% Tween 20, the balance being water, and the pH is 7.2-7.5; Preferably, the buffer further includes at least one of Nt.BstNBI reaction buffer, DNA ligase reaction buffer, and phi29 DNA polymerase reaction buffer.

7. The kit according to claim 4, wherein, The kit also includes (5) a detection reagent, preferably selected from molecular beacons; Preferably, the nucleotide sequence of the molecular beacon is shown in SEQ ID NO:4; Preferably, the molecular beacon is modified with a fluorescent group at its 5' end and with a quenching group corresponding to the fluorescent group at its 3' end; And / or, the kit further includes (6) adenosine triphosphate and / or dNTPs.

8. A method for detecting the p53 gene in a sample, characterized in that, The method includes: contacting and reacting the sample with the signal amplifier according to any one of claims 1-3, and detecting the reaction product using a detection reagent; Alternatively, the sample may be tested using the kit described in any one of claims 4-7.

9. The method according to claim 8, wherein, The method includes: (i) The hairpin probe vector immobilized with hairpin probe HP is brought into first contact with the sample. The p53 gene fragment in the sample causes the hairpin structure of the hairpin probe HP to open. The HP-1 segment in the opened hairpin probe HP is complementary to the p53 gene fragment to form a double-stranded structure. Then, the first contact product is brought into second contact with a nuclease to cleave the double-stranded structure, release the p53 gene fragment, and leave the hairpin probe HP fragment containing the HP-2 segment on the hairpin probe vector. (ii) In the presence of adenosine triphosphate, the second contact product, the lock probe and the DNA ligase are made into a third contact, so that the lock probe is complementary to the HP-2 segment in the residual hairpin probe HP fragment on the hairpin probe carrier. Then, the third contact product is made into a fourth contact with the DNA polymerase in the presence of dNTPs, so that it undergoes a rolling circle amplification reaction. (iii) The fourth contact product was detected using a detection reagent.

10. The method according to claim 9, wherein, In step (i), the conditions for the first contact include a temperature of 35-40°C and a time of 1-5 hours; And / or, in step (i), the conditions for the second contact include a temperature of 53-58°C and a time of 40-80 min; Preferably, in step (i), the content of the p53 gene in the sample is such that the concentration of the p53 gene fragment in the first contact system is not less than 5 pM; Preferably, in step (i), the amount of hairpin probe HP used is 50-100 nM relative to 1 unit of nucleating endonuclease.

11. The method according to claim 9, wherein, In step (ii), the conditions for the third contact include a temperature of 35-40°C and a time of 40-80 min; And / or, in step (ii), the conditions for the fourth contact include a temperature of 35-40°C and a time of 1-5 hours; Preferably, step (ii) further includes heating the second contact product and the lock probe at 90-100°C for 1-5 min in the presence of adenosine triphosphate before the third contact, then cooling to 20-30°C at a cooling rate not exceeding 3°C / min, and holding at this temperature for more than 2 h, so that the lock probe and the second contact product are fully hybridized. Preferably, in step (ii), the amount of adenosine triphosphate added is such that its concentration in the third contact system is 0.5-5 μM, more preferably 0.5-2.5 μM; Preferably, in step (ii), the amount of DNA ligase used is 1 × 10⁻⁶. 3 -5×10 3 Units / mL, preferably 1.5 × 10⁻⁶ 3 -2.5×10 3 Unit / mL; Preferably, step (ii) further includes terminating the rolling circle amplification reaction after the fourth contact. The preferred method for terminating the rolling circle amplification reaction includes raising the temperature of the fourth contact system to 60-70°C and holding it at that temperature for 5-15 minutes. Preferably, in step (ii), the amount of dNTPs added is such that its concentration in the fourth contact system is 50-150 μM, more preferably 50-100 μM; Preferably, in step (ii), the amount of DNA polymerase used is 1-30 units / mL, more preferably 5-20 units / mL.

12. The method according to claim 9, wherein, In step (iii), the detection reagent is selected from molecular beacons, preferably the nucleotide sequence of the molecular beacon is shown in SEQ ID NO:4; Preferably, the molecular beacon is modified with a fluorescent group at its 5' end and with a quenching group corresponding to the fluorescent group at its 3' end; Preferably, the detection of the fourth contact product in step (iii) is performed by adding a molecular beacon to the fourth contact product and reacting it at 20-30°C for 30-90 min, and then performing fluorescence detection on the reaction product to quantitatively detect the p53 gene in the sample. The preferred fluorescence detection conditions include: excitation wavelength 486 nm and emission wavelength 500-600 nm.

Images