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

Abstract

The application provides a promoter-mediated isothermal amplification method and application thereof in nucleic acid detection. The application uses a composite primer containing a promoter sequence to guide an initial isothermal amplification reaction, introduces the promoter sequence into an amplification product, and generates a target gene nucleic acid sequence containing the promoter sequence; an RNA polymerase uses the promoter to guide transcription of an RNA chain of the target gene, and the reverse transcription enzyme reversely transcribes the RNA chain to generate a large amount of cDNA, thereby continuously providing a single-stranded DNA template for subsequent isothermal amplification reaction; meanwhile, the transcription initiation process can provide a large amount of DNA single-stranded template region for synchronous isothermal amplification, thereby accelerating the DNA isothermal amplification reaction. In addition, the 5' end of the composite primer is designed with a specific primer which is reverse complementary to the target gene sequence, so that the 3' end of the newly generated nucleic acid sequence is complementary to the sequence itself to form a hairpin structure, thereby guiding and accelerating the isothermal amplification of the target gene nucleic acid sequence.

Description

Technical Field

[0001] This invention belongs to the field of gene detection technology, specifically relating to a promoter-mediated isothermal amplification method and its application in nucleic acid detection. Background Technology

[0002] With the intensification of global change, emerging human and animal diseases are showing a trend of frequent occurrence, cross-species transmission, and global spread. According to the World Health Organization (WHO), 62% of the 1,415 human diseases worldwide are zoonotic, of which 71.8% originate from wild animals. The number of emerging pathogens has surged in the past 40 years, with viruses such as Nipah virus and Ebola reported to have crossed the interspecies barrier and infected humans through animal hosts. Intensive farming practices have led to a doubling of animal density, accelerating pathogen mutation (e.g., the increased virulence of highly pathogenic avian influenza H5N1); urbanization has encroached on wildlife habitats, causing bat-borne viruses (such as Nipah virus) to spread to livestock and humans. These are important factors influencing the rapid emergence of new human and animal diseases. Trade in animal products and the movement of people have enabled the rapid cross-border spread of endemic diseases, and climate change has led to floods that activate pathogen spores in the soil, triggering their rapid spread and causing disease in humans or animals. Model predictions show that without intervention, the number of deaths from zoonotic diseases in 2050 will be 12 times that of 2020, and the frequency of spillover events will increase fourfold. Recent studies in both human and animal health have revealed an increasing trend of co-infection of emerging and old pathogens in hosts, further complicating the diagnosis and control of major and emerging diseases.

[0003] The emergence of various new infectious diseases poses a serious threat to human and farmed animal health. Some emerging pathogens have high acute mortality rates (e.g., pulmonary anthrax has a mortality rate >90%), while others cause high rates of disability due to chronic infection (e.g., brucellosis can lead to endocarditis and joint deformities). A single outbreak of some emerging diseases can cause the collapse of the entire industry chain. The prevention and control of emerging diseases faces a bottleneck due to diagnostic lag; for example, traditional pathogen culture methods take 2–5 days, while serological detection has a long window period (IgM only appears 2 weeks after infection), failing to meet the needs of early intervention. In recent years, driven by the needs of human and animal disease prevention and control, academia and industry have attempted to establish various novel rapid detection and identification methods for pathogens, promoting the prevention and control of major and emerging infectious diseases.

[0004] In recent years, newly developed multi-pathogen detection technologies, employing microfluidic chips, can simultaneously screen for 15 respiratory pathogens, solving the problem of overlapping syndromes (such as the need to differentiate between anthrax and influenza when fever is accompanied by pneumonia). Digital PCR (dPCR) technology can increase pathogen detection sensitivity to 100 copies / mL, detecting low-load samples missed by qPCR (such as chronic brucellosis bacteremia). High-throughput detection technologies based on metagenomic sequencing (mNGS) have also demonstrated significant advantages, requiring no pre-defined pathogens and completing the identification of unknown pathogens within 72 hours. These methods, with their high sensitivity and specificity, have propelled pathogen detection from the traditional morphological level to the molecular biology level, significantly improving detection efficiency and accuracy.

[0005] However, common nucleic acid detection methods, such as polymerase chain reaction (PCR), while possessing high sensitivity and specificity, rely on complex thermal cycling equipment and multiple temperature cycles, making them cumbersome and time-consuming, and difficult to meet the needs of rapid on-site detection. To address this issue, isothermal amplification technology has emerged and developed rapidly. Isothermal amplification technology can rapidly and efficiently amplify target nucleic acid sequences at a constant temperature, without the need for complex thermal cycling equipment, making it simple to operate and suitable for rapid on-site detection. For example, techniques such as sequence-dependent amplification (NASBA), strand substitution amplification (SDA), rolling circle amplification (RCA), loop-mediated isothermal amplification (LAMP), recombinase polymerase amplification (RPA), and cross-primer amplification (CPA) can achieve efficient detection in resource-constrained field environments. These isothermal amplification technologies not only significantly improve detection efficiency but also reduce equipment costs and operational complexity, providing new solutions for rapid on-site detection of human and animal pathogens.

[0006] Isothermal amplification technology allows testing personnel to quickly obtain results even without sophisticated laboratory equipment, enabling timely implementation of control measures and effectively reducing economic losses caused by disease outbreaks. These technologies have broad application prospects, especially in resource-constrained environments, providing strong technical support for the rapid detection and control of pathogenic microorganisms. The following is an introduction to several commonly used isothermal amplification techniques.

[0007] 1. Nucleic acid sequence-based amplification (NASBA) technology NASBA is an isothermal amplification technique based on an RNA template. This technique utilizes the synergistic action of reverse transcriptase, RNase H, and T7 RNA polymerase to amplify the target RNA sequence under isothermal conditions (typically 41°C). Its core principle is that reverse transcriptase converts the RNA template into cDNA, followed by RNase H degrading the RNA chain, and T7 RNA polymerase synthesizing a large amount of RNA product using the cDNA as a template. NASBA technology is characterized by high specificity, making it particularly suitable for the direct detection of RNA viruses, and is widely used in pathogen diagnosis, gene expression analysis, and food safety testing; however, this method involves a very complex and expensive enzyme system, is sensitive to inhibitors, and is subject to patent restrictions.

[0008] 2. Strand displacement amplification (SDA) SDA (Synthetic Deposition Amplification) is an isothermal nucleic acid amplification technique based on restriction endonucleases and DNA polymerases. This technique utilizes restriction endonucleases to create nicks at specific locations, followed by strand displacement activity in the DNA polymerase, which extends and displaces the downstream DNA strand from the nick. This repeated nick-extension-displacement cycle amplifies the target sequence. SDA is typically performed at a constant temperature of 50-55°C and features high amplification efficiency and rapid reaction speed, making it suitable for pathogen detection and molecular diagnostics. However, this method requires partial thermal denaturation, has a complex enzyme system, and involves relatively cumbersome steps.

[0009] 3. Rolling circle amplification (RCA) technique Recurrent DNA cloning (RCA) is an isothermal nucleic acid amplification technique based on circular DNA templates. It utilizes the highly persistent synthetic capacity of the phi29 DNA polymerase to achieve linear amplification of the target sequence under isothermal conditions of 30-37°C. The core principle of this technique is that the DNA polymerase replicates continuously along the circular template, producing ultra-long single-stranded DNA products composed of hundreds to thousands of repeating units. It features high amplification efficiency and good product uniformity, and is widely used in gene cloning, nucleic acid detection, and nanomaterial preparation. However, this method relies on circular templates and has a relatively slow amplification rate, primarily limiting its application to specific uses rather than direct diagnosis.

[0010] 4. Loop-mediated isothermal amplification (LAMP) technology LAMP is a molecular biology technique for amplifying nucleic acids at a constant temperature. LAMP technology uses 4-6 specific primers and a DNA polymerase with strand displacement activity (such as...). BstDNA polymerase (LAMP) enables the efficient amplification of target nucleic acid sequences at an isothermal temperature (typically 60-65℃). Its core principle involves designing primers to form stem-loop structures, allowing the amplification reaction to proceed rapidly under isothermal conditions without the need for complex thermal cycling equipment. LAMP technology offers advantages such as high sensitivity, specificity, ease of operation, and visualized results, and is widely used in aquatic pathogen detection; however, this method suffers from complex primer design, is prone to aerosol contamination, and is unsuitable for amplifying long fragments.

[0011] 5. Recombinase polymerase amplification (RPA) technology Isothermal amplification (RPA) is a nucleic acid amplification technique performed at room temperature (typically 37-42°C). This technique utilizes the synergistic action of recombinases, single-stranded DNA-binding proteins (SSBs), and DNA polymerases to achieve rapid and efficient amplification of target nucleic acid sequences without the need for heat cycling. Its core principle involves the recombinase binding specific primers to template DNA, followed by a chain extension reaction under the action of DNA polymerase, thereby achieving exponential amplification of the target sequence. Due to its low equipment requirements and fast reaction speed, RPA is particularly suitable for field or resource-limited environments and is currently widely used in various fields such as rapid pathogen detection, food safety monitoring, environmental microbiology detection, and clinical diagnosis. However, primer dimers can affect detection specificity, leading to a high risk of false positives. Furthermore, it is temperature-sensitive, has high-cost amplification reagents, and faces patent barriers in its application.

[0012] 6. Cross-primer amplification (CPA) technique CPA is a nucleic acid isothermal amplification technique based on strand displacement activity. This technique involves designing four or five specific primers targeting four or five regions of the target gene, utilizing primers with strand displacement properties. Bst DNA polymerase and betaine are used for amplification under isothermal conditions at around 63°C. Depending on the number of cross-primers, CPA can be divided into single-cross-priming amplification and double-cross-priming amplification, which are characterized by high specificity and ease of operation; however, this method has similar drawbacks to LAMP, including the problem of 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 techniques

[0015] In summary, with the emergence and rapid spread of various new infectious diseases, the need for rapid identification and detection of pathogenic microorganisms is becoming increasingly urgent. While traditional detection methods remain applicable in certain scenarios, their limitations are becoming increasingly apparent. Molecular detection methods, especially isothermal amplification (AFA), are becoming the mainstream technology for pathogen detection due to their high sensitivity, high specificity, and rapid response. In the future, with continuous optimization and innovation, AFA is expected to play an even more important role in the health management of humans and farmed animals. Summary of the Invention

[0016] This invention provides a promoter-based isothermal amplification (PAMP) method and its application in nucleic acid detection. The method utilizes specific primer design and enzyme reactions to achieve rapid amplification of target nucleic acids under isothermal conditions. Its key principle is the use of a composite primer containing the promoter sequence at its 3′ end to initiate the initial isothermal amplification reaction, introducing the promoter sequence into the amplification product and generating a new target gene nucleic acid sequence containing the promoter sequence. RNA polymerase, guided by the promoter, transcribes the RNA chain of the target gene, which is then used by reverse transcriptase to generate a large amount of cDNA, continuously providing single-stranded DNA templates for subsequent isothermal amplification reactions. Simultaneously, the transcription initiation process provides a large amount of single-stranded DNA template regions for concurrent isothermal amplification, accelerating the DNA isothermal amplification reaction. Furthermore, the 5′ end of the composite primer is designed with a specific primer that is inversely complementary to the target gene sequence, enabling the newly generated nucleic acid sequence's 3′ end (the specific primer) to bind complementaryly with its own sequence to form a hairpin-like structure, guiding and accelerating the isothermal amplification of the target gene nucleic acid sequence. This process is repeated until target gene nucleic acid sequences of different lengths are amplified, making them suitable for rapid on-site detection of nucleic acids.

[0017] This invention first provides a primer pair for promoter-mediated isothermal amplification, comprising: a) FP forward primer: 5′-R1-promoter-F1-3′; b) RP reverse primer: 5′-F2-promoter-R2-3′; F1 and R2 are the outer primers for isothermal amplification of the target gene, located at the 5′ end of the target gene sequence and the 5′ end of the complementary strand sequence of the target gene sequence, respectively. They 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 R2 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 strand of the target gene sequence, and F1 binds to the downstream sequence of the R1 site of the complementary strand of the target gene sequence. R1 and F2 specifically recognize the complementary sequence at the 3′ end of the nascent strand, thereby forming a hairpin structure to guide the amplification of the target gene nucleic acid sequence; Furthermore, the promoters mentioned above are promoters that can guide RNA transcription, such as the T7 promoter, SP6 promoter, T3 promoter, etc.

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

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

[0020] The primer pairs provided by this 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, comprising the above-mentioned primer pairs.

[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) Denature the nucleic acid template to be detected. As a specific example, the denaturation was carried out at a temperature of 95°C for 2 to 5 minutes. b) The isothermal amplification reaction system with added primers and denatured nucleic acid template was subjected to isothermal amplification at a temperature of 59℃~68℃; The isothermal amplification system can employ a conventional reaction system, and the composition of one specific reaction system is 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 Add sterile water to bring 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 promoter-matched transcriptases, such as T7 transcriptase, SP6 RNA polymerase, T3 RNA polymerase, etc. The reverse transcriptase is selected from M-MLV reverse transcriptase, AMV reverse transcriptase, etc.

[0023] This invention provides a primer pair for promoter-mediated amplification of target gene nucleic acid sequences. Under isothermal conditions (59–68 °C), this primer pair simultaneously unwinds double-stranded DNA and provides more amplification template. Hairpin-like structures guide isothermal amplification concurrently, enabling the amplification of low-copy numbers (e.g., single-digit or tens of copies) of target nucleic acid to millions or tens of millions of copies within tens of minutes. This achieves rapid amplification and specific detection of target genes, significantly reducing the requirements for or dependence on expensive detection instruments. This invention has wide applications, such as the rapid amplification and specific detection of target gene nucleic acid sequences. Attached Figure Description

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

[0025] Figure 2 The diagram shows the positional relationship of primers on the target gene sequence, where F refers to the forward primer, R refers to the reverse primer (also known as 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 complex primer under a DNA template, the RNA transcription and double-stranded DNA unwinding process guided by the promoter, and the reverse transcription of single-stranded RNA to generate cDNA. In the diagram, FP stands for Forward primer, and RP stands for Reverse primer (also known as BP: Backward primer). RP and BP in this invention refer to the same complex primer. MMLV, also known as M-MLV, is an abbreviation for M-MLV Reverse transcriptase. Bst E is Bst The abbreviation for DNAPolymerase.

[0027] Figure 4 This diagram illustrates the process of RNA transcription and double-stranded DNA unwinding under promoter guidance for a newly generated template with a promoter sequence, the isothermal amplification of single-stranded DNA under enzymatic action, and the formation of hairpin-like structures from single-stranded DNA. In the diagram, 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 complex primer. MMLV, also known as M-MLV, is an abbreviation for M-MLV Reversetranscriptase. Bst E is Bst An abbreviation for DNA Polymerase.

[0028] Figure 5 This diagram illustrates the isothermal amplification process of a template containing a hairpin-like structure under the action of enzymes, during which the length of the amplification product continuously increases; and the transcription process guided by the promoter, the unwinding of the DNA double helix, and the reverse transcription process, ultimately forming DNA templates of different lengths.

[0029] Figure 6 The electrophoresis results show the effect of NTP deletion on amplification products and amplification efficiency in the PAMP reaction system. The lanes represent: DNA molecular weight DL 2000 Marker, amplification temperatures of 58 ℃ and 62 ℃, and amplification products with NTP deletion alone under the same temperature conditions. The figure shows the above... Bst DNA polymerase, M-MLV reverse transcription, and T7 RNA polymerase exhibit the highest efficiency in completing PAMP at 62 °C. In the absence of NTP components alone, the T7 promoter-mediated RNA transcription process is blocked, affecting the subsequent exponential amplification reaction in PAMP (no characteristic bands are produced). This confirms that the T7 promoter sequence and T7 RNA polymerase require NTPs to synthesize RNA, thus playing a crucial mediating role in the method of this invention.

[0030] Figure 7 Electrophoresis results showing the effect of the absence of NTP components, or the absence of T7 promoter sequences or M-MLV enzyme components in the PAMP reaction system on amplification products and amplification efficiency. Figure 7 (A) Figure 7 (B) Figure 7The middle (C) figures show the PAMP amplification products corresponding to the absence of NTP, T7 RNA polymerase, or M-MLV reverse transcriptase in the reaction system. The lanes represent the DNA molecular weight DL 2000 marker, and the amplification times are 40 min, 60 min, 80 min, 100 min, and 120 min, respectively. The figure shows that the absence of NTP, T7 RNA polymerase, or M-MLV resulted in no obvious product bands within 80 minutes. This indicates that NTP, T7 RNA polymerase, and M-MLV are all important factors in improving the amplification efficiency of PAMP technology. The absence of NTP and T7 RNA polymerase has the greatest impact on the PAMP reaction (no clear bands even after 100 minutes of amplification). In contrast, the absence of M-MLV has a slightly smaller impact on the PAMP reaction efficiency (a clear band appears after 100 minutes of amplification).

[0031] Figure 8 This image shows the electrophoresis results of the PAMP amplification product after restriction endonuclease digestion to obtain a single target fragment, along with the restriction enzyme sites in the target gene. Figure 8 (A) shows the electrophoresis results, where lanes represent: DNA molecular weight DL 2000 Marker; lane 1 represents the normal amplification product of the PAMP reaction; lanes 2-5 correspond to restriction endonucleases. Alu I (Identification site AGCT). Figure 8 Restriction endonucleases can be seen in (B). Alu I has a single restriction enzyme site on the WSSV target gene amplification sequence, so a single target gene band can be obtained after enzyme digestion.

[0032] Figure 9 The image shows the electrophoresis results of the PAMP reaction temperature optimization process. The lanes represent the DNA molecular weight DL 2000 marker and the amplification products at reaction temperatures of 58 ℃, 61 ℃, 64 ℃, 67 ℃, and 70 ℃. The image shows clear amplification products within the temperature range of 61 ℃ to 67 ℃, indicating efficient PAMP amplification under these conditions.

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

[0034] Figure 11The image shows the electrophoresis results of the optimized dNTP concentration process in the PAMP reaction system. The lanes represent the DNA molecular weight DL 2000 marker, and the corresponding amplification products for 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 present at dNTP concentrations ranging from 0.8 mM to 1.6 mM; higher concentrations inhibit PAMP amplification. A concentration of 1.6 mM is recommended.

[0035] Figure 12 The image shows the electrophoresis results of the optimized betaine concentration process in the PAMP reaction system. The lanes represent the DNA molecular weight DL 2000 marker and the amplification products corresponding to 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 present at betaine concentrations ranging from 0.6 M to 1.5 M. Based on the clarity of the amplification bands, a concentration of 1.2 M is recommended.

[0036] Figure 13 This is a specificity verification diagram for the WSSV vp37 PAMP primers. The lanes represent the DNA molecular weight DL 2000 marker, and the templates are the corresponding amplification products from WSSV-positive, IHHNV-positive, HPV-positive, MBV-positive, and blank control samples. The results show that only the first lane effectively amplified the primers, while the others did not, indicating that the WSSV vp37-PAMP primers have good specificity and do not cause non-specific amplification of nucleic acids from other pathogens.

[0037] Figure 14 Electrophoresis results showing the effect of adding loop primers on the sensitivity of PAMP technology. Figure 14 (A) Figure 14 (B) Figure 14 (C) shows the effects of adding loop primers to both ends of the PAMP complex primer to promote amplification, adding only the front end of the PAMP complex primer, or adding only the back end of the PAMP complex primer on the amplification products and amplification efficiency, respectively. The concentration of the starting template was approximately 10. 3 copies / μL, used BstThe DNA polymerase was manufactured by NEB. The lanes were for a DNA molecular weight DL 2000 marker, and the amplification times were 40 min, 50 min, 60 min, 70 min, and 80 min. The results show that adding a loop primer outside the FP or RP sequence of the PAMP complex improves amplification efficiency, with adding loop primers at both ends significantly improving detection sensitivity. These results demonstrate that loop primers can improve PAMP amplification efficiency, providing a technical approach for PAMP technology optimization. However, PAMP amplification can be completed efficiently without loop primers.

[0038] Figure 15 This image shows the electrophoresis results validating the sensitivity of PAMP technology under low template concentration conditions. The lanes represent the DNA molecular weight (DL 2000) marker and the PAMP amplification products at amplification times of 40, 50, 60, 70, and 80 minutes. The results demonstrate that PAMP exhibits high sensitivity at low template concentrations (10...). 3 Under the template conditions of (copies / μL), a clear product band was observed after 50 minutes.

[0039] Figure 16 This is an electrophoresis result comparing the amplification efficiency of PAMP and LAMP techniques under low template concentration conditions. Figure 16 (A) shows PAMP primers at a low concentration (starting template concentration approximately 10). 3 Amplification results at different amplification times under template conditions (copies / μL) Figure 16 (B) shows the amplification results of ordinary LAMP primers at different amplification times under low template concentration conditions. The lanes represent the DNA molecular weight DL 2000 marker, with amplification times of 40, 50, 60, 70, and 80 minutes, corresponding to the PAMP amplification products. Furthermore, this figure also shows significant differences in the size and composition of the PAMP and LAMP amplification products. This suggests that under the same low template concentration conditions, PAMP has higher amplification efficiency than ordinary LAMP technology, with better band dispersion, clearer bands, and easier interpretation.

[0040] Figure 17 Electrophoresis results comparing the amplification efficiencies of PAMP and LAMP technologies under different temperature conditions are shown in the figure. Figure 17 (A) shows the amplification results guided by PAMP primers at different amplification temperatures. Figure 17(B) shows the amplification results guided by ordinary LAMP primers at different amplification temperatures. The lanes represent the DNA molecular weight DL 2000 Marker, and the corresponding PAMP amplification products at amplification temperatures of 50℃, 53℃, 56℃, 59℃, 62℃, 65℃, and 70℃. The results show that under the same low template concentration conditions, PAMP has better temperature suitability for amplification than ordinary LAMP technology and exhibits higher amplification efficiency.

[0041] Figure 18 Using the nucleic acid (RNA) of RNA virus CMNV as a template, the PAMP primers were detected by real-time PCR. Figure 18 (A) and LAMP primers ( Figure 18 The amplification efficiency graph in (B) shows that the number of cycles per minute on the horizontal axis represents the amplification efficiency. The results indicate that PAMP technology also exhibits good amplification efficiency for RNA templates, demonstrating higher amplification efficiency and sensitivity than LAMP technology.

[0042] Figure 19 Using the nucleic acid (RNA) of RNA virus CMNV as a template, this study used a real-time PCR instrument to detect the presence of T7 RNA polymerase alone, M-MLV polymerase alone, or other RNA polymerases alone in the PAMP reaction system. Bst The graph shows the effect of DNA polymerase on amplification products and amplification efficiency; the horizontal axis represents the cycle number, which is one cycle every 30 seconds. The initial template concentration is approximately 10. 3 copies / μL~10 4 The results from the amplification curves show that the deletion of either T7 RNA polymerase or M-MLV polymerase alone significantly reduces the amplification efficiency of the PAMP reaction, while the deletion of either T7 RNA polymerase or M-MLV polymerase alone... Bst After DNA polymerase, the PAMP reaction produced no amplification products within 240 cycles (120 minutes). The absence of T7 RNA polymerase or M-MLV polymerase alone significantly reduced the reverse efficiency; compared to normal amplification, no products were produced within 60 cycles (30 minutes). Ct The value (the number of cycles at which the "S"-shaped amplification curve intersects the baseline) only appeared after 140 cycles (70 minutes) following the deletion of M-MLV polymerase alone. Ct This value provides conclusive experimental evidence that the PAMP technology of this invention is fundamentally different from the LAMP technology. (Separate missing information) Bst DNA polymerase prevents PAMP from amplifying properly, which provides conclusive experimental evidence that the PAMP technology of this invention is fundamentally different from NASBA technology. Detailed Implementation

[0043] The existing nucleic acid sequence-dependent amplification (NASBA) technique utilizes reverse transcriptase, RNase H, and T7 RNA polymerase, along with forward and reverse primers, to mimic the replication mechanism of retroviruses in vivo for target RNA amplification, achieving a 10-12 fold increase in product within 90 minutes. However, the NASBA method cannot achieve exponential amplification of the target nucleic acid gene. This invention utilizes a complex primer containing a promoter sequence. Through isothermal amplification, sequences with reverse complementarity to the inner segments of the starting primer sequence are first formed at both ends. Specifically, the promoter-containing complex primer introduces R1 or F2 sequences at both ends of the newly amplified nucleic acid sequence, providing conditions for the spontaneous formation of hairpin-like structures at the 3′ ends, initiating subsequent exponential amplification of new nucleic acid fragments generated by the dual complex primers. Therefore, the core principle of this invention is completely different from that of NASBA. The PAMP method of this invention can achieve over a million-fold amplification of the starting template nucleic acid within 30-60 minutes, with significantly higher amplification efficiency than NASBA.

[0044] The composite primers and their amplification principles of this invention are also quite different from those of LAMP amplification primers. The pre-primer FIP in LAMP amplification is composed of "F1c-F2," meaning the FIP primer consists of the F2 fragment of the primer sequence and the F1c fragment on its complementary strand. In contrast, the composite primer FP in this invention is composed of "R1-promoter sequence-F1," meaning the FP primer consists of the F1 fragment of the primer sequence and the R1 fragment on its complementary strand. The primer compositions for BIP in LAMP and RP in PAMP are similar to FIP and FP, respectively, and will not be elaborated further. Therefore, the primer compositions of the two are clearly different. During LAMP amplification, F3 and B3 primers respectively complete the unwinding (double-stranded to single-stranded) of the new DNA strand synthesized by FIP and BIP primers. In the PAMP amplification process of this invention, on the one hand, RNA polymerase and reverse transcriptase provide the new template single strand; on the other hand, FP and RP primers themselves can complete the unwinding of their amplification products, without relying on F3 or B3 primers similar to those used in LAMP amplification for unwinding before subsequent amplification. Therefore, PAMP and LAMP amplification principles are quite different. The experimental diagrams provided in this specification further clarify the above description.

[0045] The present invention provides a method for promoter-mediated isothermal amplification of nucleic acid sequences, which utilizes the 3′ end of a composite primer containing the promoter sequence to bind to the target sequence to initiate the initial isothermal amplification reaction. The primer pairs used in this invention are designed as follows: 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′; F1 and R2 are the outer primers of the target gene, located at the 5′ end of the target gene sequence and the 5′ end of the complementary strand sequence of the target gene sequence, respectively. They are used to specifically identify the target gene binding site and guide the amplification of the target gene nucleic acid sequence.

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

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

[0048] The positional relationships of F1, R1, F2, and R2 in the target gene sequence are shown in the figure. Figure 2 .

[0049] The promoters mentioned above are promoters that can guide RNA transcription, such as the T7 promoter, SP6 promoter, and T3 promoter.

[0050] The primer pair designed in this invention utilizes the 3′ end of a composite primer containing a promoter sequence to initiate the initial isothermal amplification reaction. The promoter sequence is inserted into the amplification product, and the first round of amplification products generates a target gene nucleic acid sequence containing the promoter sequence and a sequence that is inversely complementary to the target gene sequence inside the first round of amplification primers. RNA polymerase uses the promoter sequence to guide the transcription of the target gene RNA chain, which is then used by reverse transcriptase to generate a large amount of cDNA, continuously providing single-stranded DNA templates for subsequent isothermal amplification reactions. At the same time, the transcription initiation and transcription processes provide a large number of single-stranded DNA template regions for the synchronously occurring isothermal DNA amplification, accelerating the isothermal DNA amplification reaction. More importantly, the 5′ end of the composite primer is designed with a specific primer that is inversely complementary to the target gene sequence. This guides the newly generated nucleic acid sequence 3′ end (specific primer) to bind complementaryly with its own sequence (specifically, the sequence inside the initiating primer) to form a hairpin-like structure, guiding and accelerating the isothermal amplification of the target gene nucleic acid sequence. The composite primer, containing the initiation primer fragment sequence, promoter sequence, and the reverse complementary sequence of the initiation primer's inner side, is crucial to the established method. This composite primer guides DNA polymerase to synthesize the complementary strand of the target gene. This complementary strand 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 strand. When a pair of composite primers completes the amplification of the target gene, a cycle of target gene strand amplification that triggers subsequent exponential growth is formed, ultimately amplifying target gene nucleic acid sequences of different lengths. When the starting template is RNA, reverse transcriptase, guided by the composite primer, first initiates reverse transcription of the target gene, forming cDNA containing the composite primer and complementary to the target gene, thereby initiating the amplification process of the target gene strand.

[0051] Based on the construction of the above-mentioned composite primer pairs, the present invention also provides a method for promoter-mediated isothermal amplification of target gene nucleic acid sequences, the method comprising the following steps; a) Denature the nucleic acid template to be detected. As a specific example, the denaturation was carried out at a temperature of 95°C for 2 to 5 minutes. b) The isothermal amplification reaction system with added primers and denatured nucleic acid template was subjected to isothermal amplification at a temperature of 59℃~68℃; The isothermal amplification system described above can employ a conventional reaction system. One specific reaction system has the following composition, which can also be adjusted proportionally according to the total system volume: 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 Add sterile water to bring the total reaction volume to 25 μL. 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 promoter-matched transcriptases, 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, etc.

[0052] The amplification principle of the detection method of this invention is shown in the appendix. Figure 3 ~Attached Figure 5 The specific amplification process can be broken down into the following steps: a) The F1 at the 3′ end of the FP primer and the R2 at the 3′ end of the RP primer bind complementary to 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 to produce an RNA chain under the action of RNA polymerase, thereby causing the double-stranded DNA to unwind; c) The complex primers pair with complementary RNA strands, and under the action of reverse transcriptase, the first strand of cDNA is produced; d) The RNase activity of reverse transcriptase degrades RNA: the RNA strand in the DNA hybrid strand, leaving the remaining DNA single strand; e) The composite primer binds to the complementary site of the single-stranded DNA template, and isothermal amplification occurs under the action of DNA polymerase. Steps a), b), c), and d) are repeated. The above amplification process is shown in the appendix. Figure 3 ; f) The 3′ end of the newly generated DNA single strand is the inverse complementary sequence of R1 and F2 on the composite primer. This sequence binds inversely to the R1 and F2 positions on its own sequence, forming a hairpin-like structure. The 3′ end triggers subsequent isothermal amplification. The amplification process described above is shown in the appendix. Figure 4 ; g) The double-stranded DNA template with a hairpin-like structure is transcribed again under the action of a complex primer and promoter—the process involves transcription, unwinding, and isothermal amplification, repeating steps a), b), c), d), e), and f). The above amplification process is detailed in the appendix. Figure 5 ; h) This cycle will rapidly generate a large number of target gene nucleic acid sequences of different lengths.

[0053] The reagents and materials involved in the embodiments of this invention are as follows: primers were synthesized by Sangon Biotech (Shanghai) Co., Ltd.; dNTPs, betaine, dATP, dGTP, dCTP, dTTP, and MgCl2 were purchased from Shanghai Bioengineering Co., Ltd.; and isothermal amplification was performed using... Bst DNA polymerase, RNA polymerase, and reverse transcriptase were purchased from NEB Corporation; 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 field, and are not limited to the brand or type of the specific commercially available reagents or materials in the embodiments of this invention.

[0054] The following examples illustrate the detection method and its detection effect of the present invention. These examples are for illustrative purposes only and do not constitute any limitation on the scope of protection of the present invention, which is defined in the claims.

[0055] Example 1: Verifying the principle of PAMP technology using WSSV as an example. White spot syndrome virus (WSSV) is a double-stranded DNA virus that harms shrimp farming, causing white spots on the shrimp's body surface, reduced feed intake, and high mortality (up to 100% mortality within 3-10 days after infection), making it one of the major threats to global aquaculture. Current detection methods mainly include molecular techniques (such as PCR and real-time quantitative PCR), immunological methods (such as ELISA and test strips), and histopathological observation. Molecular techniques offer high sensitivity but are equipment-dependent, immunological methods are simple to operate but have lower sensitivity, and histopathology requires experience and is time-consuming. In recent years, isothermal amplification technology has been widely used due to its significant advantages: it does not require complex thermal cycling equipment, only a constant temperature (59-68℃) is needed to complete amplification within 30-60 minutes; it has high specificity; and the results can be directly interpreted by visual observation of turbidity or fluorescence, making it particularly suitable for rapid screening in grassroots farms or resource-limited areas, providing efficient technical support for the early prevention and control of WSSV. This invention utilizes the method to achieve rapid detection of WSSV virus DNA.

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

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

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

[0059] The sequence of the 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 a 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] Without adding NTPs, the above-mentioned general PAMP reaction system was amplified for 90 minutes at 58 °C and 62 °C, respectively. (See attached image) Figure 6 The results showed that agarose gel electrophoresis revealed only weak bands in the product without NTPs, which were significantly different from the bands in the normal system. This indicates that in the absence of NTPs, the T7 promoter-mediated RNA transcription process is blocked, affecting the subsequent exponential amplification reaction. This further confirms that the T7 promoter plays a crucial mediating role in the PAMP reaction.

[0063] (2) To further verify the principle of PAMP technology, the following operations were performed: no NTPs were added to the reaction system, no T7 promoter sequence was added to the primers, and no M-MLV reverse transcriptase was added to the reaction system. All other components remained unchanged. The mixture was then incubated at 62℃ for 40, 60, 80, 100, and 120 minutes, respectively, and the amplification products were observed by agarose gel electrophoresis. (See attached image) Figure 7 Figure (A) shows that without the addition of NTPs, no target product was amplified within 80 minutes. After 100 minutes of reaction, a few product bands appeared, indicating that transcription is a crucial factor for efficient PAMP amplification. (Appendix) Figure 7Figure (B) shows that without the addition of the T7 promoter, no target product was amplified within 80 minutes, and a very small number of product bands appeared after 100 minutes of reaction, confirming that the T7 promoter-mediated RNA transcription process is key to the efficient amplification of PAMP. (Appendix) Figure 7 Figure (C) shows that without the addition of M-MLV reverse transcriptase, no target product was amplified within 80 minutes, and a clear product band appeared only after 100 minutes of reaction. 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 a template and vp37 gene as the target gene, PAMP FP and RP complex primer sequences were designed respectively. The target fragment amplified by the complex primer contained a single restriction endonuclease. Alu I's identification site (attached) Figure 8 (See Figure B). Using the FP and RP primers described above, a PAMP amplification system was prepared and amplified at 62 °C for 90 minutes. The amplified products were then added to a restriction endonuclease. Alu The enzyme digestion reaction was performed using enzyme I and buffer. After digestion, the digestion status of the amplified products was detected by agarose gel electrophoresis. (See attached image) Figure 8 As shown in (A), lanes 2-5 indicate that the amplified product was bound by restriction endonucleases. Alu After digestion with enzyme I, the amplified product is broken down into nucleic acid fragments of uniform length, thus appearing as a single target gene band on electrophoresis. This indicates that the amplified product can be... Alu I-recognition. These experimental results further confirm the amplification principle of the target fragment amplification using FP and RP primers in PAMP.

[0065] Example 2. Establishment of PAMP detection technology for WSSV (Vitiligo Syndrome Virus) (1) Optimization of PAMP reaction temperature: Using WSSV-positive nucleic acid as a template and primer sequences designed with the vp37 target gene, the amplification temperature was optimized under a standard PCR reaction system. The temperatures were set at 58 ℃, 61 ℃, 64 ℃, 67 ℃, and 70 ℃, respectively, and amplification was performed for 90 minutes. The amplification products were then observed by agarose gel electrophoresis. (See attached image) Figure 9 The results showed no significant amplification products at 58 ℃ and also no significant amplification products at 70 ℃ due to the influence of enzyme activity. However, significant amplification products were observed within the temperature range of 61 ℃ to 67 ℃, indicating highly efficient PAMP amplification under these conditions. Therefore, it is recommended that the PAMP reaction temperature be between 61 ℃ and 67 ℃.

[0066] (2) Mg in the PAMP reaction system 2+Ion concentration optimization: Using WSSV-positive nucleic acid as a template and primer sequences designed with the vp37 target gene as the template, amplification was performed at 65 °C for 90 minutes under a standard PCR reaction system. Mg concentration was optimized. 2+ Ion concentration. Set Mg 2+ Ion concentrations of 4 mM, 6 mM, 8 mM, 10 mM, and 12 mM were used, and agarose gel electrophoresis was used to examine the amplification products. (Attached) Figure 10 The display shows that when Mg 2+ Amplification products were observed at concentrations ranging from 6 mM to 12 mM. In the spirit of conservation, a concentration of 8 mM is recommended.

[0067] (3) Optimization of dNTP concentration in the PAMP reaction system: Using WSSV-positive nucleic acid as a template and primer sequences designed with the vp37 target gene, amplification was performed at 65 ℃ for 90 minutes under a standard PCR reaction system to optimize the dNTP concentration. The dNTP concentrations were set to 0.8 mM, 1.2 mM, 1.6 mM, 2 mM, and 2.4 mM, and the amplification products were observed by agarose gel electrophoresis. (See attached image) Figure 11 The results showed that amplification products were observed when the concentration of dNTPs was between 0.8 mM and 1.6 mM. Higher concentrations actually inhibited PAMP amplification, and a concentration of 1.6 mM was recommended.

[0068] (4) Optimization of betaine concentration in the PAMP reaction system: Using WSSV-positive nucleic acid as a template and primer sequences designed with the vp37 target gene as the template, amplification was performed at 65 ℃ for 90 minutes under a standard PCR reaction system. The betaine concentrations were optimized to 0.6 M, 0.9 M, 1.05 M, 1.2 M, and 1.5 M. Agarose gel electrophoresis was used to examine the amplification products. (See attached image) Figure 12 The results showed that amplification products were present at betaine concentrations ranging from 0.6 M to 1.5 M. Based on the clarity of the amplification product bands, a concentration of 1.2 M was recommended.

[0069] (5) Validation of PAMP technology specificity: Using WSSV-positive, IHHNV-positive, HPV-positive, MBV-positive nucleic acids and blank controls as templates, primer sequences were designed with the vp37 target gene as the templates, and the primer specificity was validated in an optimized PAMP reaction system. (See attached image) Figure 13 Agarose gel electrophoresis showed that only the first lane had amplification products, while the other lanes did not, 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 to both ends, the front end, or the back end of the PAMP primer sequence. The experimental design was as follows: WSSV positive nucleic acid was used as a template, and the concentration was 10 6 Copies / μL were collected using an optimized PAMP reaction system. Amplification times were set at 40, 50, 60, 70, and 80 minutes, respectively, and amplification was observed using agarose gel electrophoresis. (See attached image) Figure 14 (A) shows that PAMP at high concentrations (10) 6 With a template of (copies / μL), and loop primers added to both ends, a clear amplification band can be observed after 40 minutes. As the amplification time increases, after 80 minutes, the small fragment product decreases, and large fragment products are repeatedly replicated, mostly condensing at the agarose gel wells. Similarly, [the text abruptly ends here, likely due to an incomplete sentence or missing information]. Figure 14 Figures (B) and (C) also show a similar trend, consistent with the PAMP amplification principle. (See attached image.) Figure 14 Figures (B) and (C) show that adding loop primers only at the front or back of the primer pair resulted in a noticeable product band after 50 minutes of amplification. Therefore, adding double-loop primers to the PAMP will significantly improve amplification efficiency and detection sensitivity.

[0071] (7) Validation of PAMP sensitivity under low template concentration conditions: for example, when the WSSV positive nucleic acid template concentration is 10... 3 Copies / μL were collected using an optimized PAMP reaction system. Amplification times were set at 40, 50, 60, 70, and 80 minutes, respectively, and amplification was observed using agarose gel electrophoresis. (See attached image) Figure 15 The results showed that PAMP at low concentrations (10) 3 Under template conditions of (copies / μL), agarose gel electrophoresis showed obvious product bands after 50 minutes.

[0072] (8) Comparison of amplification efficiency between PAMP and conventional LAMP under low template concentration conditions WSSV positive nucleic acid template 10 3 Primers for PAMP and LAMP were designed using copies / μL and vp37. An optimized PAMP reaction system was used, and amplification times were set at 40 min, 60 min, 80 min, 100 min, and 120 min. Agarose gel electrophoresis was used to observe the amplification results. (See attached image) Figure 16 In Figure (A), the PAMP primers showed a clear product band at 60 minutes, with the target gene product signal increasing gradient in the swim lane, while the attached... Figure 16In the LAMP primer (B), only a weak product band was observed at 60 minutes, and a clear product band was only observed at 80 minutes, with poor separation between the bands. Therefore, under the same low template concentration conditions, PAMP has higher amplification efficiency than ordinary LAMP technology, and the bands are better dispersed, clearer, and easier to interpret.

[0073] (9) Comparison of amplification efficiency between PAMP and conventional LAMP under different temperature conditions: WSSV positive nucleic acid template 10 3 Primers for PAMP and LAMP were designed using copies / μL and vp37. An optimized PAMP reaction system was used, and amplification temperatures were set at 50 ℃, 53 ℃, 56 ℃, 59 ℃, 62 ℃, 65 ℃, and 70 ℃. Amplification was performed for 60 minutes, and the amplification status was checked by agarose gel electrophoresis. (See attached image) Figure 17 In Figure (A), the PAMP primers show a clear product band at 59 ℃ to 65 ℃, while the attached... Figure 17 The (B) LAMP primers produced only weak product bands at 59 ℃ to 62 ℃, with poor separation between the bands. Therefore, under the same low template concentration conditions, PAMP has better temperature suitability for amplification than conventional LAMP technology and has higher amplification efficiency.

[0074] Example 3. Establishment of PAMP detection technology for stealth-causing Nodamura virus (CMNV) Covert mortality nodavirus (CMNV) is a pathogen that seriously threatens shrimp farming. Detection methods mainly include reverse transcription-polymerase chain reaction (RT-PCR), real-time quantitative PCR (RT-qPCR), and histopathological observation. In recent years, isothermal amplification technology has been widely used due to its significant advantages: it requires no complex thermal cyclers, only a constant temperature (60℃~65℃), and amplification can be completed within 30~60 minutes; it has high specificity; and the results can be directly interpreted by visual observation of turbidity or fluorescence, making it particularly suitable for rapid screening in grassroots farms or resource-limited areas, providing efficient technical support for the early prevention and control of CMNV. This patented method enables rapid detection of CMNV virus RNA.

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

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

[0077] The sequence of the 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] Reaction procedure: 65 °C for 1 minute, 70 cycles, fluorescence signal acquired using FAM / SYBR Green channel.

[0081] (1) The amplification effects of PAMP and LAMP primers against CMNV were compared using a real-time PCR instrument. Figure 18 (A) Figure 18 As shown in (B), PAMP exhibits higher amplification efficiency and sensitivity.

[0082] (2) Using quantitative real-time PCR, with the nucleic acid (RNA) of RNA virus CMNV as a template, the presence of T7 RNA polymerase alone, M-MLV polymerase alone, or M-MLV polymerase alone in the PAMP reaction system was detected by quantitative real-time PCR. Bst The effect of DNA polymerase on amplification products and amplification efficiency. (Appendix) Figure 19 The results show that PAMP exhibits amplification characteristics different from LAMP and NASBA.

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

Claims

1. A primer pair for promoter-mediated isothermal amplification, characterized in that, The primer pair includes: a) FP forward primer: 5′-R1-promoter-F1-3′; b) RP reverse primer: 5′-F2-promoter-R2-3′; F1 and R2 are the outer primers for isothermal amplification of the target gene, located at the 5′ end of the target gene sequence and the 5′ end of the complementary strand sequence of the target gene sequence, respectively. They 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 R2 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 strand of the target gene sequence, and F1 binds to the downstream sequence of the R1 site of the complementary strand of the target gene sequence. R1 and F2 specifically recognize the complementary sequence at the 3′ end of the nascent strand, thereby forming a hairpin structure to guide the amplification of the target gene nucleic acid sequence; The promoter mentioned is a promoter that can guide RNA transcription, such as the T7 promoter, SP6 promoter, or T3 promoter.

2. The primer pair as described in claim 1, characterized in that, The primer pair used for promoter-mediated isothermal amplification is used to detect white spot syndrome virus; the sequence of its FP forward primer is SEQ ID NO:1, and the sequence of its RP reverse primer is SEQ ID NO:

2. The primer pair used for promoter-mediated isothermal amplification is used to detect Nodamura virus in shrimp that causes sudden death; the sequence of its FP forward primer is SEQ ID NO:3, and the sequence of its RP reverse primer is SEQ ID NO:

4.

3. The use of the primer pair according to claim 1 in the preparation of molecular detection products for isothermal amplification detection.

4. A detection product for promoter-mediated isothermal amplification, characterized in that, The test article contains the primer pair as described in claim 1.

5. A promoter-mediated isothermal amplification method for non-disease diagnosis and treatment purposes, characterized in that, The method described herein uses the primer pair as described in claim 1 for detection.

6. The method as described in claim 5, characterized in that, The method includes the following steps: 1) Denature the nucleic acid template to be tested. 2) The isothermal amplification reaction system with added primers and denatured nucleic acid template was subjected to isothermal amplification at a temperature of 59℃~68℃; 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 either M-MLV reverse transcriptase or AMV reverse transcriptase.

Images