A multi-color fluorescence channel detection method based on PCR and application thereof

Abstract

The application discloses a PCR-based multicolor fluorescence channel detection method and application, and belongs to the technical field of gene detection. The method adds heat-stable RNase HII ribonuclease and a fluorescent probe doped with RNA bases and LNA bases in a PCR reaction system, the fluorescent probe can be compatible with multicolor fluorescence channels; meanwhile, specific primers in the PCR reaction system are introduced with the same TAG as the sequence of the fluorescent probe at the 5' end, and a base bridge sequence is inserted between the TAG and the sequence of the specific primer, thereby forming specific long primers; the combination of the specific long primers and specific short primers can enable the fluorescent probe to combine with the complementary chain without competing with the extension chain of the specific primer, thereby improving the annealing efficiency of the fluorescent probe. The application utilizes RNase HII ribonuclease to cut the probe, so that the fluorescent groups and quenching groups coupled at both ends of the probe are completely separated, a strong fluorescent signal is emitted, and the application has the advantages of low cost, high specificity, multicolor channels and strong signal.

Description

Technical Field

[0001] The present invention relates to the technical field of gene detection, and in particular to a PCR-based multi-color fluorescence channel detection method and application. Background Art

[0002] Pathogens refer to organisms that can cause infectious diseases in humans or animals and plants, including viruses, mycoplasmas, chlamydia, bacteria, fungi, protozoa, parasites and other microorganisms. Pathogen detection is an important means of clinical diagnosis and prevention and control of infectious diseases. It requires rapid, comprehensive and accurate identification of pathogen species, types, drug resistance and virulence. In existing genetic testing technologies, under the basic premise of ensuring specificity, there are two main requirements for pathogen detection: one is rapid detection, including on-site rapid detection; such as existing constant temperature amplification technologies, such as LAMP and RPA, which can shorten the reaction time, and ANCA technology, which uses Ago protein for cascade enzyme cleavage, which can quickly obtain detectable fluorescent signals (Jang H, Song J, Kim S, et al. ANCA: artificial nucleic acid circuit with argonaute protein for one-step isothermal detection of antibiotic-resistant bacteria[J]. Nature Communications, 2023, 14(1): 8033.). The second is signal visualization and one-pot reaction processing; this direction is exemplified by the combination of isothermal amplification and CRISPR technology for rapid pathogen detection. For example, Joung et al. used isothermal amplification and CRISPR-Cas12b to rapidly detect the novel coronavirus, obtaining results within 40 minutes (Joung J, Ladha A, Saito M, et al. Point-of-care testing for COVID-19 using SHERLOCK diagnostics[J]. MedRxiv, 2020.). Liu et al. acylated crRNA to block the cleavage ability of CRISPR-Cas12a. After enriching the template using isothermal amplification (RPA), they used 365nm ultraviolet light to unblock the crRNA, cleaving the fluorescent probe and releasing the signal. Based on this, they achieved the integration of isothermal amplification reaction and CRISPR reaction in one pot (Liu P, Lin Y, Zhuo X, et al. UniversalcrRNA Acylation Strategy for Robust Photo-Initiated One-Pot CRISPR–Cas12aNucleic Acid Diagnostics[J]. Angewandte Chemie International Edition, 2024:e202401486.).

[0003] Single nucleotide polymorphism (SNP) is a DNA sequence polymorphism caused by the variation of a single nucleotide at the genomic level, that is, the difference of a single base in the DNA sequence. In nature, SNPs are widely present, and the detection and analysis of SNPs are of great significance in aspects such as drug development, clinical testing and gene mutation diagnosis. The detection of SNP typing is more complicated than pathogen detection. The main reason is that SNP itself only has one base difference, and the difficulty of identification is greater than that of InDel with multiple base differences and pathogen detection with large fragment differences. The main methods for SNP typing detection in the prior art are as follows:

[0004] (1) The most traditional method based on restriction endonuclease recognition uses a combination of enzyme digestion and zone electrophoresis to identify the enzyme cleavage site, and then indirectly identify whether there is a corresponding SNP mismatch at the enzyme cleavage site. This requires the use of the corresponding restriction endonuclease, which is expensive and has a low throughput.

[0005] (2) Direct sequencing-based methods, such as performing multiplex PCR on the template followed by high-depth sequencing to obtain SNP information, are high-cost and low-throughput.

[0006] (3) Technology represented by Taqman. Conventional primers are first used to amplify and enrich the template carrying the SNP, and a fluorescent probe is introduced at the same time. The fluorescent probe is complementary to the sequence where the SNP is located. When the fluorescent probe is completely complementary to the SNP, the binding efficiency of the fluorescent probe is the highest, and the fluorescent probe is rapidly hydrolyzed and releases the signal. Using two fluorescent probes corresponding to different SNPs (multi-color channels), SNP information can be obtained. However, the cost is high and the throughput is low.

[0007] (4) Probe chip-based methods. The probe end is located upstream of the 5' end of the SNP detection site and is closely adjacent to the SNP. When the template and probe bind, the dNTP modified with the fluorescent group recognizes the SNP site and extends and incorporates into the probe. Therefore, the probe cluster can enrich specific dNTPs, emit specific fluorescent signals, and then identify SNP information. The basic principle of this method is similar to that of the SNaPshot technology. The characteristics of this technology are low cost and high throughput when a sample needs to detect multiple sites. However, when a sample only needs to detect a small number of sites, the average detection cost and throughput of this technology are not as good as traditional technologies.

[0008] (5) Methods represented by HRM (High-resolution Melting Curve). The melting curves of the amplified product sequences vary due to differences in SNPs, and SNPs can be typed with the help of high-resolution machines. However, HRM has high requirements for the concentration uniformity of the DNA template, resulting in the need to normalize the DNA concentration during actual use, and the actual throughput is low. At the same time, HRM requires the use of nucleic acid dyes with high saturation and no interference with PCR reactions. These nucleic acid dyes are expensive, resulting in high implementation costs for HRM.

[0009] (6) Identify SNPs based on the principle of extension blockade. When the last base at the 3' end of the primer mismatches with the SNP site, the extension of the primer will be blocked, inhibiting PCR amplification. The specificity of extension blockade PCR is determined by the type of mismatched bases and the PCR annealing temperature. When the primer recognizes the correctly paired template, the corresponding product, namely ARMS (amplification retardation mutation system), can be obtained. The original template gene is then identified by zone electrophoresis. Another method is to introduce a TAG sequence at the 5' end of the specific primer and simultaneously add a probe coupled with a fluorescent group and a quencher group (the base sequence is the same as the TAG sequence) or incorporate a fluorescent group probe and a probe carrying a quencher group that is complementary to the fluorescent group. The initial fluorescent probe randomly curls in space and contacts the quencher group at the other end or contacts the quencher group after being complementary to the quencher probe, resulting in a low fluorescence signal. The fluorescent group and quencher group incorporated into the product chain will be isolated, and the fluorescence signal will then be increased. This type of technology uses universal probes and has the advantages of good flexibility, low cost, and high throughput. However, both technologies offer relatively limited fluorescence signal intensity, lack a clear understanding of the underlying principles of DNA polymerase mismatch recognition, and exhibit poor predictability. They often rely on touch-down PCR to partially suppress nonspecific amplification. This necessitates converting qualitative experiments into quantitative ones, making the process more complex while ensuring accuracy.

[0010] (7) RNase H-dependent PCR genotyping technology (rhAmp SNP Genotyping). RNase HII enzyme can recognize the normal complementary DNA-rN-DNA / DNA structure and cut at the 5' end of RNA. Using this feature, RNase HII enzyme can identify SNP mismatches. When the RNA base mismatches with the SNP of the template, the blocking group at the 3' end of the specific primer can block the extension of the primer, inhibit PCR amplification, and thus identify the SNP; finally, through a signal growth mechanism similar to Taqman, a fluorescent signal difference for typing is obtained. This type of technology also uses universal probes and has the advantages of good flexibility, low cost, and high throughput. However, this technology requires modifying the RNA base in the middle of the specific primer and modifying the blocking group at the 3' end. The specific primer sequence is long and the cost is high. Summary of the Invention

[0011] To address the above-mentioned problems, the present invention aims to provide a PCR-based multi-color fluorescence channel detection method and application. A thermostable RNase HII endoribonuclease and a fluorescent probe doped with RNA bases and LNA bases are added to the STEP system. The probe is cleaved by RNase HII endoribonuclease, completely separating the fluorescent group and quencher group coupled to both ends of the probe, emitting a strong fluorescent signal. This method has the advantages of low cost, high specificity, multiple color channels, and strong signal.

[0012] In order to achieve the above object, the technical solution adopted by the present invention is as follows:

[0013] In one aspect, the present invention provides a PCR-based multi-color fluorescence channel detection method, comprising the following steps:

[0014] S1: Extract the whole genome DNA of the sample to be tested;

[0015] S2: Using the DNA extracted in step S1 as a template, a simultaneous dual-enzyme PCR reaction is performed in a simultaneous dual-enzyme PCR reaction system; wherein the simultaneous dual-enzyme PCR reaction system includes 5-50 ng of DNA template, 400-800 nM of specific primers, 400-800 nM of universal reverse primers, 100-200 nM of fluorescent probe, 25-50 mU of RNase HII enzyme, 1× PCR Mix, and the remaining volume is made up with water;

[0016] S3: Place the reaction product in step S2 under excitation light, read different fluorescence signals, and complete the detection.

[0017] Furthermore, there are two fluorescent probes described in step S2, each of which is doped with an LNA base at the +7 position of the 5' end and an RNA base at the +14 position; the sequences at the +3 to +7 and +15 to +18 positions of the 5' end are complementary; and the two ends of the fluorescent probe are coupled with a fluorescent group and a quenching group, respectively.

[0018] Furthermore, the nucleotide sequences of the two fluorescent probes are:

[0019] FAM-ACCAGC / iXNA_C / ACACCC / rU / GCTG-BHQ1;

[0020] ROX-ACGTCG / iXNA_C / TCCTTC / rC / CGAC-BHQ2.

[0021] Furthermore, the specific primers described in step S2 are specific long primers or a mixture of specific long primers and specific short primers with a working concentration ratio of 1:3.

[0022] Furthermore, the specific long primer introduces a TAG identical to the fluorescent probe sequence at the 5' end of the specific primer, and inserts a base bridge sequence between the TAG and the specific primer sequence, wherein the bridge sequence is complementary to the dangling base AC at the 5' end of the fluorescent probe;

[0023] The specific short primer is the specific long primer with bridge and TAG removed.

[0024] Furthermore, the reaction program of the simultaneous dual-enzyme PCR in step S2 is as follows: hot start at 94°C for 5 min, unwinding at 94°C for 30 s, annealing and enzyme digestion at 61°C-63°C for 1 min, amplification for 35 cycles, and final extension and enzyme digestion at 63-65°C for 10 min.

[0025] Another aspect of the present invention provides a fluorescent probe for PCR reaction, wherein the fluorescent probe is doped with LNA bases and RNA bases, and both ends of the fluorescent probe are coupled to a fluorescent group and a quenching group, respectively.

[0026] In addition, the present invention also provides a PCR reaction system, including 5-50 ng of DNA template, 400-800 nM of specific primer, 400-800 nM of universal reverse primer, 100-200 nM of fluorescent probe, 25-50 mU of RNase HII enzyme, 1× PCR Mix, and the remaining volume is made up with water.

[0027] In addition, the present invention also provides a product comprising the PCR reaction system as described above.

[0028] Preferably, the product comprises a kit.

[0029] The beneficial effects of the present invention are:

[0030] 1. This invention discloses a PCR-based multicolor fluorescence channel detection method. By adding RNase HII to the PCR reaction system, and designing specific primers and fluorescent probes incorporating RNA bases and LNA bases, the RNase HII enzyme and Taq enzyme react synchronously, leveraging its endonuclease properties and its ability to generate nicks only on strands incorporating RNA bases. This strategy does not affect the Taq enzyme's polymerization reaction, while ensuring that the fluorescent probe releases a fluorescent signal completely after hydrolysis.

[0031] 2. The fluorescent probe designed in the present invention is doped with LNA bases and RNA bases, and the two ends of the fluorescent probe are respectively coupled with a fluorescent group and a quencher group. When hybridizing with the complementary chain, a DNA-rN-DNA / DNA structure is formed. The RNaseHII enzyme can recognize this structure and cut the 5' end of the RNA base, completely separating the fluorescent group and the quencher group coupled to the two ends of the probe, emitting a strong fluorescent signal.

[0032] 3. The specific long primer designed in the present invention introduces a TAG identical to the fluorescent probe sequence at the 5' end of the specific primer, and inserts two base bridge sequences between the TAG and the specific primer sequence, forming a specific long primer with a TAG sequence, a bridge sequence and a specific sequence from the 5' end to the 3' end; after the TAG sequence is blocked by introducing a 6-base pair hairpin structure, the TAG sequence and the bridge sequence together form a 6bp stem structure, the specific sequence at its 3' end specifically binds to the target sequence, and the bases other than the specific sequence are blocked. The specific primer only relies on the secondary structure brought by the long fragment specific sequence, which reduces the probability of non-specific amplification caused by the secondary structure; the blocked linker TAG sequence is not easy to induce additional primer dimers, which reduces background noise and simplifies the calculation amount for predicting primer dimers.

[0033] 4. In the present invention, when the long specific primer correctly matches the target sequence, it is extended to introduce the TAG and bridge bases into the PCR product, generating an antisense strand complementary to the TAG sequence. The fluorescent probe and the reactive strand complementary to the TAG hybridize at a temperature of 61-63°C, forming a DNA-rN-DNA / DNA structure. This enables RNase HII enzyme recognition and cleavage of the fluorescent probe.

[0034] 5. In the present invention, when some long specific primers are replaced with short specific primers in the reaction system, the extended chains and complementary chains amplified by the long and short specific primers are randomly annealed, and some products form DNA chains paired with the short specific primer extended chain and the long specific primer complementary chain. Such DNA chains will expose a hanging single chain, and the hanging single chain is complementary to the fluorescent probe. This allows the fluorescent probe to bind to the complementary chain without competing with the specific primer extended chain, thereby improving the annealing efficiency of the fluorescent probe. At the same time, the bridge structure of the long specific primer acts as a spatial buffer in the structure of the hanging single chain of the complementary chain, which can reduce the steric hindrance of the quenching group to the annealing of the fluorescent probe.

[0035] 6. Based on the PCR reaction system of the present invention, a specific PCR reaction program has been designed. Compared to conventional PCR programs, the present invention utilizes an annealing temperature of 60°C or higher during the cycling phase (RNase HII has optimal activity at 70-75°C and is active between 50°C and 75°C), allowing for simultaneous primer extension and probe hydrolysis. After cycling, a degenerate final extension and enzyme cleavage step is performed, with the final extension temperature set at 63-65°C, near the Tm of the fluorescent probe. This ensures efficient binding of the fluorescent probe to the target sequence while also taking into account the temperature requirements for RNase HII activity.

[0036] 7. The fluorescent endpoint signal finally obtained by the PCR method of the present invention can be directly analyzed visually under the excitation light source; at the same time, the fluorescent probe of the present invention is compatible with multi-color fluorescent channels, and has the advantages of flexible deployment, convenient signal reading, and simultaneous analysis of multiple targets in pathogen detection experiments; the use of a strictly one-pot reaction has the advantage of being convenient and fast for some pathogen detection technologies that require a secondary reaction after PCR for color development.

[0037] 8. This invention also systematically explains the mechanism by which SNP mismatches affect PCR reactions, proposing the TPASS (Temperature-pass Allele Specific System) theory. The thermal stability of SNP mismatches and the type of base pairing jointly influence the stringency of SNP mismatches in PCR amplification. This rigorous horizontal comparison of the strength of SNP mismatches using the critical temperature as a metric provides a clear theoretical basis for the large-scale design of highly specific SNP typing primers, eliminating the need for trial-and-error determination of the optimal annealing temperature using traditional touch-down or gradient PCR strategies. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 This is a schematic diagram of the design of two fluorescent probes in the present invention.

[0039] Figure 2 Schematic diagram of the design of two long specific primers in the present invention.

[0040] Figure 3 This is a schematic diagram of the fluorescence signal release principle of the STEP technology in the present invention.

[0041] Figure 4 This is a schematic diagram of the fluorescence signal release principle of the STEP enhancement technology in the present invention.

[0042] Figure 5 This figure illustrates the principle of the TPASS theory in the present invention, including the thermal stability of SNP pairing (ΔΔG(AT) is calculated based on the free energy of normal AT pairing; the larger the ΔΔG(AT), the lower the thermal stability of the SNP mismatch) and the specific critical temperature.

[0043] Figure 6 This is a schematic diagram of the principle of SNP typing using the three-primer method obtained by TPASS theoretical calculation in Example 1 of the present invention.

[0044] Figure 7 This is the SNP typing test result in Example 1 of the present invention.

[0045] Figure 8 This is a schematic diagram of the principle of SNP typing using the four-primer method obtained by TPASS theoretical calculation in Example 2 of the present invention.

[0046] Figure 9 This is the SNP typing test result in Example 2 of the present invention.

[0047] Figure 10 This is the SNP typing test result in Example 3 of the present invention.

[0048] Figure 11 This is the SNP typing test result in Example 4 of the present invention.

[0049] Figure 12 Schematic diagram of primer design for ALK gene functional site in Example 5 of the present invention.

[0050] Figure 13 This is the SNP typing test result in Example 5 of the present invention.

[0051] Figure 14 Schematic diagram of primer dimers for nonspecific amplification of ALK gene-specific primers and universal reverse primers in Example 5 of the present invention.

[0052] Figure 15 Schematic diagram of primer design for Sdr4 gene functional site in Example 6 of the present invention.

[0053] Figure 16 This is the SNP typing test result in Example 6 of the present invention. DETAILED DESCRIPTION

[0054] In order to enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention is further described below in conjunction with the accompanying drawings and embodiments.

[0055] The present invention provides a multi-color fluorescence channel detection method based on PCR, which specifically comprises the following steps:

[0056] S1: Extract the whole genome DNA of the sample to be tested;

[0057] Specifically, a sample to be tested is collected, and the whole genome DNA of the sample to be tested is extracted using a kit;

[0058] S2: using the DNA extracted in step S1 as a template, performing a simultaneous dual-enzyme PCR reaction in a simultaneous dual-enzyme PCR reaction system;

[0059] Specifically, the synchronous two-enzyme PCR (STEP) reaction system includes 5-50 ng of DNA template, 400-800 nM of specific primers, 400-800 nM of universal reverse primers, 100-200 nM of fluorescent probe, 25-50 mU of RNase HII enzyme, 1× PCR Mix, and the remaining volume is made up with water.

[0060] More specifically, the STEP reaction system of the present invention contains two fluorescent probes, both doped with RNA bases and LNA (locked nucleic acid) bases. Fluorescent groups and quenching groups are coupled to both ends of the fluorescent probes. The probes were synthesized by Shanghai Sangon Biotechnology Co., Ltd. The nucleotide sequences of the two fluorescent probes are:

[0061] FAM-ACCAGC / iXNA_C / ACACCC / rU / GCTG-BHQ1 (Probe-FAM);

[0062] ROX-ACGTCG / iXNA_C / TCCTTC / rC / CGAC-BHQ2 (Probe-ROX).

[0063] The 5' ends of the two fluorescent probes are coupled with fluorescent groups FAM and ROX respectively, and the 3' ends are coupled with quenching groups BHQ1 or BHQ2 respectively. The +7 position of the 5' end of the fluorescent probe is doped with LNA base (to improve the binding force between the probe and the target sequence, provide steric hindrance, and increase the specificity of the STEP results); the +14 position is doped with RNA base; the +3~+7 and +15~+18 positions of the 5' end are complementary to each other to form a 4-base-pair hairpin structure. At the same time, two dangling bases are retained at the 5' end, as shown in the attached figure. Figure 1 As shown in the figure, the LNA base increases the probe's Tm value and promotes the binding of the fluorescent probe to the complementary strand. The RNA base provides a cleavage recognition site for the RNase HII enzyme. Furthermore, a six-base interval separates the LNA and RNA bases, preventing them from coming too close to each other after binding to the complementary strand, potentially inhibiting the cleavage activity of the RNase HII enzyme. A hairpin structure is formed within the probe, with a four-base-pair stem structure that brings the fluorescent group and quencher group into close proximity, reducing fluorescence background noise. During the STEP process, conventional primers amplify the target sequence to generate a reaction strand complementary to the fluorescent probe. When the fluorescent probe hybridizes to the complementary strand, a DNA-rN-DNA / DNA structure is formed. The RNase HII enzyme recognizes this structure and cleaves the 5' end of the RNA base, completely separating the fluorescent group and quencher coupled to each end of the probe and emitting a strong fluorescent signal. It should be noted that the FAM-BHQ1 and ROX-BHQ2 in the present invention can be simply replaced with combinations including but not limited to VIC-BHQ1, AF488-BHQ1, etc. to achieve compatibility with multi-channel signals.

[0064] Furthermore, the specific primers in the present invention can all be specific long primers, or a mixture of specific long primers and specific short primers with a working concentration ratio of 1:3 (enhanced type) can be used. There are two long specific primers, and a TAG identical to the fluorescent probe sequence is introduced at the 5' end of the specific primer, and two base bridge sequences are inserted between the TAG and the specific primer sequence, forming a specific long primer with a TAG sequence, a bridge and a specific sequence from the 5' end to the 3' end; wherein the TAG sequence is identical to the fluorescent probe sequence, and the bridge sequence is complementary to the dangling base AC at the 5' end of the fluorescent probe, so that the TAG at the 5' end of the specific primer is blocked by a 6-base pair hairpin structure, as shown in the attached figure. Figure 2As shown (taking the typing primer for rice SNP site vg1224338804 as an example). After the TAG sequence is blocked by introducing a 6-base-pair hairpin structure, the TAG sequence and the bridge sequence together form a 6-bp stem structure. The specific sequence at its 3' end specifically binds to the target sequence, blocking the bases outside the specific sequence. The specific primer only relies on the long fragment specific sequence to reduce the probability of non-specific amplification caused by the secondary structure. The blocked linker TAG sequence is less likely to trigger additional primer dimers, reducing background noise and simplifying the predicted calculation of primer dimers. The primer that only contains the specific primer sequence and does not contain the bridge and TAG is the specific short primer in the present invention.

[0065] The principle of the STEP reaction system of the present invention is as follows: long specific primers and universal primers are combined to amplify the complementary product chain of the fluorescent probe. When the fluorescent probe hybridizes with the complementary chain, a DNA-rN-DNA / DNA structure is formed; RNaseHII enzyme can recognize the above structure and cut at the 5' end of the RNA base, so that the fluorescent group and quenching group coupled to both ends of the probe are completely separated, emitting a strong fluorescent signal. Figure 3 As shown. When some long specific primers are replaced with short specific primers in the reaction system, the extended chains and complementary chains amplified by the long and short specific primers are randomly annealed, and some products form DNA chains paired with the short specific primer extended chain and the long specific primer complementary chain. Such a DNA chain will expose a hanging single strand, and the hanging single strand is complementary to the fluorescent probe. This allows the fluorescent probe to bind to the complementary chain without competing with the specific primer extended chain, thereby improving the annealing efficiency of the fluorescent probe. At the same time, the bridge structure of the long specific primer acts as a spatial buffer in the structure of the hanging single strand of the complementary chain, which can reduce the spatial steric hindrance of the quenching group to the annealing of the fluorescent probe. This strategy is consistent with the principle that Taqman probes generally require a spacing of at least 1 nt between them and the specific primers, as shown in the attached Figure 4 shown.

[0066] Furthermore, based on the STEP reaction system described above, the STEP reaction procedure in the present invention is as follows: hot start at 94°C for 5 minutes, unwinding at 94°C for 30 seconds, annealing and digestion at 61-63°C for 1 minute, 35 cycles of amplification, and a final extension and digestion at 63°C for 10 minutes. Annealing temperatures above 60°C are used because RNase HII has optimal activity at 70-75°C and is active between 50°C and 75°C, allowing for simultaneous primer extension and probe hydrolysis in PCR. After the cycle is complete, a degenerate final extension and digestion step is performed, with the final extension temperature being 63-65°C, near the Tm of the fluorescent probe. This ensures efficient binding of the fluorescent probe to the target sequence while also taking into account the temperature requirements for RNase HII activity.

[0067] S3: Place the reaction product in step S2 under excitation light, read the fluorescence signals of different channels, and complete the detection.

[0068] Specifically, the emitted light is observed using filters with wavelengths of 500-540nm, 580-620nm, and 500-700nm (all filters have a cutoff depth, i.e., an OD value, of 3). After filtering background light with narrowband filters, the fluorescence signal of the corresponding fluorescent group can be purified, improving the resolution of the fluorescent signal. For example, the FAM and ROX groups used in the present invention can be excited by a 365nm light source; FAM can be fully excited by a 470nm blue LED and partially excited by a 365nm light source. When a specific fluorescent group is excited by the excitation light, amplification of the corresponding specific primer is determined, and the fluorescence brightness is positively correlated with the concentration of the amplified product. Specific detection methods include, but are not limited to, SNP typing and pathogen detection. Example 1:

[0069] In Example 1, rice was used as the research subject for SNP typing. SNP sites were selected from the rice 3K database, which includes three types of SNPs: A / T, A / C (T / G), and C / G, with five sites in each type. According to the TPASS theory, these three types of SNP mismatches are all strong mismatches. Specifically, at an annealing temperature of 63 degrees, nonspecific amplification of specific primers is completely cut off. Six classic rice varieties, Nipponbare, 9311, Minghui 63, Zhenshan 97, N22, and Casalas, were selected as samples. These six rice varieties all have high-quality reference genomes (for detailed information on the accession numbers of the mutation sites, see the website http: / / ricevarmap.ncpgr.cn / ). The genotypes of the 15 SNP sites of these six rice varieties are all known genotypes.

[0070] Table 1 Site information of three types of SNPs in Example 1

[0071]

[0072] The specific typing detection method includes the following steps:

[0073] S1: Extract genomic DNA from rice seedlings using a kit;

[0074] The genomic DNA template of the fresh sample was extracted by magnetic bead method; the DNA template was verified by PCR to be able to be used for normal amplification (the DNA template concentration was not normalized).

[0075] S2: Using the rice genomic DNA extracted in step S1 as a template, placing it in a PCR reaction system and performing PCR amplification;

[0076] The present invention adopts TPASS theory to carry out primer design, and the brief description of TPASS theory is as shown in the attached Figure 5 As shown, the thermal stability of SNP pairing (ΔΔG(AT) is calculated using the free energy of normal AT pairing as a measure; larger ΔΔG(AT) values ​​indicate lower thermal stability of the SNP mismatch) and the specificity threshold temperature are used to rationally design primers specific for strongly mismatched SNPs, achieving absolutely specific amplification. The thermal stability parameters for SNP mismatches are referenced from the work of Afek A et al. (DOI: 10.1038 / s41586-020-2843-2) and SantaLucia et al. (DOI: 10.1146 / annurev.biophys.32.110601.141800).

[0077] The results of the three-primer method for designing specific primers calculated based on the TPASS theory are shown in Table 2 below, where the underlined bases are bases with different lengths, and the wavy bases are bases that recognize the SNP sites on the specific primers. When designing specific primers using the three-primer method, the two specific primers introduce an additional base difference at the 5' end, so that the target fragments finally amplified by the specific primers with different 3' ends have different lengths, which can then be distinguished by zone electrophoresis. The principle diagram of the three-primer method for SNP typing is shown in the attached figure. Figure 6 As shown, to simplify calculations, the present invention incorporates polyA structures at the ends of longer specific primers, drawing on the polyA structure of mRNA. This introduces primer length variation. Due to the stringent mismatch nature of A bases, the introduction of A bases generally does not introduce new secondary structures that could lead to additional nonspecific amplification. The PCR reaction system is shown in Table 3, and the PCR reaction procedure is shown in Table 4.

[0078] Table 2 SNP typing primer sequences in Example 1

[0079]

[0080] Table 3 PCR reaction system in Example 1

[0081]

[0082] Table 4 PCR reaction procedure in Example 1

[0083]

[0084] S3: Perform gel electrophoresis analysis on the PCR amplification products obtained in step S2, and perform genotyping based on the analysis results.

[0085] Attachment Figure 7 The results of gel electrophoresis are shown. The seven lanes from left to right for each primer set in a, b, and c correspond to varieties such as Nipponbare, 9311, Minghui 63, Zhenshan 97, N22, Casalas, and negative control (H2O). Figure 7 In the figure, a, b, and c correspond to 5 sets of primers for each of the three types of SNPs: A / T, A / C (T / G), and C / G.

[0086] from Figure 7 As can be seen from the figure, when the SNP mismatches are A / T, A / C (T / G), and C / G, an annealing temperature of 63°C completely cuts off nonspecific amplification. Specifically, the main band is clear and bright, and the differences in the main band fragments are consistent with the haplotype differences at this SNP genotype among the six samples. This proves that the A / T, A / C (T / G), and C / G SNPs are strong mismatches. When the SNP mismatches are A / T, A / C (T / G), and C / G, fully specific typing can be performed without introducing additional artificial mismatches.

[0087] Example 2:

[0088] Example 2: Rice was used as the research object to perform SNP typing detection. A total of 5 A / G (T / C) type SNP sites were selected from the rice 3K database. According to the TPASS theory, A / G (T / C) type SNPs are weak mismatches. Specifically, at an annealing temperature of 61 degrees, the non-specific amplification of the specific primers with AC mismatches was completely cut off, while the non-specific amplification of the specific primers with TG mismatches was not cut off at an annealing temperature greater than 63°C. Six classic conventional rice varieties, Nipponbare, 9311, Minghui 63, Zhenshan 97, N22 and Casalas, were selected as samples. These six rice varieties all have high-quality reference genomes. The genotypes of the five A / G (T / C) type SNP sites of these six rice varieties are all known genotypes.

[0089] Table 5 SNP site information in Example 2

[0090]

[0091] The specific typing detection method includes the following steps:

[0092] S1: Extract genomic DNA from rice seedlings using a kit;

[0093] The genomic DNA template of the fresh sample was extracted by magnetic bead method; the DNA template was verified by PCR to be able to be used for normal amplification (the DNA template concentration was not normalized).

[0094] S2: Using the rice genomic DNA extracted in step S1 as a template, placing it in a PCR reaction system and performing PCR amplification;

[0095] Refer to the attached Figure 8 The four-primer method was used to design specific primers, and the results are shown in Table 6 below, where the wavy bases are the bases that recognize the SNP site on the specific primers; the principle of the four-primer system is the classic ARMS technology. When the SNP is A / G (T / C), the mismatch bases are AC and TG. Using the four-primer method, the recognition mismatches of the primers and the target sequence can be limited to AC. The AC mismatch is thermally unstable at high temperatures and has rigorousness, while the TG mismatch is still thermally stable at high temperatures and does not have strict rigor. Therefore, the four-primer method can also achieve rigorous specific amplification for the weak mismatch type A / G (T / C) of the SNP. The PCR reaction system in this embodiment is shown in Table 7 below, and the PCR reaction procedure is shown in Table 8 below.

[0096] Table 6 SNP typing primer sequences in Example 2

[0097]

[0098] Table 7 PCR reaction system in Example 2

[0099]

[0100] Table 8 PCR reaction procedure in Example 2

[0101]

[0102] S3: Perform gel electrophoresis analysis on the PCR amplification products obtained in step S2, and perform genotyping based on the analysis results.

[0103] Attachment Figure 9 The following are the results of gel electrophoresis. The seven lanes from left to right for each primer set correspond to varieties such as Nipponbare, 9311, Minghui 63, Zhenshan 97, N22, Casalas and negative control (H2O).

[0104] from Figure 9 As can be seen from the data, when the SNP mismatch is an A / G (T / C) type, an annealing temperature of 61°C can almost completely cut off nonspecific amplification, with minor nonspecific amplification observed at a few loci. Specifically, with the exception of the ZcA1_1814 Casaras sample, which exhibited a heterozygous band due to a small amount of contaminant strains, all main bands were clear, bright, and single. The differences in the main band fragments were consistent with the haplotype differences among the six samples at that SNP genotype. When the SNP mismatch is an A / G (T / C) type, SNP typing using the four-primer method can convert primer-template mismatches into AC or TG mismatches. AC mismatches are thermally labile and significantly inhibit nonspecific amplification at an annealing temperature of 61°C. TG mismatches are thermally stable and non-purine-purine mismatches, making them weak mismatches. The four-primer method is used to convert the specific amplification of A / G (T / C) type SNPs into the recognition of AC thermally unstable mismatches, which can perform quasi-complete specific typing without introducing additional artificial mismatches.

[0105] Example 3:

[0106] Example 3 Taking the SNP site vg1224338804 in Example 1 as an example, the STEP technology and its enhanced amplification technology in the present invention were verified.

[0107] The designed primer sequences are shown in Table 9 below, where the underlined sequence represents the TAG sequence + bridge sequence, and the wavy line represents the SNP recognition site. The conventional STEP reaction system is shown in Table 10 below, the enhanced STEP reaction system is shown in Table 11 below, and the reaction procedure is shown in Table 12 below.

[0108] Table 9 Primer sequences for SNP sites in Example 3

[0109]

[0110] Table 10 Conventional STEP reaction system in Example 3

[0111]

[0112] Table 11 Enhanced STEP reaction system in Example 3

[0113]

[0114] Table 12 STEP reaction procedure in Example 3

[0115]

[0116] The typing results obtained in this example are shown in the attached Figure 10 As shown, attached Figure 10 The corresponding 7 samples are Nipponbare, 9311, Minghui 63, Zhenshan 97, N22, Casalas and negative control (H2O) in sequence; among them, a is the signal diagram of the dual-color fluorescence channel (500-700nm) excited by the ultraviolet pen, b is the signal diagram of the red fluorescence channel (580-620nm) excited by the ultraviolet pen, and c is the signal diagram of the green fluorescence channel under the blue light gel cutting instrument (amber filter).

[0117] From the attached Figure 10 As can be seen in the figure, the fluorescence results from STEP are consistent with the electrophoresis banding results. The fluorescence signal from enhanced STEP amplification is brighter than that from conventional STEP amplification. While maintaining the total specific primer concentration, appropriately replacing some long specific primers with short ones can create a dangling single-stranded structure, reducing steric hindrance in binding between the fluorescent probe and the complementary sequence. This dangling single-stranded structure facilitates RNase HII recognition of the DNA-rN-DNA / DNA structure, improving RNase HII digestion efficiency.

[0118] In addition, from the attached Figure 10 It can also be seen that when a DNA template is combined with a primer for amplification, the amplification of primer dimers will be inhibited. Figure 10 In samples a, b, and c, the negative control (sample 7) with ddH2O added, without the inhibition of specific amplification competition, only amplified primer dimers. Its fluorescence signal intensity was greater than that of samples 1 and 3 (samples 1 and 3 were haplotype 1) but less than that of samples 2, 4, 5, and 6 (samples 2, 4, 5, and 6 were haplotype 2). Therefore, in the implementation of STEP technology, to ensure the accuracy of the results, it is best to ensure that an appropriate amount of DNA template is added to suppress the interference of primer dimers. It is also recommended that the negative and positive controls contain additional DNA template of the corresponding haplotype as a positive control.

[0119] In conventional PCR reaction systems, the final concentration of specific primers is generally controlled at 200-500 nM·μL. In this example, the total concentration of specific primers and the concentration of universal primers both reached 800 nM·μL. This example still maintained extremely high specificity at a working concentration of 800 nM·μL, demonstrating that STEP technology can maintain high reliability even under the extreme boundary conditions of high primer concentrations.

[0120] Example 4:

[0121] Example 4 Taking the SNP site vg1018748126 in Example 1 as an example, the STEP technology in the present invention is verified.

[0122] The designed primer sequences are shown in Table 13 below, where the underlined sequence is the TAG sequence + bridge sequence and the wavy line is the SNP recognition site. The conventional STEP reaction system is shown in Table 14 below, and the reaction procedure is as shown in the attached Figure 15 shown.

[0123] Table 13 Primer sequences for SNP sites in Example 4

[0124]

[0125] Table 14 Conventional STEP reaction system in Example 4

[0126]

[0127] Table 15 STEP reaction procedure in Example 4

[0128]

[0129] The SNP typing results obtained in this example are shown in the attached Figure 11 As shown, Figure 11 The corresponding 6 samples are Nipponbare, 9311, Minghui 63, Zhenshan 97, N22, and Casalas. Figure 11 This is the signal diagram of the dual-color fluorescence channel (500-700nm) excited by the ultraviolet pen.

[0130] from Figure 11 As can be seen in the figure, the fluorescence results of STEP are consistent with the electrophoresis band results. The difference from Example 3 is that the specific typing primers designed around the vg1018748126 locus have higher specificity. Specifically, the nonspecific amplification fluorescence signal triggered by the primer dimer structure is weak, essentially consistent with the background signal of the fully specific amplification.

[0131] It can also be concluded that designing specific primers and universal primer combinations with lower primer dimer background is more conducive to distinguishing false positive amplification results and ensuring the accuracy of fluorescence signal results.

[0132] Embodiment 5:

[0133] Example 5 Taking the rice ALK functional site (vg0606752888) as an example, the STEP technology and its enhanced amplification technology in the present invention were verified.

[0134] The ALK functional locus is a multi-nucleotide polymorphism (MNP) consisting of two closely adjacent SNPs, which can be used to validate the applicability of the STEP technique and TPASS theory to multiple SNPs. Based on the ALK reference genomes of six rice varieties, the genotype of variety 9311 corresponds to a low alkali digestion value, corresponding to the ZiF-ALK-LR primer. The genotypes of the other five varieties correspond to high alkali digestion values, corresponding to the ZiF-ALK-HR primer.

[0135] The specific experimental process includes the following steps:

[0136] S1: Extract genomic DNA from rice seedlings;

[0137] The genomic DNA template of the fresh sample was extracted by magnetic bead method; the DNA template was verified by PCR to be able to be used for normal amplification (the DNA template concentration was not normalized).

[0138] S2: Using the rice genomic DNA extracted in step S1 as a template, placing it in the STEP reaction system for STEP amplification;

[0139] For details, please refer to the attached Figure 12 Specific primers were designed, and the results are shown in Table 16 below. The underlined sequence represents the TAG sequence + bridge sequence, and the wavy line represents the SNP recognition site. The conventional STEP reaction system is shown in Table 17 below, the enhanced STEP reaction system is shown in Table 18 below, and the reaction procedure is shown in Table 19 below.

[0140] Table 16 Primer sequences of MNP in Example 5

[0141]

[0142] Table 17 Conventional STEP reaction system in Example 5

[0143]

[0144] Table 18 Enhanced STEP reaction system in Example 5

[0145]

[0146] Table 19 STEP reaction procedure in Example 5

[0147]

[0148] S3. Perform gel electrophoresis analysis on the STEP amplification products obtained in S2, and perform genotyping based on the analysis results according to preset conditions.

[0149] The SNP typing results obtained in this example are shown in the attached Figure 13shown. Figure 13 The corresponding seven samples from left to right are Nipponbare, 9311, Minghui 63, Zhenshan 97, N22, Casalas and negative control (H2O), among which a is the signal diagram of the dual-color fluorescence channel (500-700nm) excited by the ultraviolet pen, b is the red fluorescence channel diagram (580-620nm) excited by the ultraviolet pen, and c is the green fluorescence channel signal diagram under the blue light gel cutting instrument (amber filter).

[0150] From the attached Figure 13 As can be seen in the figure, the fluorescence results of STEP are consistent with the genotype results of the six rice samples. Specifically, under a filter of 500-700nm (OD3), the total fluorescence signal of sample 9311 amplified using both conventional and enhanced STEP techniques tends to correspond to the green fluorescence of the FAM group, while the total fluorescence signals of the other samples tend to correspond to the red fluorescence of the ROX group.

[0151] However, under the 470nm excitation channel monochromatic filter corresponding to the blue light exciter, the negative control (H2O) sample amplified using the conventional STEP technique, along with the 9311 sample, exhibited green fluorescence. Under the excitation light of an ultraviolet pen and the 580-620nm (OD3) channel monochromatic filter corresponding to red fluorescence, the negative control (H2O) sample amplified using the conventional STEP technique, along with the samples from the other five varieties, exhibited red fluorescence. This indicates that while the ZiSF-ALK primers can accurately genotype the gene, some background fluorescence noise due to primer dimers is present.

[0152] The universal reverse primer sequence in the primer combination of ZiSF-ALK (see attached Figure 14 The GG pairing (shown in Figure 2) can induce primer dimers, generating background noise in the absence of DNA template competition. The GG pairing is not located at the 3' end of the primer and has a thermal stability similar to that of the AT pairing. The thermodynamic parameters are based on the work of SantaLucia et al.

[0153] Furthermore, using enhanced STEP technology, the total fluorescence signal obtained from primer amplification of ZiSF-ALK is consistent with the haplotype corresponding to the reference genome. However, the noise signal caused by primer dimers is also enhanced to a level that can be directly observed. Therefore, when selecting primers for SNP typing or pathogen detection, attention should be paid to both specificity and background noise caused by primer dimer structures.

[0154] In addition, when the amplification efficiency of the primer is high, the interference of background noise can be controlled by reducing the primer concentration, reducing the amount of RNase HII enzyme used, reducing the number of PCR amplification cycles, selecting conventional STEP technology, reducing the excitation light intensity, or replacing the universal primer sequence.

[0155] Example 6:

[0156] Example 6 Taking the Sdr4 functional site (vg0723797131) of rice as an example, the STEP technology and its enhanced amplification technology in the present invention were verified.

[0157] The electrophoresis patterns of specific primers for the functional marker Sdr4 were verified with reference to the invention patent application publication number CN117210610A. This example converted the validated Sdr4-specific primers into fluorescent typing primers to cross-validate the specificity of the STEP technique with the repeatability of previous work.

[0158] The functional locus of Sdr4 is a complex differential locus composed of multiple single nucleotide polymorphisms (SNPs) and indels (INDELs). The Sdr4-N variant contains two adjacent 18-base repeats. ZiDF-Sdr4 can be used to validate the applicability of the STEP technique and TPASS theory to more complex MNP and INDEL conditions. Based on the Sdr4 reference genomes of six rice varieties, the genotypes of varieties 9311, Minghui 63, and Casalas correspond to the Sdr4-k haplotype, corresponding to the ZiDF-Sdr4-KR primer. The genotypes of the other three varieties correspond to the Sdr4-N haplotype, corresponding to the ZiDF-Sdr4-NR primer.

[0159] The specific experimental process includes the following steps:

[0160] S1: Extract genomic DNA from rice seedlings;

[0161] The genomic DNA template of the fresh sample was extracted by magnetic bead method; the DNA template was verified by PCR to be able to be used for normal amplification (the DNA template concentration was not normalized).

[0162] S2: Using the rice genomic DNA extracted in step S1 as a template, placing it in the STEP reaction system for STEP amplification;

[0163] For details, please refer to the attached Figure 15 Specific primers were designed, and the results are shown in Table 20 below, where the underlined sequence is the TAG sequence + bridge sequence. The conventional STEP reaction system is shown in Table 21 below, the enhanced STEP reaction system is shown in Table 22 below, and the reaction procedure is shown in Table 23 below.

[0164] Table 20 Primer sequences for the composite differential sites in Example 6

[0165]

[0166] Table 21 Conventional STEP reaction system in Example 6

[0167]

[0168] Table 22 Enhanced STEP reaction system in Example 6

[0169]

[0170] Table 23 STEP reaction procedure in Example 6

[0171]

[0172] S3. Perform gel electrophoresis analysis on the STEP amplification products obtained in S2, and perform genotyping based on the analysis results according to preset conditions.

[0173] The SNP typing results obtained in this example are shown in the attached Figure 16 shown. Figure 16 The corresponding seven samples from left to right are Nipponbare, 9311, Minghui 63, Zhenshan 97, N22, Casalas and negative control (H2O), among which a is the signal diagram of the dual-color fluorescence channel (500-700nm) excited by the ultraviolet pen, b is the red fluorescence channel diagram (580-620nm) excited by the ultraviolet pen, and c is the green fluorescence channel signal diagram under the blue light gel cutting instrument (amber filter).

[0174] From the attached Figure 16 As can be seen in the figure, the fluorescence results from STEP are consistent with the genotypes of the six rice samples. Specifically, under a 500-700 nm (OD3) filter, the total fluorescence signals amplified by both conventional and enhanced STEP techniques for the six rice varieties corresponded to the Sdr4 haplotype. Furthermore, under single-color filters in both the 500-540 nm (OD3) and 580-620 nm (OD3) channels, the fluorescence noise background of ZiDF-Sdr4 was relatively clean. This demonstrates that STEP technology is compatible with SNP typing primers for complex loci and genotyping of closely related pathogen races.

[0175] It should be noted that when the specific primer amplification efficiency during the implementation of the STEP technology is high, the fluorescent signals obtained by the conventional STEP technology and the enhanced STEP technology can reach a level that can be directly observed visually. Therefore, the conventional STEP technology can be considered to simplify the PCR system.

[0176] The basic principles, main features, and advantages of the present invention are shown and described above. Those skilled in the art should understand that the present invention is not limited to the foregoing embodiments. The foregoing embodiments and descriptions are merely illustrative of the principles of the present invention. Various changes and modifications may be made to the present invention without departing from the spirit and scope of the present invention. Such changes and modifications are intended to fall within the scope of the present invention. The scope of protection claimed in the present invention is defined by the appended claims and their equivalents.

Claims

1. A fluorescent probe for PCR reaction, characterized in that: There are two fluorescent probes, each of which is doped with an LNA base at the +7 position of the 5' end and an RNA base at the +14 position; the sequences at the +3 to +7 and +15 to +18 positions of the 5' end are complementary; and the two ends of the fluorescent probe are coupled with a fluorescent group and a quenching group respectively; The nucleotide sequences of the two fluorescent probes are: FAM-ACCAGC / iXNA_C / ACACCC / rU / GCTG-BHQ1; ROX-ACGTCG / iXNA_C / TCCTTC / rC / CGAC-BHQ2.

2. A PCR reaction system, characterized in that: Includes 5-50 ng DNA template, 400-800 nM specific primer, 400-800 nM universal reverse primer, 100-200 nM fluorescent probe, 25-50 mU RNase HII enzyme, 1× PCR Mix, and make up the remaining volume with water; The specific primer is a specific long primer or a mixture of a specific long primer and a specific short primer with a working concentration ratio of 1:3; The specific long primer is a primer that introduces a TAG sequence identical to the fluorescent probe sequence at the 5' end of the specific sequence and inserts a base bridge sequence between the TAG and the specific sequence, forming a specific long primer with a TAG sequence, a bridge sequence, and a specific sequence from the 5' end to the 3' end. The TAG sequence is identical to the fluorescent probe sequence, and the bridge sequence is complementary to the dangling base AC at the 5' end of the fluorescent probe, so that the TAG at the 5' end of the specific primer is blocked by a 6-base-pair hairpin structure. The specific short primer is the specific long primer with bridge and TAG removed; The fluorescent probe is the fluorescent probe for PCR reaction described in claim 1.

3. A product comprising the PCR reaction system according to claim 2.

4. The product according to claim 3, characterized in that The products include kits.

5. A multi-color fluorescence channel detection method based on PCR, characterized in that: The following steps are involved: S1: Extract the whole genome DNA of the sample to be tested; S2: using the DNA extracted in step S1 as a template, performing a simultaneous dual-enzyme PCR reaction in a simultaneous dual-enzyme PCR reaction system; S3: placing the reaction product in step S2 under excitation light, reading different fluorescence signals, and completing the detection; The synchronous dual-enzyme PCR reaction system adopts the PCR reaction system described in claim 2.

6. The PCR-based multi-color fluorescence channel detection method according to claim 5, characterized in that: The reaction program of the simultaneous dual-enzyme PCR in step S2 is as follows: hot start at 94°C for 5 minutes; unwinding at 94°C for 30 seconds, annealing and enzyme digestion at 61°C-63°C for 1 minute, amplification for 35 cycles; and final extension and enzyme digestion at 63-65°C for 10 minutes.

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