Promoter-mediated isothermal amplification method and application thereof in nucleic acid detection

Abstract

The invention provides a promoter-mediated isothermal amplification method and application of the promoter-mediated isothermal amplification method in nucleic acid detection. The method comprises the following steps: guiding and starting an initial isothermal amplification reaction by utilizing the 3'tail end of a composite primer containing a promoter sequence, introducing the promoter sequence into an amplification product, and newly generating a target gene nucleic acid sequence containing the promoter sequence; the RNA polymerase is guided by a promoter to transcribe an RNA chain of a target gene for reverse transcription of reverse transcriptase to generate a large amount of cDNA, a single-stranded DNA template is continuously provided for a subsequent isothermal amplification reaction, meanwhile, a transcription starting process can provide a large amount of DNA single-stranded template areas for synchronous isothermal amplification, and the DNA isothermal amplification reaction is accelerated. Besides, a specific primer which is reversely complementary with a target gene sequence is designed at the 5'tail end of the composite primer, so that the 3 'tail end of a newly generated nucleic acid sequence can be complementarily combined with the own sequence to form a hairpin-like structure, and isothermal amplification of the target gene nucleic acid sequence is guided and accelerated.

Description

Technical Field

[0001] The present invention belongs to the technical field of gene detection, and in particular relates to a promoter-mediated isothermal amplification method and its application in nucleic acid detection. Background Art

[0002] With the intensification of global change, emerging human and animal diseases are becoming more frequent, spreading across species, and spreading globally. According to the World Health Organization (WHO), 62% of the 1,415 human diseases worldwide are zoonotic, of which 71.8% originate from wildlife. Over the past 40 years, the number of emerging pathogens has surged. Examples include Nipah virus and Ebola, both of which have been reported to cross species barriers through animal hosts and infect humans. Intensive animal farming has doubled animal density, accelerating pathogen mutations (e.g., the increased virulence of highly pathogenic avian influenza H5N1). Urbanization has encroached on wildlife habitats, leading to the spread of bat-borne viruses (such as Nipah virus) to livestock and humans. These factors have become key drivers of the rapid emergence of emerging human and animal diseases. The trade in animal products and the movement of people are rapidly spreading endemic diseases across borders. Climate change-induced floods and waterlogging have activated pathogen spores in the soil, triggering their rapid spread, leading to illness in humans and animals. Model projections indicate that without intervention, the death toll from zoonotic diseases will be 12 times higher by 2050 than in 2020, and the frequency of spillover events will increase fourfold. Recently, research in both human and animal health has found that the co-infection of emerging pathogens and old pathogens in hosts has intensified, further increasing the complexity of diagnosis and prevention of major and emerging epidemics.

[0003] The emergence of various emerging diseases poses a serious threat to the health of humans and farmed animals. Some emerging pathogens have high mortality rates from acute infection (e.g., pulmonary anthrax has a mortality rate of over 90%), while others cause high disability rates from chronic infection (e.g., brucellosis can cause endocarditis and joint deformities). A single outbreak of some emerging diseases can cause industrial chains to collapse. Emerging disease prevention and control faces the bottleneck of diagnostic lags. For example, traditional pathogen culture methods typically take 2–5 days, while serological testing has a long window (IgM does not appear until 2 weeks after infection), making it difficult to meet the needs of early intervention. In recent years, driven by the need to prevent and control the spread of human and animal diseases, academia and industry have developed a variety of new rapid pathogen detection and identification methods, advancing the prevention and control of major and emerging diseases.

[0004] In recent years, newly developed multi-pathogen detection technologies, using microfluidic chips, can simultaneously screen for 15 respiratory pathogens, addressing the challenge of overlapping syndromes (e.g., fever with pneumonia requires differentiating between anthrax and influenza). Digital PCR (dPCR) technology can increase pathogen detection sensitivity to 100 copies / mL, enabling detection of low-load samples missed by qPCR (e.g., bacteremia in the chronic phase of brucellosis). High-throughput detection technologies based on metagenomic sequencing (mNGS) have also demonstrated significant advantages, requiring no pre-defined pathogens and enabling identification of unknown pathogens within 72 hours. These methods, with their high sensitivity and specificity, have advanced pathogen detection from traditional morphological to molecular biology levels, significantly improving detection efficiency and accuracy.

[0005] However, while common nucleic acid detection methods, such as the polymerase chain reaction (PCR), offer high sensitivity and specificity, they rely on complex thermal cycling equipment and multiple temperature cycles, making them cumbersome and time-consuming, making them difficult to meet the demands of rapid on-site testing. To address this issue, isothermal amplification (isothermal amplification) has emerged and rapidly developed. Isothermal amplification techniques can rapidly and efficiently amplify target nucleic acid sequences at a constant temperature, eliminating the need for complex thermal cycling equipment. These techniques, such as nucleic acid sequence-dependent amplification (NASBA), strand displacement amplification (SDA), rolling circle amplification (RCA), loop-mediated isothermal amplification (LAMP), recombinase polymerase amplification (RPA), and cross-primer amplification (CPA), enable efficient detection in resource-limited field settings. These isothermal amplification techniques not only significantly improve detection efficiency but also reduce equipment costs and operational complexity, offering new solutions for rapid on-site detection of human and animal pathogens.

[0006] Isothermal amplification techniques enable rapid detection and control of pathogens, even without sophisticated laboratory equipment. This allows for timely implementation of preventive and control measures, effectively reducing the economic losses caused by disease outbreaks. These techniques offer promising applications, particularly in resource-limited settings, providing strong technical support for the rapid detection and control of pathogens. The following describes several commonly used isothermal amplification techniques.

[0007] 1. Nucleic acid sequence-based amplification (NASBA) NASBA is an isothermal amplification technology based on RNA templates. It utilizes the synergistic action of reverse transcriptase, RNase H, and T7 RNA polymerase to amplify RNA target sequences at a constant temperature (typically 41°C). Its core principle is that reverse transcriptase converts the RNA template into cDNA, followed by RNase H degradation of the RNA strand, and T7 RNA polymerase synthesizes a large amount of RNA product using the cDNA as a template. NASBA technology boasts high specificity and is particularly suitable for the direct detection of RNA viruses. It is widely used in pathogen diagnosis, gene expression analysis, and food safety testing. However, this method's enzyme system is complex and expensive, sensitive to inhibitors, and subject to patent restrictions.

[0008] 2. Strand displacement amplification (SDA) SDA is an isothermal nucleic acid amplification technology based on restriction endonucleases and DNA polymerases. It uses a restriction endonuclease to create a nick at a specific location. A DNA polymerase with strand displacement activity then extends and displaces the downstream DNA strand from the nick, amplifying the target sequence through repeated cycles of nick-extension-displacement. SDA is typically performed at a constant temperature of 50-55°C and boasts high amplification efficiency and rapid reaction speed, making it suitable for applications in pathogen detection and molecular diagnostics. However, the method requires partial thermal denaturation, a complex enzyme system, and relatively cumbersome procedures.

[0009] 3. Rolling circle amplification (RCA) RCA is an isothermal nucleic acid amplification technique based on a circular DNA template. It leverages the high processivity of phi29 DNA polymerase to achieve linear amplification of target sequences at a constant temperature of 30-37°C. The core principle of this technique is that DNA polymerase replicates continuously along the circular template, generating ultra-long single-stranded DNA products composed of hundreds to thousands of repeating units. This technique boasts high amplification efficiency and excellent product uniformity, and is widely used in gene cloning, nucleic acid testing, and nanomaterial preparation. However, this method relies on a circular template and exhibits relatively slow amplification speed, making it primarily used in specialized applications rather than direct diagnostics.

[0010] 4. Loop-mediated isothermal amplification (LAMP) technology LAMP is a molecular biology technique that amplifies nucleic acids at a constant temperature. LAMP technology uses 4-6 specific primers and a DNA polymerase with strand displacement activity (such as BstLAMP technology utilizes DNA polymerase (DNA polymerase) to efficiently amplify target nucleic acid sequences at a constant temperature (typically 60-65°C). Its core principle is to create a stem-loop structure through primer design, enabling rapid amplification under isothermal conditions without the need for complex thermal cycling equipment. LAMP technology offers advantages such as high sensitivity, specificity, ease of use, and visual results, making it widely used in aquatic pathogen detection. However, this method is complex in primer design, prone to aerosol contamination, and unsuitable for amplifying long fragments.

[0011] 5. Recombinase polymerase amplification (RPA) technology RPA is an isothermal nucleic acid amplification technique performed at room temperature (typically 37-42°C). This technique utilizes the synergistic action of recombinase, single-stranded DNA-binding protein (SSB), and DNA polymerase to achieve rapid and efficient amplification of target nucleic acid sequences without the need for thermal cycling. Its core principle is the binding of specific primers to template DNA by the recombinase, followed by a chain extension reaction under the action of DNA polymerase, resulting in exponential amplification of the target sequence. Due to its low equipment requirements and rapid reaction speed, RPA is particularly suitable for field or resource-limited environments and has been widely used in a variety of fields, including rapid pathogen detection, food safety monitoring, environmental microbiology testing, and clinical diagnosis. However, this method is susceptible to primer dimers, which can affect detection specificity and lead to a high risk of false positives. Furthermore, RPA is temperature-sensitive and the amplification reagents are expensive, resulting in patent barriers to its use.

[0012] 6. Crossing-primer amplification (CPA) CPA is a nucleic acid isothermal amplification technology based on strand displacement activity. This technology designs 4 or 5 specific primers for 4 or 5 regions of the target gene, and utilizes strand displacement properties to amplify the target gene. Bst DNA polymerase and betaine are used for amplification at a constant temperature of approximately 63°C. CPA can be categorized as single-crossover amplification or double-crossover amplification, depending on the number of cross primers. It offers high specificity and ease of use, but similar to LAMP, it also suffers from aerosol contamination.

[0013] A comprehensive comparison of the characteristics of NASBA, SDA, RCA, LAMP, RPA, and CPA isothermal amplification techniques is shown in Table 1 .

[0014] Table 1. Comprehensive comparison of NASBA, SDA, RCA, LAMP, RPA, and CPA isothermal amplification technologies

[0015] In summary, with the emergence and rapid spread of various emerging diseases, the need for rapid identification and detection of pathogens is becoming increasingly urgent. While traditional detection methods remain applicable in certain scenarios, their limitations are becoming increasingly apparent. Molecular detection methods, particularly isothermal amplification, are becoming the mainstream technology for pathogen detection due to their high sensitivity, high specificity, and rapid response. In the future, with continued technological optimization and innovation, isothermal amplification is expected to play an even more important role in human and animal health management. Summary of the Invention

[0016] The present invention provides a promoter-mediated isothermal amplification (PAMP) method and its application in nucleic acid detection. This method utilizes specific primer design and enzymatic reactions to achieve rapid amplification of target nucleic acids under isothermal conditions. Its principle and characteristics include the use of a composite primer containing a promoter sequence at its 3' end to initiate the initial isothermal amplification reaction, introducing the promoter sequence into the amplified product and generating a new target gene nucleic acid sequence containing the promoter sequence. RNA polymerase, guided by the promoter, transcribes the target gene RNA strand, which is then reverse-transcribed by reverse transcriptase to produce a large amount of cDNA, continuously providing single-stranded DNA template for subsequent isothermal amplification reactions. Simultaneously, the transcription initiation process provides a large amount of single-stranded DNA template regions for simultaneous isothermal amplification, accelerating the DNA isothermal amplification reaction. Furthermore, a specific primer with a reverse complement to the target gene sequence is designed at the 5' end of the composite primer. This enables the 3' end of the newly generated nucleic acid sequence (specific primer) to bind to its own sequence to form a hairpin-like structure, guiding and accelerating the isothermal amplification of the target gene nucleic acid sequence. This cycle is repeated, and target gene nucleic acid sequences of different lengths are finally amplified, which are suitable for on-site rapid detection of nucleic acids.

[0017] The present invention first provides a primer pair for promoter-mediated isothermal amplification, which comprises: a) FP forward primer: 5′-R1-promoter-F1-3′; b) RP reverse primer: 5′-F2-promoter-R2-3′; Among them, F1 and R2 are external primers for isothermal amplification of the target gene, which are located at the 5' end of the target gene sequence and the 5' end of the complementary chain sequence of the target gene sequence, respectively, and are used to specifically recognize the target gene binding site and guide the amplification of the target gene nucleic acid sequence; R1 recognizes and binds to the 5' end of the target gene sequence, and the R2 sequence recognizes and binds to the downstream sequence of the F2 site of the target gene; F2 recognizes and binds to the 5' end of the complementary chain of the target gene sequence, and F1 binds to the downstream sequence of the R1 site of the complementary chain of the target gene sequence; R1 and F2 specifically recognize the complementary sequence at the 3′ end of the nascent chain to form a hairpin structure, guiding the amplification of the target gene nucleic acid sequence; Furthermore, the promoter described therein is a promoter that can guide RNA transcription, such as T7 promoter, SP6 promoter, T3 promoter, etc.

[0018] As a specific description of the embodiment, the primer pair for promoter-mediated isothermal amplification is used to detect white spot syndrome virus; the sequence of its FP forward primer is as follows: 5′-GGCGTCCTCTGCAACCTCAAAAAATTCTAATACGACTCACTATAGGGAAGGCAGTTTCTCCGTTCT-3′ (SEQ ID NO: 1), The sequence of the RP reverse primer is as follows: 5′-TTGGCGAGCAAGGCAATTTCAGCAATTCTAATACGACTCACTATAGGGCAAATCCAAGAGGCACTCCA-3′ (SEQ ID NO: 2).

[0019] Another primer pair for promoter-mediated isothermal amplification is used to detect shrimp stealing Nodamura virus; the sequence of its FP forward primer is as follows: 5′-GTTGATGATCGTGTCCATGAAGTAATTCTAATACGACTCACTATAGGGGTACAAGCATTTGATTCTAAGTACC-3′ (SEQ ID NO: 3), The sequence of the RP reverse primer is as follows: 5′-CGCGCCAAGAAATTTGGGTTAATTCTAATACGACTCACTATAGGGCGGGCTACCACTTTTGAC-3′ (SEQ ID NO: 4).

[0020] The primer pairs provided by the present invention can be used to prepare molecular detection products for isothermal amplification detection; In another aspect, the present invention provides a kit for isothermal amplification detection of white spot syndrome virus in shrimp, which comprises the above-mentioned primer pair.

[0021] In another aspect, the present invention also provides a method for promoter-mediated isothermal amplification of a target gene nucleic acid sequence, the method comprising the following steps: a) denaturing the nucleic acid template to be detected, As a specific description of the embodiment, denaturation is carried out at 95°C for 2 to 5 minutes; b) performing isothermal amplification at 59°C to 68°C in an isothermal amplification reaction system containing primers and a denatured nucleic acid template; The isothermal amplification system can adopt a conventional reaction system, wherein the components of a specific reaction system are as follows: 10× T7 RNA polymerase buffer: 1.25 μL 10× Bst Reaction Buffer: 1.25 μL dNTPs (20 mM): 1.5 μL NTP (20 mM): 1.0 μL Betaine (5 M): 6 μL MgCl2 (25 mM): 6 μL FP (20 μM): 2 μL RP (20 μM): 2 μL T7 RNA polymerase (50 U / μL) or SP6 RNA polymerase (50 U / μL): 1 μL M-MLV reverse transcriptase (200 U / μL): 0.2 μL Bst DNA polymerase (8 U / μL): 1 μL Template (pre-denatured DNA / RNA): 1 μL Sterile water was added to make up the total reaction volume to 25 μL.

[0022] The DNA polymerase is selected from Bst DNA polymerase, Klenow DNA polymerase, Vent DNA polymerase or Phi 29 DNA polymerase; The RNA polymerase is selected from a promoter-matched transcriptase, such as T7 transcriptase, SP6 RNA polymerase, T3 RNA polymerase, etc.; The reverse transcriptase is selected from M-MLV reverse transcriptase, AMV reverse transcriptase and the like.

[0023] The present invention provides a primer pair for promoter-mediated amplification of target gene nucleic acid sequences. Under constant temperature conditions (59-68°C), this primer pair not only melts double-stranded DNA but also provides more amplification template. A hairpin-like structure simultaneously guides isothermal amplification, thereby completing the reaction from a low copy number (e.g., single-digit or tens of copies) of a target nucleic acid to millions or even tens of millions of copies or more within tens of minutes. This enables rapid amplification and specific detection of the target gene, significantly reducing the requirements for, and reliance on, expensive detection equipment. The present invention has a wide range of applications, such as rapid amplification and specific detection of target gene nucleic acid sequences. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 This diagram illustrates the design and composition of primers. When the target fragment is a double-stranded nucleic acid, F1 is the first primer in the sense strand of the target gene double strand, F2 is the second primer in the sense strand of the target gene double strand, R1 is the first primer in the antisense strand of the target gene double strand, and R2 is the second primer in the antisense strand of the target gene double strand. When the target fragment is a single-stranded nucleic acid, F1 is the first primer in the single-stranded target gene, F2 is the second primer in the single-stranded target gene, R1 is the first primer in the complementary strand of the target gene, and R2 is the second primer in the complementary strand of the target gene. The T7 promoter sequence in the figure is only used to illustrate the position of the promoter in the PAMP primer; it can also be an SP6 or other promoter sequence. FP is the forward primer, and RP is the reverse primer (BP).

[0025] Figure 2 A diagram showing the positional relationship of primers on the target gene sequence, where F refers to the forward primer, R refers to the reverse primer (also called the backward primer), and c refers to the complementary fragment of the corresponding (sub) primer sequence.

[0026] Figure 3 This diagram illustrates the isothermal amplification process guided by a primer complex under a DNA template, the promoter-guided RNA transcription and double-stranded DNA melting, and the reverse transcription of single-stranded RNA to generate cDNA. FP stands for forward primer, and RP stands for reverse primer (also known as BP). RP and BP in this invention refer to the same primer complex. MMLV, also known as M-MLV, is an abbreviation for M-MLV reverse transcriptase. Bst E is Bst Abbreviation for DNA Polymerase.

[0027] Figure 4 This diagram illustrates the process of RNA transcription and double-stranded DNA melting under the guidance of a newly generated promoter-containing template, followed by isothermal amplification with the single-stranded DNA under the action of an enzyme, and the formation of a hairpin structure with the single-stranded DNA. In the figure, FP stands for forward primer, and RP stands for reverse primer (also known as BP). RP and BP in this invention refer to the same composite primer. MMLV, also known as M-MLV, is an abbreviation for M-MLV reverse transcriptase. Bst E is Bst Abbreviation for DNA Polymerase.

[0028] Figure 5 This is a schematic diagram of the subsequent isothermal amplification process of a template containing a hairpin-like structure under the action of an enzyme, and the length of the amplified product will continue to extend; and the transcription process guided by the promoter and the melting of the DNA double helix, and the reverse transcription process, and finally the formation of DNA templates of different lengths.

[0029] Figure 6 The electrophoresis results show the effect of missing NTP components on amplification products and amplification efficiency in the PAMP reaction system. The lanes are: DNA molecular weight DL 2000 Marker, amplification temperature 58 ℃, 62 ℃, and amplification products of the same temperature reaction conditions with the absence of NTP alone. Bst The three enzymes, DNA polymerase, M-MLV reverse transcriptase, and T7 RNA polymerase, were most efficient at completing PAMP at 62°C. In the absence of NTP alone, the T7 promoter-mediated RNA transcription process was blocked, affecting the subsequent exponential amplification reaction in PAMP (no characteristic bands were produced). This further confirmed that the T7 promoter sequence and T7 RNA polymerase rely on NTP to synthesize RNA, thereby playing a key mediating role in the method of the present invention.

[0030] Figure 7 The electrophoresis results show the effects of missing NTP components in the PAMP reaction system, or lack of T7 promoter sequence or M-MLV enzyme components in the primers on the amplification products and amplification efficiency. Figure 7 Middle (A), Figure 7 Middle (B), Figure 7Middle (C) shows the corresponding PAMP amplification products when the reaction system lacks NTPs, T7 RNA polymerase, or M-MLV reverse transcriptase. Lanes are DNA molecular weight marker DL 2000, and amplification times are 40, 60, 80, 100, and 120 minutes, respectively. The figure shows that no clear product bands appear within 80 minutes when the NTP component, T7 RNA polymerase, or M-MLV enzyme is missing. This indicates that NTPs, T7 RNA polymerase, and M-MLV enzyme are all important factors in improving the amplification efficiency of PAMP technology. Among them, the absence of NTPs and T7 RNA polymerase has the greatest impact on the PAMP reaction (no clear bands after 100 minutes of amplification). In comparison, the absence of M-MLV enzyme has a slightly smaller impact on the PAMP reaction efficiency (clear bands appear after 100 minutes of amplification).

[0031] Figure 8 The electrophoresis results of the PAMP amplification product obtained after restriction endonuclease digestion to obtain a single target fragment and the restriction endonuclease cleavage site in the target gene are shown. Figure 8 (A) is the electrophoresis result, where lanes are: DNA molecular weight DL 2000 Marker, 1 is the normal amplification product of PAMP reaction; 2 to 5 correspond to restriction endonucleases Alu I (recognition site AGCT). Figure 8 Restriction enzymes can be seen in (B) Alu There is a single restriction enzyme cleavage site on the WSSV target gene amplification sequence, so a single target gene band can be obtained after enzyme cleavage.

[0032] Figure 9 The following figure shows the electrophoresis results of the PAMP reaction temperature optimization process. The lanes are: DNA molecular weight DL 2000 marker, and the corresponding amplification products at reaction temperatures of 58°C, 61°C, 64°C, 67°C, and 70°C. The figure shows that clear amplification products are observed in the temperature range of 61°C to 67°C, indicating that PAMPs are efficiently amplified under these conditions.

[0033] Figure 10 Mg in the PAMP reaction system 2+ Electrophoresis results of the ion concentration optimization process, where the lanes are: DNA molecular weight DL 2000 Marker, Mg 2+ The corresponding amplification products when the ion concentrations are 4 mM, 6 mM, 8 mM, 10 mM and 12 mM. 2+ Amplification products were observed at concentrations ranging from 6 mM to 12 mM.

[0034] Figure 11The following electrophoresis results illustrate the optimization of dNTP concentrations in a PAMP reaction system. Lanes are: DNA molecular weight DL 2000 marker, and corresponding amplification products at dNTP concentrations of 0.8 mM, 1.2 mM, 1.6 mM, 2 mM, and 2.4 mM. The results show that amplification products are observed at dNTP concentrations between 0.8 mM and 1.6 mM, while higher concentrations inhibit PAMP amplification. A concentration of 1.6 mM is recommended.

[0035] Figure 12 The following is an electrophoresis diagram showing the optimization of betaine concentration in the PAMP reaction system. The lanes are: DNA molecular weight DL 2000 marker, and the corresponding amplification products at betaine concentrations of 0.6 M, 0.9 M, 1.05 M, 1.2 M, and 1.5 M. The results show that amplification products are observed at betaine concentrations ranging from 0.6 M to 1.5 M. Judging from the clarity of the amplified product bands, a concentration of 1.2 M is recommended.

[0036] Figure 13 This figure shows the specificity validation of the WSSV vp37 PAMP primers. The lanes are: DNA molecular weight DL 2000 Marker, and the corresponding amplification products when the templates are WSSV-positive nucleic acid, IHHNV-positive nucleic acid, HPV-positive nucleic acid, MBV-positive nucleic acid, and a blank control. The results show that only the first lane has amplified effectively; the others do not. This demonstrates the high specificity of the WSSV vp37-PAMP primers and avoids nonspecific amplification of nucleic acids from other pathogens.

[0037] Figure 14 This is the electrophoresis result diagram showing the effect of adding Loop primers on the sensitivity of PAMP technology. Figure 14 Middle (A), Figure 14 Middle (B), Figure 14 Middle (C) shows the effects of adding loop primers to both ends of the PAMP composite primer to promote amplification reaction, adding loop primers only to the front end, or adding loop primers only to the back end on the amplification product and amplification efficiency. The concentration of the starting template is about 10 3 copies / μL, used BstDNA polymerase was produced by NEB. Lanes are: DNA molecular weight DL 2000 Marker, and amplification times are 40 minutes, 50 minutes, 60 minutes, 70 minutes, and 80 minutes, respectively. The results show that adding loop primers to the outside of the PAMP composite primer sequence FP or RP improves amplification efficiency, and adding loop primers to both ends significantly increases detection sensitivity. This experimental result demonstrates that loop primers can improve PAMP amplification efficiency and provide a technical approach for optimizing PAMP technology. However, PAMP amplification can be completed completely and efficiently without loop primers.

[0038] Figure 15 The electrophoresis results of the sensitivity verification of PAMP technology under low template concentration conditions are as follows: DNA molecular weight DL 2000 Marker, and the corresponding PAMP amplification products under the conditions of amplification time of 40 minutes, 50 minutes, 60 minutes, 70 minutes and 80 minutes. It can be seen from the results that PAMP at low concentration (10 3 Under the template condition of 1000 copies / μL, obvious product bands appeared after 50 minutes.

[0039] Figure 16 The electrophoresis results show the comparison of the amplification efficiency of PAMP and LAMP under low template concentration conditions. Figure 16 (A) shows the PAMP primers at low concentration (the concentration of the starting template is about 10 3 Amplification results under different amplification times under the template conditions of (copies / μL), Figure 16 Middle (B) shows the amplification results of conventional LAMP primers at different amplification times under low template concentration conditions. The lanes are: DNA molecular weight DL 2000 marker, and the corresponding PAMP amplification products under amplification times of 40, 50, 60, 70, and 80 minutes. This figure also shows significant differences in the size and composition of the amplification products from PAMP and LAMP. This suggests that, under the same low template concentration conditions, PAMP has higher amplification efficiency than conventional LAMP technology, and the bands are more dispersed, clearer, and easier to identify.

[0040] Figure 17 The electrophoresis results of the comparison of the amplification efficiency of PAMP and LAMP technology under different temperature conditions are shown in Figure 2. Figure 17 (A) shows the amplification results guided by PAMP primers at different amplification temperatures. Figure 17Middle (B) shows the amplification results using conventional LAMP primers at different amplification temperatures. Lanes are: DNA molecular weight DL 2000 marker, and the corresponding PAMP amplification products at amplification temperatures of 50°C, 53°C, 56°C, 59°C, 62°C, 65°C, and 70°C. These results demonstrate that, at the same low template concentration, PAMP technology exhibits better amplification temperature compatibility and higher amplification efficiency than conventional LAMP technology.

[0041] Figure 18 The nucleic acid (RNA) of RNA virus CMNV was used as template to detect PAMP primers ( Figure 18 (A)) and LAMP primers ( Figure 18 Amplification efficiency plot (B), where the cycle count on the horizontal axis is 1 cycle per minute. The results show that PAMP technology also has good amplification efficiency for RNA templates, and has higher amplification efficiency and sensitivity than LAMP technology.

[0042] Figure 19 The RNA virus CMNV nucleic acid (RNA) was used as a template to detect the presence of T7 RNA polymerase alone, M-MLV polymerase alone, or M-MLV polymerase alone in the PAMP reaction system by fluorescence quantitative PCR. Bst The effect of DNA polymerase on the amplification product and amplification efficiency; the cycle number in the horizontal axis is 1 cycle every 30 seconds. The concentration of the starting template is about 10 3 copies / μL~10 4 From the results of the amplification curve, it can be found that the absence of T7 RNA polymerase or M-MLV polymerase alone will significantly reduce the amplification efficiency of the PAMP reaction, while the absence of Bst After DNA polymerase, the PAMP reaction did not produce any amplification products within 240 cycles (120 minutes). The absence of T7 RNA polymerase or M-MLV polymerase alone would seriously reduce the reverse efficiency; compared with the normal amplification of 60 cycles (30 minutes), Ct The value (the cycle number at which the "S"-shaped amplification curve intersects the baseline) appears only after 140 cycles (70 minutes) after the single deletion of M-MLV polymerase. Ct This also provides conclusive experimental evidence that the PAMP technology of the present invention is completely different from the LAMP technology in principle. Bst DNA polymerase will completely prevent PAMP from amplifying normally, which also provides conclusive experimental evidence that the PAMP technology of the present invention is completely different from the NASBA technology in principle. DETAILED DESCRIPTION

[0043] The core principle of existing nucleic acid sequence-dependent amplification (NASBA) technology is to use reverse transcriptase, RNase H, and T7 RNA polymerase, along with forward and reverse primers, to mimic the in vivo replication mechanism of reverse transcriptase to amplify target RNA. This method can produce 10-12 times the product in 90 minutes. However, the NASBA method cannot achieve exponential amplification of target nucleic acid genes. The present invention utilizes composite primers containing promoter sequences, which, through isothermal amplification, initially form sequences at both ends that carry reverse complementarity to the inner fragment of the starting primer sequence. Specifically, the promoter-containing composite primers introduce R1 or F2 sequences at either end of the newly amplified nucleic acid sequence, facilitating the subsequent spontaneous formation of a hairpin-like structure at the 3' end, which initiates exponential amplification of the new nucleic acid fragment generated by the dual composite primers. Therefore, the core principle of the present method is completely different from that of NASBA. The PAMP method of the present invention can achieve over a million-fold amplification of the starting template nucleic acid in 30-60 minutes, significantly exceeding NASBA in amplification efficiency.

[0044] The composite primers and their amplification principles of the present invention are also distinct from those used in LAMP amplification. The front primer FIP in LAMP amplification is composed of the "F1c-F2" structure, meaning the FIP primer consists of the F2 segment of the primer sequence and the F1c segment of its complementary strand. The composite primer FP in the present invention is composed of the "R1-promoter sequence-F1" structure, meaning the FP primer consists of the F1 segment of the primer sequence and the R1 segment of its complementary strand. The primer compositions of the BIP primer used in LAMP and the RP primer used in PAMP are similar to those of FIP and FP, respectively, and will not be further described. Therefore, the primer compositions of the two are clearly different. During LAMP amplification, the F3 and B3 primers, respectively, unzip the newly synthesized DNA strands (converting double strands to single strands) by the FIP and BIP primers. However, during the PAMP amplification process of the present invention, RNA polymerase and reverse transcriptase provide the new single-stranded template, while the FP and RP primers themselves unzip their amplification products, eliminating the need for unzipping by the F3 or B3 primers used in LAMP amplification for subsequent amplification. Therefore, the amplification principles of PAMP and LAMP are very different. The experimental drawings provided in this specification can more clearly confirm the above description.

[0045] The present invention provides a method for isothermal amplification of nucleic acid sequences mediated by a promoter, which utilizes the 3' end of a composite primer containing a promoter sequence to bind to a target sequence to initiate an initial isothermal amplification reaction. The design concept of the primer pair used in the present invention is shown in Figure 1 , the structure is as follows: a) FP forward primer: 5′-R1-promoter-F1-3′; b) RP reverse primer: 5′-F2-promoter-R2-3′; Among them, F1 and R2 are the external primers of the target gene, which are located at the 5' end of the target gene sequence and the 5' end of the complementary chain sequence of the target gene sequence, respectively, and are used to specifically identify the target gene binding site and guide the amplification of the target gene nucleic acid sequence.

[0046] R1 recognizes and binds to the 5′ end of the target gene sequence, and the R2 sequence recognizes and binds to the downstream sequence of the F2 site of the target gene; F2 recognizes and binds to the 5′ end of the complementary chain of the target gene sequence, and F1 binds to the downstream sequence of the R1 site of the complementary chain of the target gene sequence.

[0047] R1 and F2 specifically recognize the complementary sequence at the 3′ end of the nascent chain to form a hairpin structure, which also guides the amplification of the target gene nucleic acid sequence.

[0048] The positional relationship of F1, R1, F2 and R2 on the target gene sequence is shown in Figure 2 .

[0049] The promoter mentioned is a promoter that can guide RNA transcription, such as T7 promoter, SP6 promoter, T3 promoter, etc.

[0050] The primer pair designed in the present invention utilizes the 3′ end of a composite primer containing a promoter sequence to initiate an initial isothermal amplification reaction, inserting the promoter sequence into the amplified product. The first-round amplification product generates a target gene nucleic acid sequence containing the promoter sequence and a reverse complementary sequence to the target gene sequence inside the first-round amplification primer. RNA polymerase utilizes the promoter sequence as a guide to transcribe an RNA chain of the target gene, which is then reverse-transcribed by a reverse transcriptase to produce a large amount of cDNA, continuously providing a single-stranded DNA template for subsequent isothermal amplification reactions. Simultaneously, the transcription initiation and transcription processes can provide a large amount of single-stranded DNA template regions for the simultaneous isothermal DNA amplification, thereby accelerating the DNA isothermal amplification reaction. More importantly, a specific primer that is reverse complementary to the target gene sequence is designed at the 5′ end of the composite primer, guiding the 3′ end of the newly generated nucleic acid sequence (specific primer) to complementary bind with its own sequence (specifically, the inner sequence of the initiator primer) to form a hairpin-like structure, thereby guiding and accelerating the isothermal amplification of the target gene nucleic acid sequence. The key to the established method is the composite primer, which contains the sequence of the initial amplification primer fragment, a promoter sequence, and the reverse complement of the inner sequence of the initial amplification primer. This composite primer guides DNA polymerase to synthesize the complementary chain of the target gene. The complementary chain carrying the composite primer can be recognized by RNA polymerase to synthesize RNA (which is then reverse transcribed into cDNA by reverse transcriptase), and can also be recognized by another composite primer to guide DNA polymerase to synthesize the target gene chain. When a pair of composite primers completes the amplification of the target gene, a cycle of target gene chain amplification is formed, which triggers subsequent exponential amplification, ultimately amplifying target gene nucleic acid sequences of varying lengths. When the starting template is RNA, the reverse transcriptase will first initiate reverse transcription of the target gene under the guidance of the composite primer, forming cDNA containing the composite primer and complementary to the target gene, thereby initiating the amplification process of the target gene chain.

[0051] Based on the construction of the above composite primer pair, the present invention also provides a method for promoter-mediated isothermal amplification of a target gene nucleic acid sequence, wherein the method comprises the following steps: a) denaturing the nucleic acid template to be detected, As a specific description of the embodiment, denaturation is carried out at 95°C for 2 to 5 minutes; b) performing isothermal amplification at 59°C to 68°C in an isothermal amplification reaction system containing primers and a denatured nucleic acid template; The isothermal amplification system can adopt a conventional reaction system. The components of a specific reaction system are as follows, and can also be adjusted proportionally according to the total volume of the system: 10× T7 RNA polymerase buffer: 1.25 μL 10× Bst Reaction Buffer: 1.25 μL dNTPs (20 mM): 1.5 μL NTP (20 mM): 1.0 μL Betaine (5 M): 6 μL MgCl2 (25 mM): 6 μL FP (20 μM): 2 μL RP (20 μM): 2 μL T7 RNA polymerase (50 U / μL) or SP6 RNA polymerase (50 U / μL): 1 μL M-MLV reverse transcriptase (200 U / μL): 0.2 μL Bst DNA polymerase (8 U / μL): 1 μL Template (pre-denatured DNA / RNA): 1 μL Sterile water was used to make up the total reaction volume to 25 μL. Bst DNA polymerase, Klenow DNA polymerase, Vent DNA polymerase, or Phi 29 DNA polymerase; The RNA polymerase is selected from a promoter-matched transcriptase, such as T7 transcriptase (also known as T7 RNA polymerase), SP6 RNA polymerase, T3 RNA polymerase, etc.; The reverse transcriptase is selected from M-MLV reverse transcriptase, AMV reverse transcriptase and the like.

[0052] The amplification principle of the detection method of the present invention is shown in the attached Figure 3 ~Attached Figure 5 The specific amplification process can be broken down into the following steps: a) F1 at the 3' end of the FP primer and R2 at the 3' end of the RP primer complement each other with the template sequence, and isothermal amplification begins under the action of DNA polymerase, introducing a promoter sequence at one end of the amplified nucleic acid sequence; b) The promoter sequence guides transcription under the action of RNA polymerase to produce RNA chains, thereby promoting the unwinding of double-stranded DNA; c) The composite primer pairs with the complementary RNA strands and produces the first strand of cDNA under the action of reverse transcriptase; d) The RNase activity of reverse transcriptase degrades the RNA strand in the RNA:DNA hybrid, leaving the DNA single strand; e) The composite primer binds to the complementary position of the DNA single-stranded template and isothermally amplified under the action of DNA polymerase, repeating steps a), b), c), and d). The above amplification process is shown in the attached Figure 3 ; f) The 3′ end of the newly generated DNA single strand is the reverse complementary sequence of R1 and F2 on the composite primer, which binds to the reverse complementary sequence of R1 and F2 on the self sequence to form a hairpin-like structure. The 3′ end triggers the subsequent isothermal amplification. The above amplification process is shown in the attached figure. Figure 4 ; g) The double-stranded DNA template with a hairpin structure is transcribed again under the action of the composite primer and promoter, then melted and isothermally amplified, repeating steps a), b), c), d), e), and f). The above amplification process is shown in the attached Figure 5 ; h) Repeating this cycle will quickly generate a large number of target gene nucleic acid sequences of different lengths.

[0053] The information of reagents and materials involved in the examples of the present invention are as follows: primers were synthesized by Shanghai Sangon Biotechnology Co., Ltd.; dNTPs, betaine (Betaine), dATP, dGTP, dCTP and dTTP, MgCl2 were purchased from Shanghai Biotechnology Co., Ltd.; isothermal amplification Bst DNA polymerase, RNA polymerase, and reverse transcriptase were purchased from New England Biolabs; the nucleic acid dye Eva Green was purchased from Xiamen Baiweixin Biotechnology Co., Ltd. The above reagents and materials can also be selected from commonly used reagents in the art and are not limited by the brands or types of specific commercial reagents or materials in the examples of the present invention.

[0054] The following examples illustrate the detection method and detection effect of the present invention. The examples are for illustration only and do not constitute any limitation to the scope of protection of the present invention, which is described in the claims.

[0055] Example 1: Using White Spot Syndrome Virus (WSSV) as an example to verify the principle of PAMP technology White spot syndrome virus (WSSV) is a double-stranded DNA virus that harms shrimp aquaculture. It causes white spots on the shrimp's body, reduced feeding, and a high mortality rate (up to 100% within 3-10 days of infection), posing a significant threat to the global aquaculture industry. Currently, key detection methods include molecular techniques (such as PCR and real-time fluorescence quantitative PCR), immunological methods (such as ELISA and test strips), and histopathological observation. Molecular techniques are highly sensitive but equipment-dependent, immunological methods are simple but less sensitive, and histopathology requires experience and is time-consuming. The recently developed isothermal amplification technique has been widely used due to its significant advantages: it requires no complex thermal cycler and can be amplified in 30-60 minutes at a constant temperature (59-68°C). It also has high specificity, and the results can be directly interpreted by visual inspection, such as turbidity or fluorescence. This method is particularly suitable for rapid screening in small-scale farms or resource-limited areas, providing efficient technical support for the early prevention and control of WSSV. The method presented in this paper enables rapid detection of WSSV viral DNA.

[0056] 1) Confirm that the composite primer containing the starting amplification primer fragment sequence, the promoter sequence, and the reverse complementary sequence of the inner sequence of the starting amplification primer is the core of the PAMP technology.

[0057] Primer sequences were designed using white spot syndrome virus (WSSV) positive nucleic acid as a template and vp37 as the target gene sequence. The FP and RP primer sequences in WSSV-PAMP and their locations on the target gene are shown in Table 2.

[0058] Table 2. WSSV-PAMP primer sequences and their locations on target genes

[0059] That is, the sequence of the FP forward primer for detecting WSSV is as follows: 5′-GGCGTCCTCTGCAACCTCAAAAAATTCTAATACGACTCACTATAGGGAAGGCAGTTTCTTTCCGTTCT-3′, The sequence of the RP reverse primer is as follows: 5′-TTGGCGAGCAAGGCAATTTCAGCAATTCTAATACGACTCACTATAGGGCAAATCCAAGAGGCACTCCA-3′.

[0060] The positions and relationships of primers R1, F1, R2, and F2 in the target gene are as follows:

[0061] The components of the general PAMP reaction system are as follows: 10× T7 RNA polymerase buffer: 1.25 μL 10× Bst Reaction Buffer: 1.25 μL dNTPs (20 mM): 1.5 μL NTP (20 mM): 1.0 μL Betaine (5 M): 6 μL MgCl2 (25 mM): 6 μL FP (20 μM): 2 μL RP (20 μM): 2 μL T7 RNA polymerase (50 U / μL) or SP6 RNA polymerase (50 U / μL): 1 μL M-MLV reverse transcriptase (200 U / μL): 0.2 μL Bst DNA polymerase (8 U / μL): 1 μL Sterile water: 0.8 μL Template (pre-denatured DNA / RNA): 1 μL The total reaction volume was 25 μL.

[0062] No NTP was added to the universal PAMP reaction system, and the amplification was performed at 58°C and 62°C for 90 minutes respectively. Figure 6 Agarose gel electrophoresis showed that the product without NTP addition had only a faint band, which was significantly different from the product band in the normal system. This indicates that in the absence of NTP, the T7 promoter-mediated RNA transcription process is blocked, affecting the subsequent exponential amplification reaction, further confirming that the T7 promoter plays a key mediating role in the PAMP reaction.

[0063] (2) To further verify the principle of PAMP technology, the following operations were performed: no NTP was added to the reaction system, no T7 promoter sequence was added to the primer, and no M-MLV reverse transcriptase was added to the reaction system. The other components remained unchanged. Then, the reaction was incubated at 62°C for 40 minutes, 60 minutes, 80 minutes, 100 minutes, and 120 minutes, respectively. The amplification products were checked by agarose gel electrophoresis. Figure 7 Figure (A) shows that when no NTP is added to the reaction system, no target product is amplified within 80 minutes. After 100 minutes of reaction, a few product bands appear, indicating that the transcription process is an important factor for the efficient amplification of PAMP. Figure 7Figure (B) shows that without adding T7 promoter, no target product was amplified within 80 minutes. After 100 minutes of reaction, very few product bands appeared, confirming that the RNA transcription process mediated by T7 promoter is the key to the efficient amplification of PAMP. Figure 7 Figure (C) shows that without the addition of M-MLV reverse transcriptase, no target product was amplified within 80 minutes. Only after 100 minutes of reaction did a clear product band appear. This shows that the reverse transcription process initiated by reverse transcriptase is also an important factor in the efficient amplification of PAMP.

[0064] (3) Target gene product verification: Using WSSV positive nucleic acid as template and vp37 gene as target gene, the FP and RP composite primer sequences of PAMP were designed respectively. The target fragment amplified by the composite primer contains a single restriction endonuclease. Alu I recognition site (attached Figure 8 (B) shows). The PAMP amplification system was prepared using the above FP and RP primers and amplified at 62°C for 90 minutes. The amplified product was added with restriction endonucleases. Alu I and buffer, enzyme digestion reaction, after the end of enzyme digestion, the enzyme digestion of the amplified product was detected by agarose gel electrophoresis. Figure 8 In (A), lanes 2 to 5 show that the amplified products were cleaved by restriction endonucleases. Alu After enzyme digestion, the fragments are converted into nucleic acid fragments of uniform length, thus appearing as a single target gene band in the electrophoresis diagram, indicating that the amplified product can be Alu The experimental results further confirmed the amplification principle of FP and RP primers in PAMP to amplify the target fragment.

[0065] Example 2. Establishment of PAMP Detection Technology for White Spot Syndrome Virus (WSSV) (1) Optimization of PAMP reaction temperature: Using WSSV positive nucleic acid as template and vp37 target gene as primer sequence, the amplification temperature was optimized under the universal PCR reaction system. The temperature was set at 58 ℃, 61 ℃, 64 ℃, 67 ℃ and 70 ℃ respectively. The amplification was performed for 90 minutes and the amplification product was checked by agarose gel electrophoresis. Figure 9 The results showed that no significant amplification product was observed at 58°C, and no significant amplification product was observed at 70°C due to the impact of enzyme activity. However, significant amplification products were observed between 61°C and 67°C, indicating that PAMPs were highly amplified under these conditions. Therefore, a PAMP reaction temperature between 61°C and 67°C is recommended.

[0066] (2) Mg in PAMP reaction system 2+Ion concentration optimization: Using WSSV positive nucleic acid as template and vp37 target gene as primer sequence, amplify at 65℃ for 90 minutes in a universal PCR reaction system, and optimize Mg 2+ Ion concentration. Set Mg 2+ The ion concentrations were 4 mM, 6 mM, 8 mM, 10 mM and 12 mM respectively, and the amplified products were checked by agarose gel electrophoresis. Figure 10 Show, when Mg 2+ Amplification products can be obtained at concentrations between 6 mM and 12 mM. Based on the principle of economy, a concentration of 8 mM is recommended.

[0067] (3) Optimization of dNTPs ion concentration in PAMP reaction system: Using WSSV positive nucleic acid as template and vp37 target gene as primer sequence, amplify at 65℃ for 90 minutes in a universal PCR reaction system to optimize dNTPs concentration. The dNTPs concentrations were set to 0.8 mM, 1.2 mM, 1.6 mM, 2 mM and 2.4 mM respectively, and the amplification products were checked by agarose gel electrophoresis. Figure 11 It was shown that amplification products were observed when the dNTPs concentration was between 0.8 mM and 1.6 mM, but higher concentrations inhibited the amplification of PAMP. The recommended concentration was 1.6 mM.

[0068] (4) Optimization of betaine concentration in the PAMP reaction system: Using WSSV positive nucleic acid as a template and vp37 target gene as the primer sequence, amplify at 65 °C for 90 minutes in a universal PCR reaction system, optimize the betaine concentration, and set the betaine concentration to 0.6 M, 0.9 M, 1.05 M, 1.2 M, and 1.5 M, respectively. Agarose gel electrophoresis was performed to check the amplification product. Figure 12 The results showed that amplification products were observed when the betaine concentration was between 0.6 M and 1.5 M. Judging from the clarity of the amplified product bands, a concentration of 1.2 M was recommended.

[0069] (5) PAMP technology specificity verification: Using WSSV positive nucleic acid, IHHNV positive nucleic acid, HPV positive nucleic acid, MBV positive nucleic acid and blank control as templates, and vp37 target gene as primer sequences, the primer specificity was verified under the optimized PAMP reaction system. Figure 13 Agarose gel electrophoresis showed that only the first lane had an amplification product, while the other lanes had no amplification products, indicating that the WSSV vp37-PAMP primers had good specificity.

[0070] (6) Effect of adding loop primers on the sensitivity of PAMP technology: loop primers were added at both ends, front end, or back end of the PAMP primer sequence. The experimental design was as follows: WSSV positive nucleic acid was used as template, the concentration was 10 6 copies / μL, using the optimized PAMP reaction system, the amplification time was set to 40 minutes, 50 minutes, 60 minutes, 70 minutes and 80 minutes respectively, and the amplification was checked by agarose gel electrophoresis. Figure 14 (A) shows that PAMP at high concentration (10 6 Under the condition of 100 copies / μL) template, loop primers were added at both ends, and obvious amplification bands were observed in 40 minutes. As the amplification time prolonged, the small fragment products decreased after 80 minutes, and large fragment products were repeatedly replicated and mostly condensed in the agarose gel sample well. Figure 14 Figures (B) and (C) also show similar trends, which are consistent with the PAMP amplification principle. Figure 14 Figures (B) and (C) show that only when a loop primer is added to the front or back end of the primer pair does a clear product band appear after 50 minutes of amplification. Therefore, adding a double-loop primer to the PAMP significantly improves amplification efficiency and detection sensitivity.

[0071] (7) Sensitivity verification of PAMP technology under low template concentration conditions: For example, when the WSSV positive nucleic acid template is 10 3 copies / μL, using the optimized PAMP reaction system, the amplification time was set to 40 minutes, 50 minutes, 60 minutes, 70 minutes and 80 minutes respectively, and the amplification was checked by agarose gel electrophoresis. Figure 15 It was shown that PAMP at low concentration (10 3 Under the condition of 1000 copies / μL) template, agarose gel electrophoresis showed that there was a clear product band at 50 minutes.

[0072] (8) Comparison of amplification efficiency between PAMP and conventional LAMP technology under low template concentration conditions WSSV positive nucleic acid template 10 3 copies / μL, vp37 designed PAMP and LAMP primers respectively, and used the optimized PAMP reaction system. The amplification time was 40 minutes, 60 minutes, 80 minutes, 100 minutes and 120 minutes respectively, and the amplification was checked by agarose gel electrophoresis. Figure 16 In the figure (A), the PAMP primers have obvious product bands at 60 minutes, and the target gene product signal in the lane increases gradually. Figure 16In (B), the LAMP primers produce only faint product bands at 60 minutes, but distinct product bands emerge at 80 minutes, with poor separation between the bands. Therefore, at the same low template concentration, PAMP technology exhibits higher amplification efficiency than conventional LAMP technology, with better band dispersion, clearer bands, and easier identification.

[0073] (9) Comparison of amplification efficiency between PAMP and conventional LAMP technology under different temperature conditions: WSSV positive nucleic acid template 10 3 copies / μL, vp37 designed PAMP and LAMP primers respectively, used the optimized PAMP reaction system, and the amplification temperatures were 50 ℃, 53 ℃, 56 ℃, 59 ℃, 62 ℃, 65 ℃ and 70 ℃ respectively. After amplification for 60 minutes, the amplification status was checked by agarose gel electrophoresis. Figure 17 In the figure (A), the PAMP primers have obvious product bands at 59 ℃ to 65 ℃, and the attached Figure 17 (B) LAMP primers produce only faint product bands at 59°C–62°C, and the bands are poorly resolved. Therefore, at the same low template concentration, PAMP has a better amplification temperature profile than conventional LAMP technology and exhibits higher amplification efficiency.

[0074] Example 3. Establishment of PAMP Detection Technology for CMNV Covert mortality nodavirus (CMNV) is a pathogen that poses a serious threat to the shrimp aquaculture industry. Detection methods primarily include reverse transcription-polymerase chain reaction (RT-PCR), real-time quantitative PCR (RT-qPCR), and histopathological observation. Isothermal amplification technology, recently developed, has gained widespread adoption due to its significant advantages: it requires no complex thermal cycler and can complete amplification within 30 to 60 minutes at a constant temperature (60°C–65°C). It also exhibits high specificity, and results can be directly interpreted by visual inspection using methods such as turbidity or fluorescence. This method is particularly suitable for rapid screening in low-income farms or resource-limited areas, providing effective technical support for the early prevention and control of CMNV. This patented method enables rapid detection of CMNV RNA.

[0075] (1) Design of relevant primers: Select the gene fragment to be amplified based on the RNA sequence and design specific primers based on the sequence. The FP and RP primer sequences in CMNV-PAMP and their locations on the target gene are shown in Table 3.

[0076] Table 3. CMNV-PAMP primer sequences and their locations on target genes

[0077] That is, the sequence of the FP forward primer for detecting CMNV is as follows: 5′-GTTGATGATCGTGTCCATGAAGTAATTCTAATACGACTCACTATAGGGGTACAAGCATTTGATTCTAAGTACC-3′, The sequence of the RP reverse primer is as follows: 5′-CGCGCCAAGAAATTTGGGTTAATTCTAATACGACTCACTATAGGGCGGGCTACCACTTTTGAC-3′.

[0078] The positions and relationships of primers R1, F1, R2, and F2 in the target gene are as follows:

[0079] The optimized reaction system of CMNV-PAMP is as follows: 10× T7 RNA polymerase buffer: 1.0 μL 10× Bst Reaction Buffer: 1.0 μL dNTPs (20 mM): 1.5 μL NTP (20 mM): 1.0 μL Betaine (5 M): 6 μL MgCl2 (25 mM): 5 μL FP (20 μM): 2 μL RP (20 μM): 2 μL T7 RNA polymerase (50 U / μL) or SP6 RNA polymerase (50 U / μL): 1 μL M-MLV reverse transcriptase (200 U / μL): 0.35 μL M-MLV Buffer: 0.2 μL Bst DNA polymerase (8 U / μL): 1.25 μL Eva green: 0.8 μL Sterile water: 0.9 μL Template (total RNA from CMNV-positive tissue, pre-denatured at 95°C for 3 minutes): 1 μL The total reaction volume was 25 μL.

[0080] The reaction program was as follows: 65°C for 1 minute, 70 cycles, and the fluorescence signal was collected using the FAM / SYBR Green channel.

[0081] (1) The amplification effects of PAMP and LAMP primers against CMNV were compared by fluorescence quantitative PCR. Figure 18 Middle (A), Figure 18 Middle (B) shows that PAMP exhibits higher amplification efficiency and sensitivity.

[0082] (2) Fluorescence quantitative PCR was used to detect the absence of T7 RNA polymerase alone, M-MLV polymerase alone, or M-MLV polymerase alone in the PAMP reaction system using the RNA virus CMNV nucleic acid (RNA) as a template. Bst The effect of DNA polymerase on amplification products and amplification efficiency. Figure 19 The results showed that PAMP exhibited different amplification characteristics from LAMP and NASBA.

[0083] The starting template of the amplification method of the present invention can be a DNA template or an RNA template. The amplification method provided by the present invention can be used for rapid amplification and specific detection of target gene nucleic acid sequences, and is suitable for rapid detection of various nucleic acids including pathogenic nucleic acids.

Claims

1. A primer pair for promoter-mediated isothermal amplification, characterized in that: The primer pair comprises: a) FP forward primer: 5′-R1-promoter-F1-3′; b) RP reverse primer: 5′-F2-promoter-R2-3′; Among them, F1 and R2 are external primers for isothermal amplification of the target gene, which are located at the 5' end of the target gene sequence and the 5' end of the complementary chain sequence of the target gene sequence, respectively, and are used to specifically recognize the target gene binding site and guide the amplification of the target gene nucleic acid sequence; R1 recognizes and binds to the 5' end of the target gene sequence, and the R2 sequence recognizes and binds to the downstream sequence of the F2 site of the target gene; F2 recognizes and binds to the 5' end of the complementary chain of the target gene sequence, and F1 binds to the downstream sequence of the R1 site of the complementary chain of the target gene sequence; R1 and F2 specifically recognize the complementary sequence at the 3′ end of the nascent chain to form a hairpin structure, guiding the amplification of the target gene nucleic acid sequence.

2. The primer pair according to claim 1, wherein The promoter is a promoter that can guide RNA transcription.

3. The primer pair according to claim 2, wherein The promoter is T7 promoter, SP6 promoter or T3 promoter.

4. The primer pair according to claim 1, wherein The primer pair for promoter-mediated isothermal amplification is used to detect white spot syndrome virus; the sequence of the FP forward primer is SEQ ID NO: 1, and the sequence of the RP reverse primer is SEQ ID NO:

2.

5. The primer pair according to claim 1, wherein The primer pair for promoter-mediated isothermal amplification is used to detect shrimp-killing Nodamura virus; the sequence of the FP forward primer is SEQ ID NO: 3, and the sequence of the RP reverse primer is SEQ ID NO:

4.

6. Use of the primer pair according to any one of claims 1 to 5 in the preparation of a molecular detection product for isothermal amplification detection.

7. A detection product for promoter-mediated isothermal amplification, characterized in that: The detection product comprises the primer pair according to any one of claims 1 to 5.

8. A promoter-mediated isothermal amplification method, characterized in that: The method is to use the primer pair described in any one of claims 1 to 5 for detection.

9. The method according to claim 8, wherein The method comprises the following steps: 1) Denaturing the nucleic acid template to be detected, 2) The isothermal amplification reaction system containing the primers and the denatured nucleic acid template is subjected to isothermal amplification at a temperature of 59°C to 68°C.

10. The method according to claim 9, wherein The DNA polymerase used in the isothermal amplification reaction system is Bst DNA polymerase, Klenow DNA polymerase, Vent DNA polymerase, or Phi 29 DNA polymerase; The RNA polymerase is T7 transcriptase, SP6 RNA polymerase, or T3 RNA polymerase; The reverse transcriptase is M-MLV reverse transcriptase or AMV reverse transcriptase.

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