Loop-mediated isothermal amplification method and kit for detecting equine herpesvirus type 1 and equine herpesvirus type 4

Abstract

The invention relates to the field of molecular biology diagnosis, in particular to a loop-mediated isothermal amplification method and kit for detecting equine herpes viruses 1 and 4, and the method comprises the following steps: S1, designing and constructing loop-mediated isothermal amplification (LAMP) primer groups aiming at gB gene specific sequences of equine herpes viruses 1 and 4, each primer group comprising six primers F3, B3, FIP, BIP, LoopF and LoopB; s2, preparing an LAMP (loop-mediated isothermal amplification) reaction system, wherein the reaction system comprises the primer group, a buffer solution, dNTPs (deoxyribonucleoside triphosphates), divalent metal ions and DNA (deoxyribonucleic acid) polymerase; s3, adding nucleic acid of a sample to be detected into the reaction system; s4, carrying out LAMP reaction under the condition of constant temperature; and S5, detecting an LAMP reaction amplification product. The loop-mediated isothermal amplification method for detecting the equine herpes viruses 1 and 4 and the kit thereof can be used for efficiently and accurately detecting the equine herpes viruses 1 and 4, so that the limitations of the traditional detection method in the aspects of time, operation complexity and sensitivity are overcome; and a powerful technical means is provided for rapid diagnosis and prevention and control of horse herpes virus diseases.

Description

Technical Field

[0001] This invention relates to the field of molecular biological diagnostics, and more particularly to a loop-mediated isothermal amplification method and kit for detecting equine herpesvirus types 1 and 4. Background Technology

[0002] Equine herpesvirus 1 (EHV-1) and equine herpesvirus 4 (EHV-4) are the main pathogens causing nasal pneumonia and viral abortion in equines. EHV-1, in particular, can cause a serious neurological disease—equine herpesvirus encephalopathy (EHM). This disease is widespread globally, causing significant economic losses to the horse breeding and racing industries. Given its substantial impact on animal health and the economy, the World Organisation for Animal Health (OIE) has listed equine herpesvirus infection as a notifiable disease, highlighting the importance of its prevention and control. Timely and accurate diagnosis of equine herpesvirus infection is crucial for disease surveillance, outbreak control, and the implementation of effective interventions.

[0003] Currently, laboratory diagnostic methods for equine herpesvirus mainly include virus isolation and identification, serological diagnostic methods, and molecular biological detection techniques.

[0004] Virus isolation and identification is the traditional "gold standard" method, which involves isolating viruses through cell culture and then identifying them. However, this method is time-consuming, usually taking several days or even weeks to complete, and is cumbersome to operate, with stringent requirements for experimental conditions. Its sensitivity is also affected by viral titer and the time of sampling, making it difficult to meet the needs of rapid diagnosis.

[0005] Serological diagnostic methods, such as virus neutralization tests, complement fixation tests, and enzyme-linked immunosorbent assays (ELISA), are mainly used to detect the level of antibodies produced by the body against equine herpesvirus (EHV), thereby determining whether an infection has occurred. While serological methods are relatively simple to perform, they have significant limitations. First, antibody production usually lags behind viral infection; in the early stages of infection, antibody levels are low or even negative, easily leading to false negatives and hindering early diagnosis. Second, serological methods may exhibit cross-reactivity, particularly between EHV-1 and EHV-4, affecting diagnostic specificity. Furthermore, serological methods struggle to distinguish between vaccine-immunized antibodies and naturally occurring antibodies, limiting their application in disease surveillance and control.

[0006] Molecular biology detection techniques based on conventional PCR, such as polymerase chain reaction (PCR), can directly detect viral nucleic acids, exhibiting high sensitivity and specificity, and significantly shortening the detection cycle compared to virus isolation and identification. However, traditional PCR techniques still have certain limitations. First, PCR amplification requires precise temperature cycling control and relies on expensive PCR instruments and other sophisticated equipment, resulting in high equipment costs and limiting its widespread application in grassroots laboratories and field settings. Second, the traditional PCR amplification and electrophoresis detection process still requires considerable operational time and experimental skills, with the overall detection time typically exceeding several hours, making it difficult to meet the demand for rapid detection. Furthermore, for some complex samples, PCR amplification may be inhibited, affecting the sensitivity and accuracy of the detection.

[0007] In recent years, loop-mediated isothermal amplification (LAMP) technology has attracted much attention as a novel nucleic acid amplification technique due to its unique advantages. LAMP technology performs nucleic acid amplification under isothermal conditions, eliminating the need for temperature cycling and making it simple and rapid to operate. LAMP technology utilizes multiple specific primers working synergistically, resulting in high amplification efficiency, strong specificity, and sensitivity comparable to or even exceeding that of PCR technology. LAMP amplification products are abundant and can be detected using various simple methods such as visual observation and fluorescent staining, without the need for complex instruments. These advantages make LAMP technology demonstrate enormous application potential in the field of rapid detection of pathogenic microorganisms.

[0008] Despite the significant advantages of LAMP technology, there are currently no publicly reported methods for detecting equine herpesvirus (EHV) types 1 and 4, either domestically or internationally. Existing molecular diagnostic methods for EHV primarily rely on traditional PCR technology, while the application of LAMP technology in this field remains unexplored. Therefore, there is an urgent need to develop a rapid, sensitive, highly specific, and easy-to-use LAMP detection method for EHV to overcome the shortcomings of existing detection technologies and meet the pressing need for rapid diagnosis and control of EHV. Summary of the Invention

[0009] To address the technical problems existing in the prior art, this invention provides a loop-mediated isothermal amplification method and kit for detecting equine herpesvirus 1 and 4, which can be used for efficient and accurate detection of equine herpesvirus 1 and 4, thereby overcoming the limitations of traditional detection methods in terms of time, operational complexity and sensitivity, and providing a powerful technical means for the rapid diagnosis and prevention of equine herpesvirus disease.

[0010] This invention discloses a loop-mediated isothermal amplification method for detecting equine herpesviruses (EHV) types 1 and 4. Specifically, in step S1, the method first designs and constructs LAMP primer sets targeting specific sequences of the gB genes of EHV-1 and EHV-4. Each primer set precisely contains six primers: F3, B3, FIP, BIP, LoopF, and LoopB. These six primers are not simply arranged linearly, but work synergistically to form a sophisticated amplification mechanism. The FIP and BIP primers each contain two target sequence regions, complementary to different sites on the template DNA, and form hairpin structures within their own structures. This design significantly improves the specificity and efficiency of amplification. The F3 and B3 primers, acting as outer primers, are responsible for the initiation strand substitution reaction; the LoopF ​​and LoopB loop primers further accelerate the amplification process. The precise sequences and synergistic effects of these six primers are one of the key technical features for achieving specific amplification.

[0011] In step S2, the LAMP reaction system needs to be prepared. This reaction system is like a carefully formulated miniature biochemical reactor, containing all the necessary components to support the LAMP reaction. The core components include the specific primer set designed in step S1, which is crucial for ensuring the specificity of the detection target; the buffer solution is used to maintain the required pH and ionic environment to ensure optimal enzyme activity; dNTPs (deoxynucleoside triphosphates) are the basic raw materials for DNA synthesis; and divalent metal ions, such as magnesium ions (Mg²⁺ ions). 2+ ), is an essential cofactor for Bst DNA polymerase to exert its enzymatic activity; while Bst DNA polymerase is a DNA polymerase with strand displacement activity, capable of efficiently synthesizing and amplifying DNA under isothermal conditions, and is the core enzyme of LAMP technology. These components work together to construct a reaction environment that can efficiently and specifically amplify the target viral nucleic acid.

[0012] Step S3 involves adding the nucleic acid from the sample to be tested into the reaction system; this is the step of introducing the sample into the detection system. The quality and quantity of nucleic acid extraction directly affect the sensitivity and accuracy of subsequent detection.

[0013] Subsequently, in step S4, the reaction system containing the sample nucleic acids is placed under isothermal conditions for LAMP reaction. "Isothermal conditions" are a significant feature that distinguishes LAMP technology from traditional PCR technology. Unlike PCR, which requires precise temperature cycling control, LAMP only needs to maintain the reaction system at a constant temperature (e.g., the optimized 63°C of this invention) for continuous and efficient DNA amplification by the Bst DNA polymerase. This isothermal characteristic greatly simplifies the operation process, reduces dependence on instruments, and makes it more suitable for rapid on-site detection. In this isothermal reaction process, six primers work synergistically to amplify the gB gene sequence of the target virus exponentially.

[0014] Finally, in step S5, the LAMP amplification products need to be detected to determine whether the reaction was successful and whether the sample contains the target viral nucleic acid. Detection methods can be varied, including visual observation (a positive reaction produces a visible white precipitate), fluorescence staining (e.g., using SYBR Green I or calcein dye for visual detection), gel electrophoresis, and real-time fluorescence monitoring. Different detection methods are suitable for different application scenarios, and users can choose the appropriate method based on their actual needs. Product detection is the key step in ultimately determining whether a sample is positive.

[0015] According to the present invention, in a loop-mediated isothermal amplification method for detecting equine herpesvirus types 1 and 4, the primer set in step S1 is:

[0016] The LAMP primer set for EHV-1 detection contains the following primers:

[0017] The F3 primer is located at positions 389-408 and has the sequence 5'-ATCAGCACGTACAGTTGAACT-3'.

[0018] Primer B3, located at positions 578-597, has the sequence 5'-GCGATCCCCTGCATAATCAC-3':

[0019] FIP primers, with F1c located at positions 373-392 and F2 located at positions 324-342, have a sequence of 5'-TCTAGCAGGGCCGGTGTCCTC-GGAAGACGCCGAGTTTCTG-3';

[0020] BIP primers, B1c is located at positions 496-515 and B2 is located at positions 553-571, with a sequence of 5'-CAGCGGAAATACAGCGCCGCA-ATTGTCCACGTTGACCACG-3';

[0021] Loop F primers, located at positions 347-363, have a sequence of 5'-CCGCGGTGTACACCTC-3';

[0022] Loop B primer, located at positions 520-536, has the sequence 5'-GCTCCACGCTCTCAGGT-3';

[0023] or,

[0024] The LAMP primer set for EHV-4 detection contains the following primers:

[0025] The F3 primer is located at positions 408-426 and has the sequence 5'-GGCAGAGTACCTCCGAGAT-3'.

[0026] Primer B3 is located at positions 637-655 and has the sequence 5'-CGGTCGATAGGGCAAAGGA-3'.

[0027] FIP primers, with F1c located at positions 487-511 and F2 located at positions 442-460, have a sequence of 5'-TGGCCCCAGGAGTAGCAAACT-GCTTACCACGACGACGAAG-3';

[0028] BIP primers, with B1c located at positions 547-567 and B2 located at positions 605-622, have a sequence of 5'-GTCGGATGGATGCCATGGAG-CAGACGCGCTTCTACCT-3';

[0029] Loop F primers, located at positions 461-479, have the sequence 5'-AGGTCGAGCTCCACCTCGT-3';

[0030] Loop B primers, located at positions 572-594, have the sequence 5'-ACACATCAACCTCTGCAACTGC-3'.

[0031] The above technical solution specifies the exact nucleotide sequences and their positions on the viral gB genes of the LAMP primer set used for EHV-1 and EHV-4 detection. This technical feature is key to achieving the high specificity of the detection effect of this invention. The primer set was designed based on in-depth analysis and screening of the specific sequences of the gB genes of equine herpesviruses type 1 and 4. The technical advantage lies in the fact that this primer set can specifically recognize and bind to the gB gene target sequences of EHV-1 and EHV-4, avoiding cross-reactions with the nucleic acid sequences of other related viruses (such as equine arteritis virus, porcine pseudorabies virus, bovine rhinotracheitis virus, equine influenza virus, etc.), thereby ensuring the high specificity of the LAMP amplification reaction. Specifically, primers F3 and B3 serve as outer primers, initiating and assisting the LAMP reaction; primers FIP and BIP serve as inner primers, containing F1c-F2 and B1c-B2 structures, capable of forming a loop structure and driving the self-circulating amplification of the LAMP reaction; primers LoopF ​​and LoopB serve as loop primers, accelerating the formation of the loop structure and further improving amplification efficiency. The interrelationship among these technical features lies in the fact that the design of these primer sequences ensures that, under the isothermal amplification conditions of the subsequent step S4, the LAMP reaction can only be efficiently initiated and induced to produce specific amplification products when the sample to be tested contains an EHV-1 or EHV-4gB gene sequence that specifically matches the primer set. This lays the foundation for achieving highly specific detection in subsequent technical solutions.

[0032] According to a method for detecting equine herpesvirus types 1 and 4 using loop-mediated isothermal amplification, in step S4, the isothermal condition is 63°C and the LAMP reaction time is 40 minutes.

[0033] The aforementioned technical solution further specifies the isothermal conditions for the LAMP reaction as 63℃ and the reaction time as 40 minutes. The technical advantage lies in the fact that these reaction conditions are optimized parameters that significantly shorten the detection time while ensuring efficient LAMP reaction, enabling rapid detection. 63℃ is the optimal temperature range for Bst DNA polymerase activity, ensuring maximum enzyme activity for efficient strand displacement and DNA synthesis. The 40-minute reaction time is sufficient to effectively amplify the target nucleic acid, achieving rapid detection. The interrelationship and synergistic effect between these technical features lies in the fact that this optimal reaction temperature and time, combined with the aforementioned defined specific primer set, ensure that under optimal enzyme activity conditions, the specific primers can efficiently bind to and amplify the gB gene sequence of the target virus, thereby obtaining detectable amplified products in a short time and achieving rapid and efficient detection.

[0034] According to a loop-mediated isothermal amplification method for detecting equine herpesvirus types 1 and 4 of the present invention, the concentrations of each primer in the primer set are as follows: the concentrations of outer primers F3 and B3 are 0.2 μmol / L, the concentrations of inner primers FIP and BIP are 1.6 μmol / L, and the concentrations of loop primers LoopF ​​and LoopB are 0.4 μmol / L.

[0035] The above technical solution defines the optimal concentration ratio of primers in the LAMP reaction system: outer primers F3 and B3 at 0.2 μmol / L, inner primers FIP and BIP at 1.6 μmol / L, and loop primers LoopF ​​and LoopB at 0.4 μmol / L. The technical advantage lies in the fact that this primer concentration ratio is optimized to balance multiple stages of the LAMP reaction, including initiation, self-circulating amplification, and loop formation, achieving optimal amplification efficiency and specificity. The high concentration of inner primers FIP and BIP ensures rapid initiation and efficient self-circulating amplification of the LAMP reaction; the low concentration of outer primers F3 and B3 helps improve reaction specificity; and the moderate concentration of loop primers LoopF ​​and LoopB accelerates loop formation, further improving amplification efficiency. The interrelationship and synergy among the technical features lies in the fact that the optimal primer concentration ratio is matched with the aforementioned specific primer sequences and the aforementioned optimal reaction temperature and time, together constructing an efficient, rapid and specific LAMP reaction system. Under the set reaction conditions, various primers can work synergistically to maximize the driving of the LAMP amplification reaction of the target gene.

[0036] According to the present invention, a loop-mediated isothermal amplification method for detecting equine herpesvirus types 1 and 4 is provided, wherein the divalent metal ion is Mg. 2+ The concentration of the substance is 3.0 mmol / L; the reaction system also contains betaine at a concentration of 0.4 mol / L; and the concentration of the dNTPs is 2.5 mmol / L.

[0037] The above technical solution limits the optimal concentrations of key components in the LAMP reaction system, including Mg. 2+ The concentrations were 3.0 mmol / L for Mg, 0.4 mol / L for betaine, and 2.5 mmol / L for dNTPs. The technical advantage lies in the fact that these components and their concentrations were optimized to provide the best biochemical environment for the LAMP reaction, ensuring enzyme activity, template stability, and reaction efficiency. 2+As an essential cofactor for Bst DNA polymerase, its optimal concentration ensures full enzyme activity; betaine reduces DNA secondary structure, decreases the inhibitory effect of GC-enriched sequences on amplification, and improves amplification efficiency and sensitivity; dNTPs, as raw materials for DNA synthesis, have an optimal concentration that ensures a sufficient supply of DNA for synthesis. The interrelationship and synergistic effect among these technical features lie in the fact that these reaction system components, together with the primer set, reaction conditions, and Bst DNA polymerase defined in the aforementioned scheme, work together to construct an optimized LAMP reaction system. Mg 2+ Betaine and dNTPs work together to create a suitable reaction environment, enabling Bst DNA polymerase to efficiently utilize specific primers and rapidly amplify the nucleic acid of the target virus under isothermal conditions, ultimately achieving rapid, sensitive, and specific detection results.

[0038] According to a loop-mediated isothermal amplification method for detecting equine herpesvirus types 1 and 4 according to the present invention, in step S5, the detection method of the amplification product is selected from one or more of the following:

[0039] Magnesium pyrophosphate precipitate can be observed with the naked eye;

[0040] Staining with the fluorescent dye SYBR GREEN I;

[0041] Calcein staining;

[0042] Agarose gel electrophoresis;

[0043] Real-time fluorescence monitoring.

[0044] The above technical solutions list various detection methods for LAMP amplification products, including visual observation of magnesium pyrophosphate precipitation, SYBR Green I staining, calcein staining, agarose gel electrophoresis, and real-time fluorescence monitoring. The advantage of this technology is that it provides users with diverse detection options, allowing them to flexibly choose the appropriate detection method based on experimental conditions and needs. Visual observation of magnesium pyrophosphate precipitation is simple and quick, requiring no special instruments, making it suitable for rapid on-site detection in grassroots laboratories. Fluorescent dye staining and calcein staining methods enable visual detection, improving the intuitiveness of the results. Agarose gel electrophoresis can verify the specificity of the amplification products, and real-time fluorescence monitoring can achieve quantitative or semi-quantitative detection, shortening the detection time. The interrelationship and synergistic effect among these technical features lies in the fact that these detection methods are selected based on the characteristics of the LAMP reaction amplification products. The LAMP reaction produces a large amount of magnesium pyrophosphate precipitate and characteristic ladder-like DNA bands. The detection methods listed above can effectively identify these characteristic products, thereby determining whether the LAMP reaction has been successfully carried out and achieving the detection of equine herpesvirus. Users can flexibly choose appropriate detection methods according to their actual situation to meet different detection needs and experimental conditions.

[0045] According to a method for detecting equine herpesvirus types 1 and 4 using loop-mediated isothermal amplification, in step S2, the DNA polymerase is Bst DNA polymerase.

[0046] The above technical solution specifies that the DNA polymerase used in the LAMP reaction is Bst DNA polymerase. The technical advantage lies in the fact that Bst DNA polymerase is a DNA polymerase with strand displacement activity, a key enzyme for achieving isothermal LAMP amplification. Bst DNA polymerase exhibits high activity and stability under isothermal conditions of 63°C, enabling efficient strand extension and displacement, thereby driving the self-circulating amplification of the LAMP reaction and achieving exponential nucleic acid amplification. The interrelationship and synergistic effect among the technical features lies in the compatibility of Bst DNA polymerase with the aforementioned specific primer set and the aforementioned isothermal reaction conditions, jointly constructing the core technical solution of this invention. The strand displacement activity of Bst DNA polymerase ensures that the LAMP reaction can proceed efficiently under isothermal conditions, while the specific primers ensure the specificity of the amplification reaction, ultimately achieving rapid, efficient, and specific detection of equine herpesvirus.

[0047] According to the present invention, a loop-mediated isothermal amplification method for detecting equine herpesviruses types 1 and 4 is provided, wherein the total volume of the LAMP reaction system is 25 μL; and the limit of detection for EHV-1 nucleic acid is 9.4 × 10⁻⁶. -5 The limit of detection for EHV-4 nucleic acid testing is 1.559 × 10 ng / μL.-4 ng / μL.

[0048] The above technical solution limits the total volume of the LAMP reaction system to 25 μL and discloses the lowest detection limits for nucleic acid detection of EHV-1 and EHV-4. The technical advantages are that the 25 μL reaction volume aligns with the trend towards micro-reactions, saves reagent costs, and facilitates high-throughput operation; the published detection limit data (EHV-1: 9.4 × 10⁻⁶) is also relevant. -5 ng / μL, EHV-4 was 1.559×10 -4 The results (ng / μL) directly demonstrate that the LAMP method of this invention has high detection sensitivity, exceeding that of traditional PCR methods, enabling the detection of samples with low viral loads and improving early diagnosis rates. The interrelationship and synergistic effect among the technical features lie in the fact that the reaction volume and detection limit data directly reflect the optimized combination of the aforementioned technical solutions. The optimized reaction system and conditions enable efficient amplification and high-sensitivity detection even with minimal reaction volume, thus proving the superiority of the technical solution of this invention.

[0049] According to a method for detecting equine herpesvirus 1 and 4 using loop-mediated isothermal amplification, in step S2, a premix solution is first prepared, the premix solution containing all reagents except template DNA; in step S3, a positive control and a negative control are further provided, wherein the positive control is a known EHV-1 or EHV-4 positive sample DNA, and the negative control is template-free DNA.

[0050] The above technical solution defines the preparation method of the premixed LAMP reaction reagent and the setting of positive and negative controls. The technical advantages are that the preparation of the premixed solution simplifies experimental procedures, reduces human error, and improves the reproducibility and stability of the experiment; the setting of positive and negative controls allows for monitoring the experimental process, verifying the reliability of the results, and ensuring the accuracy of the detection results. The interrelationship and synergistic effect among these technical features lies in the fact that the preparation of the premixed solution and the setting of controls further optimize and improve the aforementioned defined LAMP detection method, making it easier to operate, more reliable, and more suitable for practical applications. The premixed solution simplifies the reagent preparation process and reduces operational difficulty, while the control settings provide quality control, ensuring the accuracy of the detection results.

[0051] Based on the above, the present invention also provides a kit for detecting loop-mediated isothermal amplification of equine herpesvirus types 1 and 4, comprising:

[0052] A set of LAMP primers designed for the gB gene specific sequence of equine herpesvirus type 1 and / or type 4, the primer set containing six primers: F3, B3, FIP, BIP, LoopF ​​and LoopB;

[0053] LAMP reaction buffer;

[0054] dNTPs mixture;

[0055] Divalent metal ion solution;

[0056] betaine solution;

[0057] Bst DNA polymerase;

[0058] The kit is used to detect equine herpesvirus type 1 and / or type 4 under a constant temperature of 63°C. The detection results are determined by observing the amplification products generated by the LAMP reaction.

[0059] It also includes one or more of the following components:

[0060] SYBR GREEN I dye or calcium chlorophyll developing solution;

[0061] Positive control;

[0062] Negative control.

[0063] The above-described technical solution defines a loop-mediated isothermal amplification (LAMP) kit for detecting equine herpesvirus types 1 and 4. The kit contains various components required to implement the method of this invention and may optionally include dyes and controls. The technical advantage lies in that this kit standardizes and commercializes the LAMP detection method, making it convenient for users, lowering the technical threshold, and facilitating the promotion and application of the detection method of this invention. The kit contains various components with optimized proportions; users only need to follow the instructions to quickly and conveniently detect equine herpesvirus. The interrelationship and synergistic effect among the technical features lies in the fact that this kit is an integrated embodiment of the method of this invention, integrating all key components into a single kit to form a complete product. This allows users to more easily utilize the technology of this invention for equine herpesvirus detection, accelerating the commercial application and widespread adoption of this technology. The kit format ensures the stability of reagent quality and the consistency of detection results, further enhancing the practical value of the technical solution of this invention.

[0064] The loop-mediated isothermal amplification method for detecting equine herpesvirus types 1 and 4 of the present invention can produce the following technical effects:

[0065] 1. Speed: Utilizing loop-mediated isothermal amplification (LAMP) technology, the reaction proceeds under isothermal conditions, eliminating the need for complex temperature cycling. Furthermore, LAMP technology itself possesses extremely high amplification efficiency. Under optimized reaction conditions, nucleic acid amplification and detection (steps S4 and S5) can be completed in a relatively short time. Compared to traditional methods, detection time is significantly reduced, resulting in a substantial improvement in detection efficiency. This is of great significance for the rapid diagnosis of acute infectious diseases. If real-time fluorescence monitoring is employed, results can be obtained in an even shorter time, further accelerating the detection speed.

[0066] 2. High Sensitivity: Due to its unique primer design and amplification mechanism, LAMP technology can exponentially amplify target DNA in a short time, generating a large number of amplification products. Experimental data show that this technology has higher sensitivity than conventional PCR methods. Its high sensitivity allows this method to detect samples with low viral loads, effectively avoiding false negative results caused by low viral loads, improving the diagnostic rate of early infection, and providing strong support for early disease control.

[0067] 3. High Specificity: In step S1, this method designs and constructs LAMP primer sets specifically targeting the gB gene sequences of EHV-1 and EHV-4. The gB gene is an important gene of EHV, and the gB gene sequence differs among different types of EHV. The design of the specific primer set ensures that the primers can only precisely bind to and initiate amplification with the gB gene sequences of EHV-1 and EHV-4, without cross-reacting with other related viruses (such as equine arteritis virus, porcine pseudorabies virus, bovine rhinotracheitis virus, equine influenza virus, etc.), thereby ensuring the accuracy and reliability of the detection results and effectively avoiding false positive results.

[0068] 4. Ease of Operation: The LAMP reaction is an isothermal reaction (step S4), requiring only conventional laboratory equipment such as a constant-temperature water bath or metal bath for amplification, eliminating the need for expensive PCR instruments and precise temperature cycling control. Furthermore, the LAMP reaction system is simple to prepare, and the operation steps are straightforward and require no complex professional skills. Product detection methods are diverse; visual observation and visualization staining methods require no special equipment, further simplifying the operation process and making it more convenient and faster. This ease of operation makes this method more readily applicable in resource-constrained environments such as grassroots laboratories, veterinary stations, and even livestock farms, enabling rapid on-site detection. Attached Figure Description

[0069] To more clearly illustrate the technical solutions in this invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of this invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0070] Figure 1 This is a schematic diagram of the EHV-1LAMP primer sequence and position of the present invention;

[0071] Figure 2 This is a schematic diagram of the EHV-4LAMP primer sequence and position of the present invention;

[0072] Figure 3 This is a chart showing the composition and concentration of the optimal reaction system for EHV-1LAMP according to the present invention;

[0073] Figure 4 This is a chart showing the composition and concentration of the optimal reaction system for EHV-4LAMP according to the present invention;

[0074] Figure 5 This is a schematic diagram of the method flow of the present invention;

[0075] Figure 6 This is a visualization of the EHV-1 LAMP product SYBR GREEN I staining results from the present invention;

[0076] Figure 7 This is a visualization of the EHV-1LAMP product calcein staining detection results of the present invention;

[0077] Figure 8 This is a visualization of the EHV-4LAMP product SYBR GREEN I staining results from this invention;

[0078] Figure 9 This is a visualization of the EHV-4LAMP product calcein staining detection results of the present invention;

[0079] Figure 10 This is a graph showing the sensitivity verification results of the EHV-1LAMP method of this invention;

[0080] Figure 11 This is a graph showing the sensitivity verification results of the EHV-4LAMP method of this invention;

[0081] Figure 12 This is a graph showing the specificity verification results of the EHV-1LAMP method of the present invention;

[0082] Figure 13 This is a graph showing the specificity verification results of the EHV-4LAMP method of the present invention;

[0083] Figure 14 This is a real-time fluorescence monitoring curve of the EHV-1LAMP of the present invention;

[0084] Figure 15 This is a real-time fluorescence monitoring curve of the EHV-4LAMP of the present invention;

[0085] Figure 16 This is a typical electrophoresis pattern of the EHV-1 LAMP reaction product of the present invention;

[0086] Figure 17 This is a typical electrophoretic pattern of the EHV-4LAMP reaction product of the present invention. Detailed Implementation

[0087] Embodiments of the present invention are described in detail below. Examples of these embodiments are shown in the accompanying drawings, wherein the same or similar reference numerals denote the same or similar elements or elements having the same or similar functions throughout. The embodiments described below with reference to the accompanying drawings are exemplary and are only used to explain the present invention, and should not be construed as limiting the present invention.

[0088] Combination Figures 1 to 17 As shown, this embodiment aims to detail the specific implementation process of the loop-mediated isothermal amplification (LAMP) method for detecting equine herpesvirus type 1 (EHV-1) and equine herpesvirus type 4 (EHV-4), including primer design, optimization of the LAMP reaction system, reaction steps and result determination, and to verify the effectiveness and technical effect of the method.

[0089] 1. Primer design

[0090] To achieve specific detection of EHV-1 and EHV-4, we designed specific LAMP primer sets targeting the conserved gB gene sequences of both viruses. The primer design process utilized LAMP Primer Explorer V4 software, which assists in designing efficient LAMP primer sets containing six primers: F3, B3, FIP, BIP, LoopF, and LoopB.

[0091] Primer sequences designed for the EHV-1 (GenBank accession number AY464052) gB gene sequence and their locations in the genome are as follows: Figure 1 As shown in Table 1.

[0092] Primer sequences designed for the EHV-4 (GenBank accession number NF80567) gB gene sequence and their locations in the genome are as follows: Figure 2 As shown in Table 2.

[0093] The primer sequences shown in Tables 1 and 2 were synthesized by Shanghai Bioengineering Co., Ltd. The primer sequences were designed to ensure specific amplification of EHV-1 and EHV-4, avoiding cross-reactions with other equine respiratory pathogens or related herpesviruses, thus guaranteeing the accuracy of the test results.

[0094] 2. Optimization of the LAMP reaction system

[0095] To achieve optimal LAMP amplification efficiency and detection sensitivity, the LAMP reaction system was optimized in this embodiment. The optimization process mainly investigated reaction temperature, primer concentration, and Mg... 2+ The effects of key factors such as concentration, betaine concentration, and dNTP concentration on the LAMP reaction were investigated. Optimal concentrations and reaction conditions for each component were determined through single-factor experiments. The final optimal reaction systems for EHV-1 and EHV-4 LAMP are shown below. Figure 3 and Figure 4 As shown in Tables 3 and 4.

[0096] In Tables 3 and 4, the 10×LAMP Buffer contains 200 mM Tris-HCl (pH 8.8), 100 mM KCl, 100 mM (NH4)2SO4, 1% Tween 20, and BstDNA polymerase, specifically Bacillus stearothermophilus DNA polymerase, purchased from New England Biolabs. In practice, a premix containing all reagents except the template DNA can be prepared in advance to simplify the procedure and reduce human error.

[0097] 3. LAMP reaction steps

[0098] The LAMP reaction operation procedure of the present invention is simple and quick, and the specific steps are as follows:

[0099] 3.1 Reagent Preparation: Prepare EHV-1 or EHV-4 LAMP reaction solution according to the reaction system shown in Table 3 or Table 4. A premix containing 10×LAMP Buffer, dNTPs, MgSO4, betaine, primer mixture, and Bst DNA polymerase can be prepared in advance.

[0100] 3.2 Sample Addition: Add 2 μL of the nucleic acid DNA sample to 23 μL of LAMP reaction premix, bringing the final reaction volume to 25 μL. To ensure the reliability of the experimental results, the following controls must be included in the experiment:

[0101] (1) Positive control: DNA from known EHV-1 or EHV-4 positive samples was used as a template to verify the effectiveness of the LAMP reaction system;

[0102] (2) Negative control: Use nuclease-free water instead of template DNA to eliminate reagent contamination and non-specific amplification.

[0103] 3.3 Isothermal Amplification: Place the reaction tubes in a constant-temperature water bath or metal bath and react at 63°C for 40 minutes. Isothermal conditions are one of the core advantages of LAMP technology. It eliminates the need for temperature-controlled equipment such as PCR instruments; a regular water bath or metal bath is sufficient for experimental needs, greatly simplifying operation and reducing equipment costs.

[0104] 3.4 Product Detection: LAMP amplification products can be detected using various methods. This embodiment uses the following three methods:

[0105] Method 1: Visual observation: The LAMP reaction produces a large amount of magnesium pyrophosphate precipitate. A white turbidity is visible in the positive reaction tube, while the negative reaction tube and negative control tube remain clear. This method is simple and intuitive, and results can be determined without any special equipment.

[0106] Method 2, Visualization with Fluorescent Dye (SYBR GREEN I Staining): After the reaction, add 1 μL of diluted SYBR GREEN I dye (e.g., 1000-fold diluted commercial SYBR GREEN I solution) to the reaction tube. In the positive reaction tube, due to the presence of amplification products, the dye intercalates into the DNA and emits green fluorescence, which is clearly visible to the naked eye under blue light excitation. The negative reaction tube and negative control tube remain orange or show weak background fluorescence.

[0107] Method 3, Agarose Gel Electrophoresis: Take 5 μL of the reaction product and perform 1.5% agarose gel electrophoresis at 80V for 20 minutes. Positive reaction samples show typical ladder-like bands composed of multiple bands on the gel, which is a characteristic feature of LAMP amplification products. Negative reaction samples and negative control samples do not show obvious bands.

[0108] Figure 6 and Figure 7 The visualization results of the EHV-1 LAMP products are presented. For example... Figure 6 As shown, using SYBR GREENI staining, the positive reaction tube showed a bright green (left), while the negative control remained its original orange (right). Figure 7 The test results A and Figure 7 The test results B show the results of calcein staining under fluorescent and ultraviolet light respectively. Positive samples show light green or bright green fluorescence (left), and negative samples show orange-red (right).

[0109] Figure 8 and Figure 9Similar visual detection results for EHV-4LAMP products were presented, verifying the good applicability and reproducibility of the detection method for both viruses. These visualizations intuitively demonstrate that the detection method provided by this invention is fast, convenient, and easy to interpret, making it particularly suitable for rapid on-site detection applications.

[0110] Figure 16 and Figure 17 Typical electrophoretic patterns of EHV-1 and EHV-4 LAMP reaction products are shown. Unlike the single band of a specific size produced by traditional PCR, the LAMP reaction produces a ladder-like pattern of multiple bands formed by repeating structural units of different lengths, which is a characteristic feature of LAMP products. This result further confirms that this method can successfully amplify the target sequence and generate typical LAMP reaction products.

[0111] Method 4: Real-time fluorescence monitoring: SYBR Green I fluorescent dye was added to the LAMP reaction system, and the reaction process was monitored in real time using a real-time fluorescence PCR instrument. For example... Figure 14 and Figure 15 As shown, the LAMP reactions of EHV-1 and EHV-4 exhibited significant fluorescence signal enhancement at approximately 15-20 minutes and 10-16 minutes, respectively, indicating the generation of a large amount of amplified products at this time. This method not only allows for real-time interpretation of results, shortening the detection time, but also enables semi-quantitative analysis, further improving the practicality of the detection.

[0112] 4. Result Determination

[0113] The LAMP reaction results can be determined using the product detection methods described above.

[0114] Visual inspection: A white precipitate appears in the positive reaction tube, while the negative reaction tube and negative control tube are clear.

[0115] SYBR GREEN I staining: positive reaction tubes show green fluorescence, while negative reaction tubes and negative control tubes show orange fluorescence.

[0116] Agarose gel electrophoresis: positive reaction samples show ladder-like bands, while negative reaction samples and negative control samples show no bands.

[0117] If the positive control shows a positive result, the negative control shows a negative result, and the result of the sample to be tested is consistent with the positive or negative control, then the test result is valid.

[0118] 5. Verification of technical effectiveness

[0119] To verify the effectiveness and technical advantages of the LAMP method established in this invention, we conducted the following experiments:

[0120] 5.1 Sensitivity Validation: EHV-1 and EHV-4 standard DNA templates were serially diluted and detected using both the LAMP method of this invention and conventional PCR, respectively. The detection sensitivity of the two methods was compared. Experimental results showed that the limit of detection for nucleic acid detection using the EHV-1 LAMP method was 9.4 × 10⁻⁶. -5 The limit of detection for nucleic acid detection using the EHV-4LAMP method is 1.559 × 10⁶ ng / μL. -4 The sensitivity is improved by at least one order of magnitude compared to conventional PCR methods, demonstrating that the LAMP method of this invention has the advantage of high sensitivity.

[0121] like Figure 10 As shown, the EHV-1LAMP method was used to detect a series of serially diluted templates, demonstrating its high sensitivity. Lanes 1-6 correspond to the templates undiluted to 10⁻⁶ ppm. -5 The diluted template is visible at 10 -5 Dilution (9.4 × 10⁻⁶) -5 Even at concentrations of ng / μL, distinct ladder-like bands can still be observed, demonstrating that this method has extremely high detection sensitivity.

[0122] like Figure 11 As shown, the EHV-4LAMP method also exhibits high sensitivity characteristics, at 1.559 × 10⁻⁶. -4 The target sequence can still be detected at a dilution of ng / μL.

[0123] 5.2 Specificity Verification: Using the LAMP primer set of this invention, LAMP detection was performed on the nucleic acids of EHV-1, EHV-4, and other related viruses (including equine arteritis virus, porcine pseudorabies virus, bovine rhinotracheitis virus, equine influenza virus, etc.). The results showed that the LAMP method of this invention only produced positive amplification signals for EHV-1 and EHV-4 samples, and there was no cross-reaction with other related viruses, proving that this method has excellent specificity.

[0124] Figure 12 and Figure 13 The results of the LAMP method's specificity validation are shown. As illustrated, only EHV-1 or EHV-4 positive samples (lane 1 or lane 2) show obvious ladder-like bands, while other related viruses such as equine arteritis virus (EAV), porcine pseudorabies virus (PRV), bovine rhinotracheitis virus (IBRV), equine influenza virus type 1 (H7N7) and equine influenza virus type 2 (H3N8) did not produce amplification products, demonstrating that this method has excellent specificity and can reliably distinguish different viruses.

[0125] 5.3 Rapidity Validation: The LAMP method of this invention can complete amplification and result determination within 40 minutes at a constant temperature of 63℃. The entire detection process is short, simple, and quick, requiring no complex temperature cycling or precision instruments, significantly improving detection efficiency, and is especially suitable for rapid on-site detection and high-throughput screening. If a real-time fluorescence monitoring method is used, detection results can be obtained within 15-20 minutes, further improving detection speed.

[0126] Furthermore, to facilitate user operation and further improve the standardization and reproducibility of the detection method, this invention provides a loop-mediated isothermal amplification kit for detecting equine herpesvirus types 1 and 4. This kit may contain the following components:

[0127] Component 1: EHV-1LAMP primer mixture and / or EHV-4LAMP primer mixture (selected as needed, containing F3, B3, FIP, BIP, LoopF, LoopB primers);

[0128] Component 2: LAMP reaction buffer (10× concentration);

[0129] Component 3: dNTPs mixture (10 mM);

[0130] Component 4: MgSO4 solution (100mM);

[0131] Component 5: Betaine solution (10M);

[0132] Component 6: Bst DNA polymerase (8 U / μL);

[0133] Component 7 (optional): SYBR GREEN I dye or calcein colorimetric solution (for visual detection);

[0134] Component 8 (optional): Positive control (EHV-1 or EHV-4 DNA standard);

[0135] Component 9 (optional): Negative control (nuclease-free water).

[0136] The concentrations and ratios of each component in the kit are consistent with the optimized LAMP reaction system described above. Users only need to follow the instructions provided with the kit to quickly and accurately detect equine herpesvirus types 1 and 4.

[0137] In summary, this embodiment details a method for detecting equine herpesvirus types 1 and 4 based on LAMP technology. This method utilizes a specifically designed primer set, optimized reaction system, and isothermal conditions to achieve efficient amplification and rapid detection of EHV-1 and EHV-4 nucleic acids. Detection results can be determined through various methods such as visual observation, fluorescent staining, or agarose gel electrophoresis. It boasts significant advantages such as speed, sensitivity, specificity, and ease of operation, overcoming the shortcomings of traditional detection methods, such as long processing time, complex operation, and insufficient sensitivity. This provides strong technical support for the rapid diagnosis and control of equine herpesvirus diseases. Furthermore, the kit developed based on this method will further promote the application of this technology in grassroots laboratories and field testing, showing broad application prospects.

[0138] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit them; those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A loop-mediated isothermal amplification method for detecting equine herpesvirus types 1 and 4, characterized in that, Includes the following steps: S1. Design and construct loop-mediated isothermal amplification (LAMP) primer sets targeting the gB gene specific sequences of equine herpesvirus type 1 and type 4. Each primer set contains six primers: F3, B3, FIP, BIP, LoopF, and LoopB. S2. Prepare the LAMP reaction system, which includes the primer set, buffer, dNTPs, divalent metal ions and DNA polymerase; S3. Add the nucleic acid of the sample to be tested into the reaction system; S4. The LAMP reaction is carried out under isothermal conditions; S5. Detect the LAMP reaction amplification products.

2. The loop-mediated isothermal amplification method for detecting equine herpesvirus types 1 and 4 according to claim 1, characterized in that, In step S1, the primer set is: The LAMP primer set for EHV-1 detection contains the following primers: The F3 primer is located at positions 389-408 and has the sequence 5'-ATCAGCACGTACAGTTGAACT-3'. Primer B3, located at positions 578-597, has the sequence 5'-GCGATCCCCTGCATAATCAC-3': FIP primers, with F1c located at positions 373-392 and F2 located at positions 324-342, have a sequence of 5'-TCTAGCAGGGCCGGTGTCCTC-GGAAGACGCCGAGTTTCTG-3'; BIP primers, B1c is located at positions 496-515 and B2 is located at positions 553-571, with a sequence of 5'-CAGCGGAAATACAGCGCCGCA-ATTGTCCACGTTGACCACG-3'; Loop F primers are located at positions 347-363 and have the sequence 5'-CCGCGGTGTACACCTC-3'. Loop B primers are located at positions 520-536 and have the sequence 5'-GCTCCACGCTCTCAGGT-3'. or, The LAMP primer set for EHV-4 detection contains the following primers: The F3 primer is located at positions 408-426 and has the sequence 5'-GGCAGAGTACCTCCGAGAT-3'. Primer B3 is located at positions 637-655 and has the sequence 5'-CGGTCGATAGGGCAAAGGA-3'. FIP primers, with F1c located at positions 487-511 and F2 located at positions 442-460, have a sequence of 5'-TGGCCCCAGGAGTAGCAAACT-GCTTACCACGACGACGAAG-3'; BIP primers, with B1c located at positions 547-567 and B2 located at positions 605-622, have a sequence of 5'-GTCGGATGGATGCCATGGAG-CAGACGCGCTTCTACCT-3'; Loop F primers, located at positions 461-479, have the sequence 5'-AGGTCGAGCTCCACCTCGT-3'; Loop B primers are located at positions 572-594, with the sequence 5'-ACACATCAACCTCTGCAACTGC-3'.

3. The loop-mediated isothermal amplification method for detecting equine herpesvirus types 1 and 4 according to claim 1, characterized in that, In step S4, the constant temperature condition is 63°C, and the LAMP reaction time is 40 minutes.

4. The loop-mediated isothermal amplification method for detecting equine herpesvirus types 1 and 4 according to claim 1, characterized in that, The concentrations of each primer in the primer set are as follows: the concentrations of outer primers F3 and B3 are 0.2 μmol / L, the concentrations of inner primers FIP and BIP are 1.6 μmol / L, and the concentrations of loop primers LoopF ​​and LoopB are 0.4 μmol / L.

5. The loop-mediated isothermal amplification method for detecting equine herpesvirus types 1 and 4 according to claim 1, characterized in that, The divalent metal ion is Mg. 2+ The concentration of the substance is 3.0 mmol / L; the reaction system also contains betaine at a concentration of 0.4 mol / L; and the concentration of the dNTPs is 2.5 mmol / L.

6. The loop-mediated isothermal amplification method for detecting equine herpesvirus types 1 and 4 according to claim 1, characterized in that, In step S5, the detection method for the amplification product is selected from one or more of the following: Magnesium pyrophosphate precipitate can be observed with the naked eye; Staining with the fluorescent dye SYBR GREEN I; Calcein staining; Agarose gel electrophoresis; Real-time fluorescence monitoring.

7. The loop-mediated isothermal amplification method for detecting equine herpesvirus types 1 and 4 according to any one of claims 1 to 6, characterized in that, In step S2, the DNA polymerase is Bst DNA polymerase.

8. The loop-mediated isothermal amplification method for detecting equine herpesvirus types 1 and 4 according to claim 2, characterized in that, The total volume of the LAMP reaction system was 25 μL; and the limit of detection for EHV-1 nucleic acid was 9.4 × 10⁻⁶. -5 The limit of detection for EHV-4 nucleic acid testing is 1.559 × 10 ng / μL. -4 ng / μL.

9. The loop-mediated isothermal amplification method for detecting equine herpesvirus types 1 and 4 according to claim 2, characterized in that, In step S2, a premix solution is first prepared, which contains all reagents except template DNA. In step S3, a positive control and a negative control are also set up, wherein the positive control is a known EHV-1 or EHV-4 positive sample DNA, and the negative control is template-free DNA.

10. A kit for detecting equine herpesvirus types 1 and 4 using loop-mediated isothermal amplification, characterized in that, include: A set of LAMP primers designed for the gB gene specific sequence of equine herpesvirus type 1 and / or type 4, the primer set containing six primers: F3, B3, FIP, BIP, LoopF ​​and LoopB; LAMP reaction buffer; dNTPs mixture; Divalent metal ion solution; betaine solution; Bst DNA polymerase; The kit is used to detect equine herpesvirus type 1 and / or type 4 under constant temperature conditions of 63°C. The detection results are determined by observing the amplification products generated by the LAMP reaction. It also includes one or more of the following components: SYBR GREEN I dye or calcium chlorophyll developing solution; Positive control; Negative control.

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