A kit for improving the stability of CY5 signal in a direct amplification PCR system of a blood sample and application

Abstract

This invention relates to a kit and application for improving the stability of CY5 signals in direct amplification PCR systems of blood samples, belonging to the field of gene detection technology. The invention provides a CY5-modified probe carrying a protecting group, which is formed by coupling an activated protecting group containing ester and hydrazone bonds with a CY5-modified probe. It also provides a PCR detection method using the aforementioned CY5-modified probe carrying a protecting group for non-disease diagnosis or treatment purposes. The probe designed in this invention can break ester bonds at the high temperature (90-95℃) of the PCR reaction. Simultaneously, as the temperature increases, the pH changes; for every 10℃ increase in temperature, the pKa value decreases by approximately 0.3. Under suitable pH conditions, the protecting group itself breaks, further reducing the steric hindrance effect of the probe and releasing the target probe. This solves the problems of severe signal interference, low detection sensitivity, and poor specificity of CY5 fluorescent probes in direct amplification PCR of blood samples.

Description

Technical Field

[0001] This invention belongs to the field of gene detection technology, and specifically relates to a kit and its application for improving the stability of CY5 signal in direct amplification PCR systems of blood samples. Background Technology

[0002] Molecular diagnostics is one of the core technologies in modern medical diagnostics. Among them, PCR technology, due to its high sensitivity and specificity, is widely used in various scenarios such as genotyping, pathogen detection, and genetic disease screening. In recent years, the rapid development of direct amplification PCR technology has propelled molecular diagnostics into the "hands-free" era. The core advantage of this technology is that it eliminates the need for nucleic acid extraction from blood samples. Raw blood samples can be directly added to the PCR reaction system, achieving an integrated operation from sample processing to amplified product detection. This not only significantly shortens the detection process time and reduces the risk of sample contamination during nucleic acid extraction, but also effectively increases detection throughput and reduces detection costs. It has irreplaceable application value in clinical rapid diagnosis and emergency prevention and control scenarios.

[0003] The core breakthrough of blood direct amplification PCR technology lies in the development of anti-inhibition polymerase and the optimization of the lysis system. At present, domestic and foreign manufacturers have carried out a lot of pioneering work in the research and development of anti-inhibition polymerase, which has basically solved the problem of inhibition of amplification reaction by common PCR inhibitors such as heme, heparin and protein in blood samples, making direct PCR amplification of blood samples possible.

[0004] However, in multiplex detection scenarios using direct amplification PCR in blood, especially when using the CY5 fluorescence channel as the detection channel, significant signal interference challenges remain. This has led to the current standard human genomic typing kits primarily using hydrophilic FAM / VIC / ROX channels, with the hydrophobic CY5 fluorescence channel rarely introduced into direct amplification PCR systems. CY5 fluorescent dyes have significant potential for multiplex PCR detection due to their advantages such as long emission wavelengths (above 650 nm), high fluorescence intensity, and low overlap with other fluorescence channels. However, their application in direct amplification systems is severely limited by the following factors: 1. Background fluorescence quenching and autofluorescence interference: The iron porphyrin structure of hemoglobin in blood samples exhibits strong absorption and fluorescence quenching capabilities at wavelengths above 650 nm, leading to significant attenuation of the CY5 fluorescence signal. This results in unstable PCR amplification curves, abnormal baselines, and affects the accuracy of the detection results. 2. Non-specific adsorption interference: CY5 fluorescent probes are highly hydrophobic. After blood samples are lysed, a large number of cell membrane fragments and denatured proteins with hydrophobic groups are released. CY5 probes easily undergo non-specific adsorption with these substances, preventing them from fully binding to the target nucleic acid, significantly inhibiting fluorescence signal intensity and reducing detection sensitivity. 3. Oxidative damage to the fluorescent group: Blood samples contain various oxidatively active substances that can participate in the oxidation reaction of the CY5 fluorescent group, leading to structural damage and loss of luminescence, further exacerbating signal interference.

[0005] To address the aforementioned challenges in applying the CY5 channel to direct amplification PCR in blood, several improvement schemes have been proposed in the existing technology. However, all of these have significant drawbacks and cannot meet the actual needs of clinical testing, as detailed below:

[0006] 1. Adding surfactants to the reaction system: Adding surfactants such as BSA (bovine serum albumin), Tween, and PEG to the PCR reaction system attempts to improve the solubility of the CY5 probe and reduce its non-specific adsorption. However, high concentrations of surfactants alter the viscosity and ionic strength of the reaction system, interfering with the PCR reaction kinetics. This not only inhibits the efficiency of PCR amplification but also affects the specific binding of the probe to the target nucleic acid. Overall, the improvement effect is not significant and cannot fundamentally solve the signal interference problem.

[0007] 2. Hydrophilic modification of CY5 probes: Hydrophilic groups are coupled to the CY5 fluorescent group using chemical modification techniques to increase the probe's hydrophilicity and reduce its non-specific adsorption to hydrophobic substances in blood. However, this method has limited applicability to direct amplification of blood samples. Because blood samples contain a large amount of cell membrane debris, even with hydrophilic modification of the CY5 probe, it is still difficult to completely avoid the specific adsorption of the probe to cell membrane debris, resulting in unstable fluorescence signals and poor detection repeatability.

[0008] 3. Replace fluorescent probe types: Use other types of long-wavelength fluorescent probes, such as CY5.5 and Alexa Fluor 680, to replace the CY5 probe in an attempt to avoid fluorescence quenching interference from hemoglobin. However, the amplification signal intensity of these fluorescent probes is lower than that of conventional CY5 probes. In low-concentration target detection scenarios, this can easily lead to false negative results, especially in multiplex PCR reaction systems, where the insufficient signal intensity is even more pronounced and cannot meet the sensitivity requirements of multiplex detection.

[0009] 4. Sample pretreatment or nucleic acid extraction: Blood samples are pretreated by heating or nucleic acid is extracted in advance to remove interfering substances in the blood before PCR detection. Although this method can reduce interference to some extent, it increases the number of detection steps, which not only prolongs the detection time but also increases the risk of sample contamination. This contradicts the core advantages of blood direct amplification PCR—"extraction-free and rapid"—and cannot meet the needs of rapid clinical testing.

[0010] In summary, current technologies cannot effectively address the signal interference problem of the CY5 fluorescent channel in direct amplification PCR of blood, thus limiting its application in blood direct amplification multiplex detection. Therefore, developing a CY5 probe modification technology that can fundamentally solve the above-mentioned technical deficiencies and is compatible with the blood direct amplification PCR system has significant clinical implications and application value. Summary of the Invention

[0011] To address the shortcomings of existing technologies and to solve the problems of severe signal interference, low detection sensitivity, and poor specificity of CY5 fluorescent probes in direct amplification PCR of blood samples, this invention aims to design and provide a temperature-responsive protective group-modified CY5 probe, as well as a kit for improving the stability of CY5 signals in direct amplification PCR systems of blood samples. By designing a temperature-responsive protective group containing hydrazone and ester bonds to modify the CY5 probe, the high-temperature conditions of the PCR reaction are used to achieve controlled cleavage of the protective group, while reducing interference from blood components on the CY5 probe, thereby improving the accuracy, sensitivity, and stability of direct amplification PCR detection of blood samples.

[0012] The technical principle of this invention is as follows: The protecting group designed in this invention is an organic molecule containing hydrazone and ester bonds. The ester bonds can be broken at the high temperature (90-95℃) of the PCR reaction. Simultaneously, as the temperature increases, the pH changes; for every 10℃ increase in temperature, the pKa value decreases by approximately 0.3. Under suitable pH conditions, the protecting group itself breaks down, further reducing the steric hindrance effect of the probe and releasing the target probe. Furthermore, during the heating process, various proteins in the blood preferentially denature and cell membranes lyse, making them less likely to bind to the CY5 probe, further reducing the binding efficiency with the target probe, increasing the effective concentration in the system, and reducing the interference of blood components on the target probe.

[0013] To achieve the above objectives, the present invention adopts the following technical solution:

[0014] On one hand, the present invention provides a CY5-modified probe carrying a protecting group, wherein the CY5-modified probe carrying the protecting group is formed by coupling an activated protecting group containing ester and hydrazone bonds with a CY5-modified probe.

[0015] Existing protecting groups are mostly single-structure (containing only hydrazone bonds or only ester bonds), which cannot achieve stepwise and controllable cleavage under high-temperature PCR conditions, and cannot simultaneously meet the dual requirements of "reducing blood interference" and "probe activity release". The dual-structure protecting group designed in this invention first undergoes ester bond cleavage at 90-95℃, followed by hydrazone bond cleavage as the system pH changes, releasing probe activity stepwise. This avoids non-specific binding of the probe to blood components at room temperature and precisely activates the probe during the PCR reaction.

[0016] A CY5-modified probe carrying a protecting group, the structural formula of which is shown in Formula I below:

[0017] Formula I.

[0018] Secondly, the present invention provides a method for preparing a CY5-modified probe carrying a protecting group, comprising the following steps:

[0019] Weigh p-hydroxybenzaldehyde and methyl hydrazine formate, and carry out a reflux reaction in a solvent to obtain a hydrazone intermediate;

[0020] The above-mentioned hydrazone intermediate was reacted with ethyl bromoacetate in the presence of a catalyst to obtain a precursor containing protecting groups with ester and hydrazone structures;

[0021] After purifying the above-mentioned protecting group precursor, an activating reagent was added for activation to obtain an active protecting group that can be coupled with the CY5 probe.

[0022] Weigh the above-mentioned active protecting group and perform a coupling reaction with the Cy5-modified probe, purify it, and obtain the CY5-modified probe carrying the protecting group.

[0023] The preparation method described herein, wherein the heating reflux reaction conditions are: time 6-8 h, temperature 75-85 °C; these reaction conditions ensure that p-hydroxybenzaldehyde and methyl hydrazine formate react fully, improve the yield of hydrazone intermediates, and avoid the formation of byproducts.

[0024] The mass ratio (or molar ratio) of p-hydroxybenzaldehyde and methyl hydrazine carbamate is 1:1 to 1.2;

[0025] The solvent is at least one of anhydrous ethanol, anhydrous methanol, isopropanol, acetonitrile, N,N-dimethylformamide, dimethyl sulfoxide, tetrahydrofuran, and 1,4-dioxane; the amount of anhydrous ethanol as a solvent is 5-15 mL / mmol of p-hydroxybenzaldehyde.

[0026] The catalyst is at least one selected from potassium carbonate, sodium carbonate, cesium carbonate, sodium bicarbonate, triethylamine, N,N-diisopropylethylamine (DIPEA), pyridine, sodium hydroxide, and potassium hydroxide; the molar ratio of the catalyst to ethyl bromoacetate is 1.0 to 1.5:1.

[0027] The activating agent is at least one of N-hydroxysuccinimide, 1-hydroxybenzotriazole, and 1-hydroxy-7-azabenzotriazole; the molar ratio of the activating agent to the protecting group precursor is 1.0 to 1.5:1.

[0028] The conditions for the coupling reaction are: pH 7.5~8.5, reaction time 12~16 h.

[0029] Thirdly, the present invention provides the application of a CY5-modified probe carrying a protective group in direct amplification PCR detection of blood for non-disease diagnosis or treatment purposes.

[0030] Fourthly, the present invention provides the application of the CY5-modified probe carrying a protective group in the preparation of a kit for improving the stability of CY5 signal in a direct amplification PCR system of blood samples.

[0031] In existing technologies, improving the CY5 signal in blood-based direct amplification PCR systems mainly involves optimizing PCR buffer components or increasing probe dosage. However, these methods cannot fundamentally solve the problem of blood components interfering with the probe and are prone to non-specific amplification and large fluctuations in fluorescence signals. In contrast, the modified probe of this invention can reduce the non-specific binding of blood components to the probe from the source by utilizing the temperature-responsive cleavage characteristics of the protecting group, thereby stabilizing the CY5 channel fluorescence signal, reducing signal fluctuations, and improving detection repeatability. When applied to reagent kit preparation, it can give the kit the core advantages of "nucleic acid extraction-free, high sensitivity, and high stability." Compared with existing kits, it has significant differentiation and innovation, and the market demand for the kit is broad, indicating a promising industrialization prospect.

[0032] Fifthly, the present invention provides a PCR detection method for non-disease diagnosis or treatment purposes using the CY5-modified probe carrying a protective group, the PCR detection method comprising the following steps:

[0033] Prepare the PCR amplification system as follows: 0.1-0.2 μL PCR polymerase, 0.2-0.3 μL dNTP Mix, 8-12 μL buffer, 0.3-0.7 μL upstream primer, 0.3-0.7 μL downstream primer, 0.2-0.5 μL CY5-modified probe with a protecting group, 1-3 μL sample to be tested, and purified water to make up to 20 μL.

[0034] The reaction was performed according to the PCR amplification procedure, and the fluorescence signal of the CY5 channel was detected.

[0035] The PCR detection method described above uses a raw blood sample that has not undergone nucleic acid extraction as the test sample.

[0036] The buffer solution contains Mg 2+ Or the buffer solution contains no Mg 2+ Buffer solution and Mg 2+ Combinations of solutions.

[0037] The PCR detection method described above includes the following PCR amplification program: 90-95℃ for 10 min pre-denaturation; 90-95℃ for 15 s, 60℃ for 34 s, 10 cycles for pre-amplification; 95℃ for 15 s, 60℃ for 34 s, 50 cycles for fluorescence acquisition.

[0038] By combining the modified probes with an optimized PCR system and segmented amplification program, a complete blood direct amplification PCR detection protocol was formed. The pre-denaturation stage at 95℃ for 10 min effectively lyses blood cell membranes and denatures blood proteins, mitigating interference from blood inhibitors and triggering initial breakage of protective groups. Compared to existing conventional pre-denaturation programs (95℃ for 2-3 min), this protocol is more suitable for blood direct amplification. The 10-cycle pre-amplification stage achieves preliminary enrichment of low-abundance target nucleic acids, reducing signal interference in subsequent fluorescence acquisition stages. The 50-cycle fluorescence acquisition stage, with fluorescence acquisition during the 60℃ annealing extension stage, ensures the specificity and accuracy of the fluorescence signal. This detection method is simple to operate, time-efficient, and low-cost, requiring no specialized nucleic acid extraction equipment or technicians. It can be widely applied to blood sample testing in various non-disease diagnostic scenarios, addressing the technical pain points of cumbersome operation, low sensitivity, and unstable signals compared to existing methods.

[0039] In a sixth aspect, the present invention provides a kit for improving the CY5 signal in a direct amplification PCR system of blood samples, comprising the CY5-modified probe carrying a protective group.

[0040] When the target gene is the Mthfr677 gene, the CY5-modified probe carrying the protective group is: NH2-CY5-AATCGGCTCCCGC-MGB. The upstream primer is 5-AGGCTGACCTGAAGCACTTGAA-3, and the downstream primer is 5-CTCAAAGAAAAGCTGCGTGATGA-3. Depending on the target gene, the nucleic acid sequence fragment matching the target gene sequence in the CY5-modified probe carrying the protective group can be replaced. Accurate detection can be achieved under PCR amplification conditions of 90-95℃.

[0041] The kit may further include an inhibitory PCR polymerase and a PCR buffer (containing Mg). 2+ The mixture of dNTPs, upstream and downstream specific primers, and enzyme-free and nucleic acid-free purified water has been precisely optimized in proportion and can be directly used to prepare PCR reaction systems without the need for additional reagents, making it convenient to use.

[0042] In a seventh aspect, the present invention provides the application of the kit in the detection of target genes in blood samples using a PCR amplification method.

[0043] Compared with the prior art, the present invention has the following beneficial effects:

[0044] 1. Fundamentally solves the signal interference problem of CY5 probe in blood direct amplification: This invention modifies the CY5 probe with a temperature-responsive protecting group before the PCR reaction. The protecting group can block the hydrophobic region of the CY5 probe, reducing its non-specific adsorption to cell membrane debris and denatured proteins in the blood. Under the high temperature of PCR, the protecting group breaks down and releases the active probe. At the same time, the interfering proteins in the blood are preferentially denatured and inactivated. The dual effect significantly reduces the interference of blood components on the CY5 probe, avoiding problems such as background fluorescence quenching and signal attenuation, so that the CY5 channel can be stably used in blood direct amplification PCR.

[0045] 2. Controllability and thoroughness of protecting group breakage: The protecting group contains both hydrazone bonds and ester bonds. The high temperature of PCR (90-95℃) can trigger the breakage of ester bonds, while the pH change caused by the increase in temperature triggers the breakage of hydrazone bonds. The dual breakage mechanism ensures that the protecting group is completely broken and detached during the PCR reaction, completely eliminating the steric hindrance effect, so that the target probe can fully bind to the target nucleic acid and improve the detection sensitivity.

[0046] 3. Does not affect PCR reaction efficiency and detection specificity: The protecting group of this invention only breaks at the high temperature of PCR and remains stable at room temperature. It does not interfere with the viscosity and ionic strength of the PCR reaction system, nor does it affect the activity of the anti-inhibitory polymerase. Furthermore, the cleavage products of the protecting group do not inhibit PCR amplification and probe binding, ensuring the specificity and accuracy of the detection.

[0047] 4. Simple operation and compatible with existing detection systems: The probe preparation process of this invention is simple and can be achieved through conventional chemical synthesis and purification methods. It does not require modification of existing blood direct amplification PCR instruments and reaction systems, and can be directly adapted to existing detection platforms without adding additional detection steps, thus retaining the core advantages of blood direct amplification PCR: "pickup-free, rapid, and low-cost".

[0048] 5. Wide range of applications: The probes of this invention can be designed with corresponding nucleic acid sequences according to different target genes, and are suitable for direct amplification PCR detection of blood of multiple genes, especially for multiplex PCR detection scenarios. It can give full play to the advantages of the CY5 fluorescence channel and improve the throughput and accuracy of multiplex detection. For example, it can be applied to the genotyping detection of genes related to folic acid metabolism such as MTHFR 677 site and 1298 site, providing reliable technical support for clinical diagnosis.

[0049] 6. Avoid oxidative damage to fluorescent groups: The protecting group can provide a certain degree of protection to the CY5 fluorescent group at room temperature, reducing the oxidative damage to the CY5 fluorescent group by oxidative active substances in the blood, and further ensuring the stability and intensity of the fluorescence signal. Attached Figure Description

[0050] Figure 1Graph showing signal values ​​from PCR amplification using different probes;

[0051] Figure 2 This is a schematic diagram of the reaction process of the present invention. Detailed Implementation

[0052] The present invention will be further described below with reference to the accompanying drawings and embodiments.

[0053] Example 1: Synthesis and purification of CY5-modified probes carrying protecting groups

[0054] 1. Design and synthesis of temperature-responsive protecting groups:

[0055] (1) Using p-hydroxybenzaldehyde and methyl hydrazine as raw materials, the molar ratio of p-hydroxybenzaldehyde and methyl hydrazine is 1:1.05. The reaction is carried out in anhydrous ethanol under reflux for 6-8 hours at 80℃ to obtain the hydrazone intermediate. The amount of anhydrous ethanol used as solvent is 10 mL / mmol of p-hydroxybenzaldehyde.

[0056] (2) The hydrazone intermediate was reacted with ethyl bromoacetate under the catalysis of potassium carbonate to obtain a precursor containing a protecting group with ester bond and hydrazone bond structure; the molar ratio of potassium carbonate to ethyl bromoacetate catalyst was 1.2:1.

[0057] (3) After purifying the protecting group precursor by silica gel column chromatography, it is activated by N-hydroxysuccinimide (NHS) (the molar ratio of the activating reagent N-hydroxysuccinimide to the protecting group precursor is 1.2:1) to obtain a protecting group that can be coupled to the Cy5 probe, as shown in Formula I below. Alternatively, the structure of this protecting group can be purchased directly and activated by NHS. The structural formula of the protecting group is shown in Formula I below:

[0058] Formula I.

[0059] 2. Modification and purification selection of Cy5 probes:

[0060] Commercially synthesized Cy5 probes (with an amino modification site at the 5' end) were prepared by reacting the aforementioned active protecting group with the Cy5 probe in phosphate buffer (pH 8.0) at room temperature for 12-16 hours, allowing the protecting group to couple to the probe via an amide bond. After the reaction, the modified probe was purified using high-performance liquid chromatography (HPLC), and the target product with a purity greater than 95% was collected, yielding the Cy5-modified probe with the protecting group.

[0061] This embodiment uses the Mthfr677 gene as an example. The synthesized sequence (Anhui General Biosynthesis) is Sequence 1: NH2-CY5-AATCGGCTCCCGC-MGB (the nucleic acid sequence is shown as SEQ ID NO.1: AATCGGCTCCCGC). The synthesized sequence: the protecting group -CO-NH-CY5-AATCGGCTCCCGC-MGB is used as the probe of this invention.

[0062] Comparative Example 1:

[0063] Taking the Mthfr677 gene as an example, the synthesized sequence FAM-AATCGGCTCCCGC-MGB was used as the modified probe.

[0064] Comparative Example 2:

[0065] Taking the Mthfr677 gene as an example, the synthesized sequence: VIC-AATCGGCTCCCGC-MGB was used as the modified probe.

[0066] Comparative Example 3:

[0067] Taking the Mthfr677 gene as an example, the synthesized sequence CY5-AATCGGCTCCCGC-MGB was used as the modified probe.

[0068] Comparative Example 4:

[0069] Taking the Mthfr677 gene as an example, the synthesized sequence: ROX-AATCGGCTCCCGC-MGB was used as the modified probe.

[0070] Example 2: Comparison of baseline signal and amplification performance of different labeled probes

[0071] (1) Taking the human Mthfr677 gene as an example, it was synthesized by Anhui General Biotechnology Co., Ltd. Primer sequences were designed: the nucleic acid sequence of the upstream primer is shown in SEQ ID NO.2: 5-AGGCTGACCTGAAGCACTTGAA-3 and the nucleic acid sequence of the downstream primer is shown in SEQ ID NO.3: 5-CTCAAAGAAAAGCTGCGTGATGA-3.

[0072] The blood PCR amplification reagent was purchased from Zhuhai Baorui Biotechnology Co., Ltd., product name: 50×2*Superstart Direct Premix (Probe qPCR), catalog number: MD201.

[0073] Configure the amplification system according to the instructions, as shown in Table 1 below:

[0074] Table 1 Amplification System Product Name Volume (uL) 50×Superstart Direct Enzyme 0.3 <![CDATA[2×Superstart Direct Buffer (Mg 2+ free)]]> 10 <![CDATA[250mM MgCl2]]> 0.4 50×dNTP Mix 0.25 upstream primer 0.5 Downstream primer 0.5 probe* 0.3 blood 2 Purified water To 20

[0075] Wherein, * represents the addition of probes with different signal markers as in Example 1 and Comparative Examples 1-4 for comparison.

[0076] PCR amplification program: 95℃ for 10 min, pre-denaturation; 95℃ for 15 s, 60℃ for 34 s, 10 cycles for pre-amplification; 95℃ for 15 s, 60℃ for 34 s, 50 cycles for fluorescence acquisition.

[0077] The results are as follows Figure 1 The graph shows the signal values ​​of PCR amplification using different probes. It can be seen that the fluorescence amplification curve of the probe of this invention exhibits a typical "S" shape, with a steep rise and a significantly higher fluorescence signal peak than other existing modification methods (probes modified with a single hydrazone bond, probes modified with a single ester bond, and probes without a protecting group). Furthermore, the inflection point of the amplification curve is earlier. This result directly corroborates that the signal values ​​(fluorescence signal intensity, Ct value) of the probe of this invention are superior to other modification methods.

[0078] (2) Using the same method as in Example 2, the baseline was set to 3-15, the threshold was set to 100000, and the Ct value was generated. The specific results are shown in Table 2 below:

[0079] Table 2 Ct values signal channel probe Ct value FAM Comparative Example 1 Synthesized Sequence 2 35.21 VIC Comparative Example 2 Synthesized Sequence 3 35.31 CY5 Sequence 4 synthesized in Comparative Example 3 Under ROX Sequence 5 synthesized in Comparative Example 4 36.84 Protecting group +CY5 Sequence 6 synthesized in Example 1 35.44

[0080] The test results above show that the baseline of conventionally synthesized CY5 probes is unstable and drifts, easily leading to abnormal Ct values ​​when using fixed thresholds and baselines. This phenomenon did not occur when using FAM, VIC, and ROX probes. Therefore, the selection range is limited when using the CY5 channel for direct blood amplification. Meanwhile, probes with added protecting groups significantly reduce interference from blood.

[0081] Example 3: Effect of different temperatures on the improved probe

[0082] The PCR reaction temperatures in Example 2 were set to 85℃, 90℃, and 95℃ for comparative experiments. The CY5-modified probe with a protective group designed in Example 1 was used as the probe. All other reaction conditions were the same as in Example 2. The signal intensity (relative) was measured, and the results are shown in Table 3 below.

[0083] Table 3 Signal strength results data Temperature (°C) 85 90 95 Signal strength (relative) 54.32% 85.63% 98.27%

[0084] As can be seen, the relative signal intensity of the probe of this invention shows a significant upward trend with the increase of PCR reaction temperature (pre-denaturation and denaturation stages), which confirms the core technical principle of this invention—the protective group of the probe of this invention contains a dual structure of hydrazone bonds and ester bonds, and its breaking efficiency is positively correlated with the PCR reaction temperature: when the temperature reaches 95℃, the ester bond breaks completely, and at the same time, the pH of the system changes with the increase of temperature, which makes it easier to trigger the breaking of hydrazone bonds, the removal of the protective group is more thorough, the probe activity is released more fully, the binding efficiency with the target nucleic acid is higher, and the fluorescence signal intensity also increases accordingly.

[0085] At temperatures of 85℃ and 90℃, the ester and hydrazone bonds are not fully broken, resulting in more residual protecting groups, significant steric hindrance, and ineffective release of probe activity, leading to low signal intensity. The signal intensity is best at 95℃.

[0086] Example 3: Response of the modified probe to different pH values

[0087] The modified Cy5 probe synthesized in Example 1 was incubated in buffer solutions at pH 6.5, 7.5, 8.0, and 9.0 for 30 seconds, respectively. Its fluorescence signal was detected, and the fluorescence intensity recovery rate was calculated as shown in Formula 1.

[0088] Fluorescence intensity recovery rate (%) = F_experimental group / F_control group × 100% (Formula 1)

[0089] The results are shown in Table 4 below. With the untreated probe as the control group, the fluorescence intensity recovery rate of the probe exceeded 90% in the pH range of 6.5-7.5; while under the condition of pH greater than 9.0, the fluorescence intensity recovery rate was less than 40%. This result verifies the precise response characteristics of the protecting group to the pH range of the PCR system.

[0090] Table 4. Signal intensity results after incubation at different pH levels pH 6.5 7.5 8.5 9.0 Signal strength recovery rate 93.28% 95.02% 66.76% 39.85%

[0091] In summary, the schematic diagram of the reaction process of this invention is as follows: Figure 2As shown, the specific explanation is as follows: (1) First, during the PCR heating, DNA denaturation, and enzyme activation process, blood components and cells undergo denaturation or lysis. Because the CY5 probe has a protective group, it does not interact with the components in the blood. (2) As the temperature rises and the time increases, the protective group of the probe and the ester bond coupled to the probe undergo hydrolysis, releasing the target probe to bind to the PCR product. (3) As the temperature rises, the pH value of the buffer system drops from 9.0 to about 7.5, causing the hydrazone bond (–N=N–) inside the protective group to break, releasing the target probe. The release process of the protective group exhibits a biphasic dynamic characteristic: the initial stage is a rapid release period, releasing most of the active probe; then it enters a slow release period, with the remaining protective group gradually released. This biphasic release characteristic is completely consistent with the pre-amplification stage of the PCR reaction, ensuring that the probe can gradually recover its activity during the binding process with the target sequence.

Claims

1. A CY5 modified probe carrying a protecting group, characterized in that, The CY5-modified probe carrying the protecting group is formed by coupling the activated protecting group containing ester and hydrazone bonds with the CY5-modified probe.

2. A CY5 modified probe carrying a protecting group, characterized in that, The structural formula of the protecting group is shown in Formula I below: Formula I.

3. The method for preparing a CY5-modified probe carrying a protecting group as described in claim 1, characterized in that, Includes the following steps: Weigh p-hydroxybenzaldehyde and methyl hydrazine formate, and carry out a reflux reaction in a solvent to obtain a hydrazone intermediate; The above-mentioned hydrazone intermediate was reacted with ethyl bromoacetate in the presence of a catalyst to obtain a precursor containing protecting groups with ester and hydrazone structures. After purifying the above-mentioned protecting group precursor, an activating reagent was added for activation to obtain an active protecting group that can be coupled with the CY5 probe. Weigh the above-mentioned active protecting group and perform a coupling reaction with the Cy5-modified probe, purify it, and obtain the CY5-modified probe carrying the protecting group.

4. The preparation method according to claim 2, characterized in that, The conditions for the heating reflux reaction are: time 6-8 hours, temperature 75-85℃; The molar ratio of p-hydroxybenzaldehyde to methyl hydrazine is 1:1 to 1.2; The solvent is at least one of anhydrous ethanol, anhydrous methanol, isopropanol, acetonitrile, N,N-dimethylformamide, dimethyl sulfoxide, tetrahydrofuran, and 1,4-dioxane. The catalyst is at least one of potassium carbonate, sodium carbonate, cesium carbonate, sodium bicarbonate, triethylamine, N,N-diisopropylethylamine, pyridine, sodium hydroxide, and potassium hydroxide. The activating agent is at least one of N-hydroxysuccinimide, 1-hydroxybenzotriazole, and 1-hydroxy-7-azabenzotriazole; The conditions for the coupling reaction are: pH 7.5~8.5, reaction time 12~16 h.

5. The application of the CY5-modified probe carrying a protective group as described in claim 1 in direct amplification PCR detection of blood for non-disease diagnosis or treatment purposes.

6. The application of the CY5-modified probe carrying a protective group as described in claim 1 in the preparation of a kit for improving the stability of CY5 signal in a direct amplification PCR system of blood samples.

7. A PCR detection method for non-disease diagnosis or treatment purposes using a CY5-modified probe carrying a protecting group as described in claim 1, characterized in that, The PCR detection method includes the following steps: Prepare the PCR amplification system: 0.1~0.2 uL PCR polymerase, 0.2~0.3 uL dNTP Mix, 8~12 uL buffer, 0.3~0.7 uL upstream primer, 0.3~0.7 uL downstream primer, 0.2~0.5 uL CY5 modified probe with a protecting group, 1~3 uL sample to be tested, and purified water to make up to 20 uL.

8. The PCR detection method as described in claim 7, characterized in that, The sample to be tested is a raw blood sample that has not undergone nucleic acid extraction; The buffer comprises Mg 2+ or the buffer comprises no Mg 2+ The buffer is combined with a Mg 2+ containing solution; The PCR amplification program is as follows: 90~95℃ for 10 min, pre-denaturation; 90~95℃ for 15 s, 60℃ for 34 s, 10 cycles for pre-amplification; 95℃ for 15 s, 60℃ for 34 s, 50 cycles for fluorescence acquisition.

9. A kit for improving the stability of CY5 signal in a direct amplification PCR system of blood samples, characterized in that, A CY5-modified probe containing a protecting group as described in claim 1.

10. The application of the kit as described in claim 9 in the detection of the target gene in blood samples using a PCR amplification method.

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