Method for detecting equine herpesvirus type 1 and equine herpesvirus type 4 through visual loop-mediated isothermal amplification based on color change

Abstract

The invention relates to a method for detecting equine herpes viruses type 1 and type 4 through visual loop-mediated isothermal amplification based on color change. The method comprises the following steps: S1, extracting nucleic acid of a sample to be detected; s2, preparing a reaction system containing an LAMP primer group designed for horse herpes virus, DNA polymerase and a color indicator; s3, performing amplification reaction on the reaction system under a constant-temperature condition; and S4, judging a detection result by observing the color change of the reaction system. The method aims at overcoming the defects that in the prior art, an horse herpes virus detection method is tedious in operation and long in consumed time, or professional equipment is needed and the like, and particularly provides a more efficient and convenient horse herpes virus detection means aiming at the requirements of on-site rapid detection and basic application.

Description

Technical Field

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

[0002] Equine nasal pneumonia, caused by equine herpesvirus 1 (EHV-1) and equine herpesvirus 4 (EHV-4), is widespread globally, causing significant economic losses to the horse breeding and racing industries. This disease not only triggers respiratory illnesses and viral abortions in horses, but in severe cases can also lead to neurological disorders. Its harmfulness has been given high priority by the World Organisation for Animal Health (OIE), and it is listed in the list of legally reportable diseases. The International Animal Health Code clearly stipulates the reporting and control measures for equine nasal pneumonia; rapid and accurate diagnosis is a crucial link in disease control and the inspection and quarantine of imported racehorses.

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

[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 complex to operate, requiring high levels of expertise and skilled personnel. It is difficult to meet the needs of rapid detection, especially in the early stages of an outbreak, where delays can lead to the spread of the epidemic.

[0005] Serological methods, such as neutralization tests, complement fixation tests, and enzyme-linked immunosorbent assays (ELISA), primarily detect antibody levels in equine serum to aid in diagnosis. While relatively simple to perform, serological methods have the following limitations: First, antibody production usually lags behind viral infection, potentially leading to false negatives in the early stages of infection; second, serological methods are prone to cross-reactivity, making it difficult to distinguish between different types of herpesvirus infections, thus reducing diagnostic specificity; furthermore, serological methods cannot directly detect the virus, only reflecting the body's immune response, and cannot serve as direct evidence of early infection or viral carriage.

[0006] Molecular biology methods, especially polymerase chain reaction (PCR) technology, have become an important means of detecting equine herpesvirus due to their high sensitivity and specificity. PCR technology can directly detect viral nucleic acid, with higher sensitivity than virus isolation and identification and serological methods, and good specificity. However, traditional PCR methods still have certain limitations: First, PCR amplification requires precise temperature cycling control and relies on expensive PCR instruments and other specialized equipment, limiting its application in grassroots laboratories and field settings; second, the detection of PCR amplification products usually requires agarose gel electrophoresis, which is cumbersome, time-consuming, and may introduce contamination risks during the electrophoresis process, making it difficult to meet the needs of rapid, on-site detection.

[0007] To overcome the shortcomings of traditional diagnostic methods, loop-mediated isothermal amplification (LAMP) technology has emerged. LAMP is a novel DNA amplification technique that operates under isothermal conditions, eliminating the need for temperature-sensitive equipment like PCR instruments. It boasts significant advantages such as rapid amplification speed, high efficiency, high sensitivity, and strong specificity. LAMP technology has been successfully applied to the rapid detection of various pathogenic microorganisms, demonstrating promising application prospects. However, the interpretation of traditional LAMP results typically requires analysis using instruments such as agarose gel electrophoresis or turbidimeters. While this simplifies the process compared to PCR electrophoresis, it still necessitates certain experimental procedures and equipment, limiting the rapid and widespread application of LAMP technology at the grassroots level and in field settings. This is particularly true in port inspection and quarantine departments and grassroots farms lacking specialized equipment and personnel, where the application of traditional LAMP methods remains an obstacle.

[0008] Therefore, in order to further simplify the interpretation of LAMP detection results and improve the convenience and on-site applicability of detection, it is urgent to develop a visual LAMP detection method that does not require electrophoresis or special instruments and can judge the results simply by observing color changes with the naked eye. Summary of the Invention

[0009] To address the technical problems existing in the prior art, this invention provides a method for detecting equine herpesvirus 1 and 4 based on color-changing visualization loop-mediated isothermal amplification. This method overcomes the shortcomings of existing equine herpesvirus detection methods, such as cumbersome operation, long time consumption, or the need for specialized equipment. It provides a more efficient and convenient means of detecting equine herpesvirus, especially for the needs of rapid on-site detection and grassroots applications.

[0010] This invention discloses a method for visualizing equine herpesvirus 1 and 4 using color-change-based loop-mediated isothermal amplification (LAMP). The core of this method lies in utilizing color changes to visualize the detection results. Specifically, the method first requires extracting nucleic acids, such as DNA or RNA, from the sample to be tested. Subsequently, a reaction system is prepared, containing a LAMP primer set specifically designed for equine herpesvirus, a DNA polymerase capable of catalyzing DNA amplification, and a color indicator to indicate the amplification result. After preparing the reaction system, it is placed under isothermal conditions for amplification. During this isothermal amplification process, if equine herpesvirus 1 or 4 nucleic acid is present in the sample, the LAMP primer set will specifically recognize and bind to the specific gene sequence of the viral nucleic acid. Under the action of the DNA polymerase, efficient DNA amplification is performed using the viral nucleic acid as a template, resulting in a large amount of DNA amplification products. Finally, the detection result is determined by visually observing the color change of the color indicator in the reaction system. The role of the color indicator is to interact with the DNA amplification products generated in the LAMP amplification reaction, thereby causing an observable color change in the reaction system. If the expected color change occurs, it indicates the presence of equine herpesvirus type 1 or 4 in the sample, resulting in a positive test result; conversely, if there is no significant color change, the test result is negative. The entire process does not rely on complex instruments or equipment; the result can be intuitively determined solely by the color change.

[0011] According to the method for detecting equine herpesvirus 1 and 4 based on color-change visualization loop-mediated isothermal amplification of the present invention, the LAMP primer set includes primers F3, B3, FIP, BIP, Loop F and Loop B, which specifically target the gB gene of equine herpesvirus 1 and / or 4.

[0012] The aforementioned technical solution defines the primer set used in the LAMP reaction as six primers: F3, B3, FIP, BIP, Loop F, and Loop B. Furthermore, the design target of this primer set is limited to the gB gene of equine herpesvirus type 1 and / or 4. Technically, there is a close technical correlation between this primer set limitation and the selection of the gB gene target. The gB gene (glycoprotein B gene) is a highly conserved and species-specific gene segment in the genomes of equine herpesvirus types 1 and 4. It plays a crucial role in viral adsorption, penetration, and fusion, and is an essential gene for viral infection and replication. By selecting the gB gene as the target for primer design and employing the six primers required for LAMP technology (F3, B3, FIP, BIP, Loop F, Loop B), highly specific recognition and amplification of equine herpesvirus types 1 and 4 are achieved. These six primers work synergistically. During the LAMP reaction, the FIP and BIP primers hybridize with the inner and outer regions of the target sequence, respectively, initiating a strand substitution reaction to form a dumbbell-shaped structure. Subsequently, the B3 and F3 primers further promote amplification from the outside, while the Loop F and Loop B primers accelerate the self-loop reaction and subsequent cyclic amplification by hybridizing with specific regions of the loop structure. This multi-primer synergy and the high conservation of the gB gene ensure the specificity of the method, avoiding cross-reactions with other equine viruses or non-specific nucleic acid sequences, ensuring the accuracy of the detection results, and thus laying the molecular biological foundation for the accurate diagnosis of equine herpesvirus types 1 and 4. Experimental data (e.g.) Figure 14 and Figure 15 The specific experimental results shown include, Figure 14 This is a result of EHV-1LAMP specific experiments. Figure 15 The results of the EHV-4LAMP specificity experiment also fully verified that the primer set has good specificity for EHV-1 and EHV-4, and no cross-reactivity with other equine viruses and other animal viruses, further confirming the technical effects that can be achieved by the above technical features.

[0013] According to the method for detecting equine herpesvirus 1 and 4 by visual loop-mediated isothermal amplification based on color change according to the present invention, the isothermal condition in step S3 is 60-65℃ for 30-50 minutes, and after the reaction is completed, enzyme inactivation treatment is performed by heating at 80℃ for 2 minutes.

[0014] The above technical solution specifies the isothermal conditions for the LAMP amplification reaction, specifically a reaction time of 60-65℃ for 30-50 minutes, followed by enzyme inactivation treatment at 80℃ for 2 minutes after the reaction. These optimized temperature, time, and enzyme inactivation conditions are carefully selected and coordinated to achieve efficient, stable, and controllable LAMP amplification, ensuring the reliability of the detection results. Firstly, isothermal reaction is a core characteristic of LAMP technology, avoiding the complexity of temperature cycling in traditional PCR technology. 60-65℃ is the optimal temperature range for the catalytic activity of Bst DNA polymerase (a DNA polymerase with strand displacement activity commonly used in LAMP reactions). Within this temperature range, Bst DNA polymerase can efficiently perform DNA polymerization and strand displacement reactions, driving exponential amplification in the LAMP reaction. The 30-50 minute reaction time is sufficient to produce detectable amounts of amplified products, ensuring rapid detection. The precise control of reaction temperature and time achieves a balance between amplification efficiency and detection speed. Secondly, the enzyme inactivation treatment at 80℃ for 2 minutes is a crucial step in controlling the termination of the LAMP reaction and preventing non-specific amplification and contamination. Heating to 80℃ effectively inactivates Bst DNA polymerase, terminating the ongoing LAMP reaction and avoiding false positive results caused by over-reaction or continuous amplification. Simultaneously, enzyme inactivation also reduces the risk of contamination of the subsequent experimental environment by the amplification products. Therefore, the coordination between reaction temperature, reaction time, and enzyme inactivation treatment ensures the high efficiency, stability, controllability, and reliability of the LAMP reaction, ultimately achieving rapid and accurate viral nucleic acid amplification detection.

[0015] According to the method for detecting equine herpesvirus 1 and 4 based on color-change visualization loop-mediated isothermal amplification of the present invention, the color indicator is SYBR Green I fluorescent dye or calcein chromogenic solution.

[0016] The above technical solution specifies that the color indicator used in this invention is either SYBR Green I fluorescent dye or calcein chromogenic solution. The choice of these two color indicators is closely related to the purpose of visual detection. They interact with DNA amplification products during or after the LAMP reaction through different mechanisms of action, producing visible color changes, thus enabling result interpretation without complex instruments. SYBR Green I fluorescent dye is an intercalating dye whose molecular structure can intercalate between base pairs of double-stranded DNA. When SYBR Green I intercalates into double-stranded DNA, its fluorescence intensity is significantly enhanced, while its fluorescence is very weak in the free state. In the LAMP reaction, as DNA amplification products accumulate, the content of double-stranded DNA in the system increases significantly. Adding SYBR Green I results in a large amount of intercalation into double-stranded DNA, emitting strong green fluorescence, making positive results easy to observe. Calcein chromogenic solution is a metal indicator; its main component, calcein, is orange-red in the free state. When it reacts with metal ions (such as zinc ions, Zn...), it... 2+ After chelation, it exhibits green fluorescence. In the LAMP reaction system, a certain amount of manganese ions (Mn) is usually added. 2+ In the reaction system, free manganese ions chelate with calcein, inhibiting its fluorescence. As the LAMP reaction proceeds, dNTPs are consumed, and pyrophosphate ions (PPi) are produced as a byproduct. PPi can bind to manganese ions, reducing the concentration of free manganese ions and releasing calcein bound to manganese ions, thus restoring its fluorescence and producing a color change. Therefore, SYBR Green I and calcein chromogenic solution utilize the principles of DNA embedding and metal ion indicator, respectively, to specifically interact with LAMP amplification products, converting the nucleic acid amplification results into visually perceptible color signals. This achieves visualized detection, simplifies the result interpretation process, and eliminates the need for complex equipment such as electrophoresis.

[0017] According to the method for detecting equine herpesvirus 1 and 4 based on color change visualization loop-mediated isothermal amplification of the present invention, when the color indicator is SYBR Green I fluorescent dye, the dye is added after the reaction in step S3. The positive reaction shows green fluorescence, and the negative reaction shows no obvious color change; or by direct observation, the solution in the positive reaction tube becomes white and turbid, while the solution in the negative reaction tube remains transparent.

[0018] The above technical solution specifically defines the method of result interpretation and color change when using SYBR Green I fluorescent dye as a color indicator. Technically, this interpretation method is closely related to the mechanism of action of SYBR Green I and the characteristics of LAMP amplification products, achieving simple and intuitive result interpretation. First, since SYBR Green I is an intercalation dye, it needs to bind to double-stranded DNA to produce strong fluorescence; therefore, it needs to be added after the LAMP reaction. Adding the dye after the reaction minimizes the potential impact of the dye on the reaction process, ensuring amplification efficiency. Second, the above solution describes two observation methods: observing fluorescence under UV light and directly observing turbidity with the naked eye. Under UV light, the positive reaction tube contains a large amount of double-stranded DNA amplification products, and SYBR Green I intercalates to emit strong green fluorescence, while the negative reaction tube contains little or no non-specific amplification, resulting in weak or no fluorescence and no obvious color change. This difference in fluorescence signal provides a highly sensitive visual interpretation method. Furthermore, a significant characteristic of the LAMP reaction is the production of a large amount of DNA amplification products, including stem-loop structures and DNA fragments of varying lengths, forming a visible white turbid precipitate. In the positive reaction tube, the accumulation of a large amount of amplification products causes the solution to become white and turbid, while the solution in the negative reaction tube remains clear. This difference in turbidity provides a simple basis for interpretation by direct visual observation, allowing for rapid result determination even without auxiliary equipment such as UV lamps. Therefore, observing green fluorescence under a UV lamp and directly observing white turbidity complement each other, fully utilizing the characteristics of SYBR Green I and the features of LAMP amplification products, achieving sensitive, intuitive, and visual result interpretation without the need for complex equipment. Experimental data (e.g.) Figure 6 The SYBRGreen I visualization of LAMP results (with positive samples on the left and negative samples on the right) also confirms the effectiveness of this interpretation method.

[0019] According to the method for detecting equine herpesvirus 1 and 4 based on color change visualization loop-mediated isothermal amplification of the present invention, when the color indicator is calcein chromogenic solution, the chromogenic solution is added in step S2, wherein the calcein chromogenic solution is a mixture of 0.25 mmol / L calcein and 50 mmol / L manganese chloride; the positive reaction shows a light green color, and the negative reaction shows an orange-red color; or when observed under ultraviolet light, the positive reaction tube shows strong green fluorescence, and the negative reaction tube shows weak fluorescence or no fluorescence.

[0020] The aforementioned technical solution specifically defines the component concentrations (0.25 mmol / L calcein and 50 mmol / L manganese chloride), addition steps (S2, added before the reaction), and color interpretation criteria for positive and negative results (light green / orange-red under fluorescent light, strong / weak fluorescence under ultraviolet light) when using calcein chromogenic solution as a color indicator. Technically, these specific features work together to precisely control the interaction between the color indicator and the LAMP reaction system, achieving stable, reliable, and easily interpretable visual detection results. Firstly, the calcein chromogenic solution is explicitly defined as a mixture of 0.25 mmol / L calcein and 50 mmol / L manganese chloride. This concentration ratio has been precisely optimized to balance the color development effect and the compatibility of the reaction system. The 0.25 mmol / L calcein concentration ensures sufficient intensity of the visual color change signal after the reaction, while excessively high concentrations may result in an overly dark background color, affecting interpretation. The 50 mmol / L manganese chloride concentration provides sufficient Mn... 2+ The ions ensure effective quenching of calcein fluorescence in the initial reaction state, forming a clear color baseline. Furthermore, upon the occurrence of a positive reaction, calcein is effectively released through chelation with pyrophosphate ions (PPi), producing a noticeable color change. The concentration ratio of calcein and manganese chloride is coordinated to guarantee the sensitivity and effectiveness of the chromogenic solution. Secondly, the above scheme explicitly states that the chromogenic solution is added in step S2, i.e., during the preparation of the LAMP reaction system. Pre-addition of the chromogenic solution before the reaction allows the color change to reflect the progress of the LAMP reaction in real time. This pre-addition method simplifies the operation steps, avoids the need for additional indicator addition after the reaction, and improves the convenience of detection. Simultaneously, pre-addition allows the color change to be directly correlated with the amount of amplified product generated, providing a dynamic and visual detection method. Furthermore, the above scheme details the color interpretation standards for positive and negative reactions under fluorescent and ultraviolet lamps. Under fluorescent lamps, the positive reaction appears light green, and the negative reaction appears orange-red; this color contrast is clear and easily distinguishable by the naked eye. Under ultraviolet light, the positive reaction tube exhibits strong green fluorescence, while the negative reaction tube shows weak or no fluorescence. Ultraviolet light observation further enhances the signal intensity of positive results, improving detection sensitivity and interpretation accuracy. This is especially true for weakly positive samples, where ultraviolet light observation provides a more reliable basis for judgment. The two observation methods, fluorescent and ultraviolet light, complement each other, offering flexible choices for different detection conditions and needs. Therefore, the specific component concentrations of the calcein chromogenic solution, the method of pre-reaction addition, and the clearly defined color interpretation criteria—these technical features are interconnected and synergistic, jointly constructing a stable, sensitive, and convenient visual LAMP detection system. This system enables rapid result interpretation without complex instruments and ensures the reliability and accuracy of the detection results. Experimental data (such as...) Figure 7 and Figure 9 The LAMP results for calcein visualization shown are as follows, in which Figure 7 Visualize LAMP results for calcein in EHV-1. Figure 9 The LAMP results for visualizing calcein in EHV-4 also fully validated the technical effects achieved by these technical features.

[0021] According to the method of visual loop-mediated isothermal amplification detection of equine herpesvirus 1 and 4 based on color change according to the present invention, the sample to be tested is a nasopharyngeal swab or a blood sample, and DNA extraction is performed in step S1 using a commercial DNA extraction kit or conventional methods.

[0022] The above technical solution specifies that the sample type to be tested is either a nasopharyngeal swab or a blood sample, and indicates that nucleic acid extraction in step S1 can be performed using a commercially available DNA extraction kit or a conventional method. This limitation on sample type and nucleic acid extraction method is closely related to the practical application scenarios and ease of operation of this invention, making it more practical. Nasopharyngeal swabs and blood are commonly used and representative sample types for clinical diagnosis of equine herpesvirus infection. Nasopharyngeal swabs are easy to collect and can reflect respiratory infection status; blood samples can be used to detect viremia. Choosing these two sample types allows the method of this invention to be directly applied to clinical sample testing, meeting actual diagnostic needs. Simultaneously, the above solution clearly indicates that nucleic acid extraction can be performed using a commercially available DNA extraction kit or a conventional method. Commercially available DNA extraction kits are easy to operate and have high extraction efficiency, suitable for scenarios with high requirements for detection speed and ease of operation, such as rapid on-site testing. Conventional DNA extraction methods (such as phenol-chloroform extraction, boiling methods, etc.) are lower in cost and suitable for routine laboratory testing. This flexible choice of nucleic acid extraction method allows the method of this invention to adapt to different application scenarios and laboratory conditions, improving the universality and operability of the method. The combination of limitations on sample type and nucleic acid extraction method enables the method of this invention to be effectively applied to the clinical diagnosis of equine herpesvirus types 1 and 4, and has good practicality and operability.

[0023] According to the method for detecting equine herpesvirus 1 and 4 based on color change visualization loop-mediated isothermal amplification of the present invention, the total volume of the reaction system in step S2 is 25 μL, including 2.5 μL 10×Thermopol buffer, 8 mmol / L MgSO4, 1.4 mmol / L dNTPs, 8 U Bst DNA polymerase, 0.2 μmol / L F3 and B3 primers, 1.6 μmol / L LFIP and BIP primers, 0.8 μmol / L LLoop F and Loop B primers, and template DNA.

[0024] The above technical solution specifies in detail the components and concentrations of the LAMP reaction system, including the concentrations of Thermopol buffer, MgSO4, dNTPs, Bst DNA polymerase, and various primers, and limits the total reaction volume to 25 μL. The precise ratios of these components and concentrations were determined through experimental optimization; they work synergistically to ensure the high efficiency and stability of the LAMP reaction, guaranteeing reliable detection results. First, Thermopol buffer provides a suitable reaction environment for Bst DNA polymerase, maintaining stable pH and ionic strength to ensure optimal enzyme activity. MgSO4 is an important divalent metal ion in the LAMP reaction; as a cofactor of Bst DNA polymerase, it participates in the enzyme's catalytic reaction and affects DNA stability. An optimized MgSO4 concentration of 8 mmol / L provides the best enzyme activity and amplification efficiency. dNTPs (deoxynucleoside triphosphates) are the raw materials for DNA synthesis; a concentration of 1.4 mmol / L meets the requirements for large-scale DNA synthesis in the LAMP reaction. 8 U of Bst DNA polymerase provides sufficient enzyme activity to drive the LAMP reaction rapidly and efficiently. Regarding primer concentrations, F3 and B3 primers are at low concentrations (0.2 μmol / L), FIP and BIP primers at high concentrations (1.6 μmol / L), and Loop F and Loop B primers at moderate concentrations (0.8 μmol / L). This primer concentration ratio is optimized based on the characteristics of the LAMP reaction and the primer action mechanism, balancing the reaction's specificity and efficiency. The higher concentrations of FIP and BIP, as inner primers, are beneficial for initiating the reaction and forming dumbbell-shaped structures; the lower concentrations of F3 and B3, as outer primers, improve reaction specificity; and the moderate concentration of the Loop primers accelerates the formation of loop structures and cyclic amplification. The total reaction volume is limited to 25 μL to facilitate precise reagent addition and heat conduction, ensuring the homogeneity and stability of the reaction system. The types, concentrations, and volumes of each component in the reaction system are coordinated to create an optimized LAMP reaction environment, ensuring the high efficiency, specificity, and stability of the reaction, and providing a reliable basis for subsequent visualization detection of nucleic acid amplification.

[0025] The present invention relates to a method for detecting equine herpesvirus 1 and 4 based on color-change visualization loop-mediated isothermal amplification. This method allows for direct observation of color changes to determine the detection results without the need for agarose gel electrophoresis, turbidimeters, or other special instruments and equipment. It is suitable for rapid on-site detection in primary laboratories or at the grassroots level.

[0026] The above technical solution emphasizes that the method of this invention does not require special instruments such as agarose gel electrophoresis or turbidimeters; results can be determined simply by observing color changes with the naked eye, highlighting its application value in rapid detection in grassroots laboratories or on-site settings. This technical feature is closely related to the core advantages of visualized LAMP detection, giving it significant advantages in application scenarios. Traditional nucleic acid detection methods, such as PCR and conventional LAMP, typically require equipment such as agarose gel electrophoresis or turbidimeters for result interpretation. Agarose gel electrophoresis is cumbersome and time-consuming, requiring equipment such as electrophoresis apparatus and gel imaging systems; while turbidimeters can detect the amplification process in real time, they also require specialized instruments and are relatively complex to operate. The method of this invention introduces color indicators, transforming the results of nucleic acid amplification into visible color changes, eliminating the need for the aforementioned complex instruments and allowing direct determination of positive or negative results with the naked eye. This feature of requiring no special instruments greatly simplifies the operation process, reduces detection costs, and significantly improves the convenience and accessibility of the method, making it highly suitable for application in grassroots laboratories or resource-scarce areas. Especially in rapid on-site testing scenarios, such as port inspection and quarantine, and the early stages of an epidemic, obtaining test results quickly is crucial. The method of this invention eliminates the need for bulky instruments and equipment, is simple and fast to operate, and provides intuitive result interpretation, effectively meeting the needs of rapid on-site testing and providing technical support for the rapid prevention and control of equine herpesvirus types 1 and 4. Therefore, the interrelated technical characteristics of requiring no special equipment, visual interpretation, and applicability to grassroots and on-site rapid testing give this method significant application advantages and market prospects.

[0027] The method for detecting equine herpesvirus 1 and 4 based on color-change visualization loop-mediated isothermal amplification according to the present invention has a detection limit of 9.4 × 10⁻⁶ for equine herpesvirus 1 nucleic acid. -5 The detection limit for equine herpesvirus 4 nucleic acid is 1.559 × 10 ng / μL. -4 ng / μL.

[0028] The above technical solution specifies the lowest detection limits for equine herpesvirus 1 and 4 nucleic acids. Specifically, the detection limit for equine herpesvirus 1 is 9.4 × 10⁴. -5 The detection limit for equine herpesvirus 4 nucleic acid is 1.559 × 10 ng / μL. -4 The concentration of ng / μL further quantifies the high sensitivity of the method of this invention. Experimental data (e.g.) Figure 10 , Figure 11 , Figure 12 and Figure 13 The results of the sensitivity comparison experiment are shown, in which, Figure 10 This is the sensitivity result of EHV-1 conventional PCR amplification. Figure 11This is the sensitivity result of EHV-4 conventional PCR amplification. Figure 12 The results show the sensitivity of EHV-1 LAMP amplification. Figure 13 The sensitivity results of EHV-4 LAMP amplification also confirm that the LAMP method has higher sensitivity than the conventional PCR method. This high sensitivity makes the method of this invention valuable for early diagnosis of equine herpesvirus types 1 and 4, detection of latent infections, and environmental monitoring. It can improve the detection rate of diseases, reduce the rate of missed diagnoses, and provide a more effective technical means for disease control. Therefore, high sensitivity is a significant advantage of the method of this invention compared to traditional PCR and other methods, and a key technical feature that makes it competitive in the field of animal disease diagnosis.

[0029] The method for detecting equine herpesvirus 1 and 4 based on color-change visualization loop-mediated isothermal amplification of the present invention can achieve the following technical effects:

[0030] 1. Simple and intuitive detection operation: Because it uses color indicators for visual interpretation, complex procedures such as agarose gel electrophoresis or turbidimeters are unnecessary. Detection results can be directly determined by visually observing color changes. (Reason: The color indicator interacts directly with the DNA amplification products to produce color changes, simplifying the result interpretation process and avoiding reliance on complex instruments.) Therefore, the method of this invention significantly reduces the difficulty of operation, allowing even non-professionals to quickly master the detection operation after simple training.

[0031] 2. Rapid and efficient detection speed: Loop-mediated isothermal amplification (LAMP) technology inherently features isothermal amplification and high efficiency. Combined with optimized reaction systems and conditions, the entire detection process (from reaction to result interpretation) can be completed within 1 hour. (Reason: LAMP technology performs DNA amplification under isothermal conditions, eliminating the need for temperature cycling like PCR, resulting in high amplification efficiency and fast reaction speed. The optimized reaction system further enhances reaction efficiency.) Compared to traditional virus isolation and identification and PCR methods, detection time is significantly reduced, enabling rapid diagnosis.

[0032] 3. High Sensitivity Detection Capability: The inherent high sensitivity of LAMP technology enables this method to detect samples with low viral loads. Experimental data demonstrates that the LAMP method established in this invention has a significantly higher limit of detection (LOD) for EHV-1 and EHV-4 nucleic acids than conventional PCR methods. (Reason: LAMP technology utilizes multiple primer sets for synergistic action, enabling efficient amplification of target DNA. Even with extremely low viral nucleic acid content in the sample, detectable amplification products can still be generated.) High sensitivity ensures detection accuracy and reduces the false negative rate, making it particularly suitable for detection in early infection or low viral load situations.

[0033] 4. Highly Specific Detection Results: The LAMP primer set is designed based on the conserved sequences of the specific gB genes of equine herpesviruses 1 and 4, ensuring high detection specificity. Experimental data show that this method has no cross-reactivity with other equine viruses and animal viruses. (Reason: The specific primer design ensures that the LAMP amplification reaction only amplifies the specific gene sequences of equine herpesviruses 1 and 4, avoiding false positive results caused by non-specific amplification.) High specificity guarantees the reliability of the detection results, avoids cross-reactivity with other viruses, and improves diagnostic accuracy.

[0034] 5. Low equipment dependence and low cost: The LAMP reaction is carried out under isothermal conditions, eliminating the need for expensive equipment such as PCR instruments; only a constant-temperature water bath or other simple isothermal equipment is required. Results are interpreted by visually observing color changes, eliminating the need for electrophoresis apparatus, gel imaging systems, or turbidimeters. (Reason: The isothermal amplification characteristic of LAMP technology reduces the need for sophisticated instruments; the visual detection method avoids the need for complex result interpretation equipment.) This significantly reduces detection costs, making this method easier to popularize and apply, especially in resource-constrained grassroots laboratories and field environments.

[0035] 6. Strong Field Applicability: The method's ease of operation, speed, efficiency, lack of special equipment requirements, and visualized results make it ideal for grassroots laboratories, port inspection and quarantine departments, and rapid on-site testing. It is particularly suitable for the rapid inspection and quarantine of imported racehorses, providing a convenient and reliable technical means for disease control and port quarantine. (Reason: The combined effect of the above-mentioned technical benefits allows this method to eliminate reliance on specialized laboratories and complex equipment, meeting the needs of rapid on-site testing and possessing strong practicality and promotional value.)

[0036] In summary, the technical solution of this invention overcomes the shortcomings of existing technologies by combining loop-mediated isothermal amplification technology with color indicator visualization detection methods, and realizes a simple, rapid, sensitive, specific, economical, and field-applicable method for detecting equine herpesvirus types 1 and 4, which has significant technical effects and application value. Attached Figure Description

[0037] 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.

[0038] Figure 1 This is a table showing the EHV-1 LAMP primer sequences and their positions according to the present invention;

[0039] Figure 2 This is a table showing the EHV-4LAMP primer sequences and their positions according to the present invention;

[0040] Figure 3 This is a diagram of the optimal reaction system for the EHV-1 visualization LAMP of the present invention;

[0041] Figure 4 This is a diagram of the optimal reaction system for the EHV-4 visualization LAMP of the present invention;

[0042] Figure 5 This is a flowchart of the method for detecting equine herpesvirus types 1 and 4 based on color-changing visualization loop-mediated isothermal amplification according to the present invention.

[0043] Figure 6 This is a visualization of the SYBR GREEN I LAMP detection results of EHV-1 of the present invention (comparison of positive and negative samples);

[0044] Figure 7 This is a visualization of the LAMP detection results of EHV-1 for calcein in this invention (comparison of positive and negative samples under sunlight and ultraviolet light);

[0045] Figure 8 This is a visualization of the SYBR GREEN I LAMP detection results of EHV-4 according to the present invention (comparison of positive and negative samples);

[0046] Figure 9 This is a visualization of the LAMP detection results of EHV-4 calcein in this invention (comparison of positive and negative samples under sunlight and ultraviolet light);

[0047] Figure 10 This is a graph showing the sensitivity results of EHV-1 conventional PCR amplification according to the present invention;

[0048] Figure 11 This is a graph showing the sensitivity results of EHV-4 conventional PCR amplification according to the present invention;

[0049] Figure 12 This is a graph showing the sensitivity results of EHV-1 LAMP amplification according to the present invention;

[0050] Figure 13 This is a graph showing the sensitivity results of EHV-4LAMP amplification according to the present invention;

[0051] Figure 14 This is a graph showing the EHV-1 LAMP specific detection results of the present invention (compared with other viral nucleic acids);

[0052] Figure 15 This is a diagram of the EHV-4LAMP specific detection results of the present invention (compared with other viral nucleic acids);

[0053] Figure 16 This is a graph showing the repeatability test results of the EHV-1LAMP of the present invention;

[0054] Figure 17 This is a graph showing the repeatability test results of the EHV-4LAMP of this invention. Detailed Implementation

[0055] The following is combined Figures 1 to 17 Embodiments of the present invention are described in detail below, examples of which are illustrated 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.

[0056] This embodiment aims to describe in detail the specific operation steps, reaction system, result interpretation, and technical effects of visual LAMP detection of equine herpesvirus type 1 (EHV-1) and type 4 (EHV-4) using calcein chromogenic solution as a color indicator.

[0057] (I) Materials and Reagents

[0058] 1. Template DNA:

[0059] EHV-1 reference strain DNA (Catalog No.: ATCC VR-743);

[0060] EHV-4 reference strain DNA (Catalog No.: ATCC VR-772);

[0061] Negative control DNA: DNA extracted from the blood of healthy horses that does not contain equine herpesvirus nucleic acid;

[0062] Other viral nucleic acids: Equine arteritis virus (EAV), porcine pseudorabies virus (PRV), bovine rhinotracheitis virus (IBRV), H7N7 subtype equine influenza virus (H7N7 EIV), and H3N8 subtype equine influenza virus (H3N8 EIV) nucleic acids, used for specific experiments.

[0063] 2. Primers: based on Figure 1 and Figure 2 Based on the sequences in Tables 1 and 2, LAMP primers were synthesized by Shanghai Bioengineering Co., Ltd.

[0064] EHV-1 LAMP primers: F3, B3, FIP, BIP, Loop F, Loop B (sequences see...) Figure 1 (Table 1);

[0065] EHV-4LAMP primers: F3, B3, FIP, BIP, Loop F, Loop B (sequences see...) Figure 2 Table 2).

[0066] 3. Enzymes and reagents:

[0067] 10×Thermopol buffer (New England Biolabs, Ipswich, MA, USA);

[0068] Magnesium sulfate (MgSO4) (Sigma-Aldrich, St. Louis, MO, USA);

[0069] dNTPs mixture (Promega, Madison, WI, USA);

[0070] Bst DNA polymerase (large fragment) (New England Biolabs, Ipswich, MA, USA);

[0071] Calcein colorimetric solution: a mixture of 0.25 mmol / L calcein (Sigma-Aldrich, St. Louis, MO, USA) and 50 mmol / L manganese chloride (MnCl2) (Sigma-Aldrich, St. Louis, MO, USA), prepared immediately before use;

[0072] DNA Extraction Kit: Blood DNA Extraction Kit from Tiangen Biotech (Beijing) Co., Ltd.

[0073] DL2000 DNA Marker (TaKaRa, Dalian, China).

[0074] (II) Experimental Methods

[0075] 1. Sample DNA extraction (step S1):

[0076] Sample preparation: Collect nasopharyngeal swabs or blood samples from racehorses suspected of being infected with equine herpesvirus.

[0077] DNA Extraction: Using the blood DNA extraction kit from Tiangen Biotech (Beijing) Co., Ltd., DNA was extracted from nasopharyngeal swab washings and blood samples according to the kit instructions. The extracted DNA was dissolved in 50 μL of elution buffer and stored at -20°C for later use.

[0078] Quality control: The concentration and purity of the extracted DNA were measured using a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA) to ensure that the DNA quality met the requirements of the subsequent LAMP reaction.

[0079] 2. Preparation of the LAMP reaction system (step S2):

[0080] HV-1LAMP reaction system (25μL): according to Figure 3 Table 3 shows the preparation of the EHV-1 visualization LAMP reaction system. The specific components and final concentrations are as follows: 2.5 μL 10×Thermopol buffer (1×), 8 mmol / L MgSO4, 1.4 mmol / L dNTPs, 8 U Bst DNA polymerase, 0.2 μmol / L F3 and B3 primers, 1.6 μmol / L FIP and BIP primers, 0.8 μmol / L Loop F and Loop B primers, 1 μL calcein chromogenic solution, and 2 μL template DNA. Finally, nuclease-free water is added to a final volume of 25 μL.

[0081] EHV-4LAMP reaction system (25μL): according to Figure 4 The EHV-4 visualization LAMP reaction system was prepared as shown in Table 4. The specific components and final concentrations were the same as those in the EHV-1 system, only the EHV-4 specific LAMP primer set needed to be replaced.

[0082] Control settings: For each batch of testing, a positive control (EHV-1 or EHV-4 reference strain DNA) and a negative control (DNA from a healthy horse without viral nucleic acid or water without nuclease) are set up.

[0083] 3. LAMP amplification reaction (step S3):

[0084] Place the prepared reaction system into a PCR tube and put it into a constant temperature water bath (Shanghai Yiheng Scientific Instruments Co., Ltd.) or a constant temperature metal bath (Hangzhou Mikai Instruments Co., Ltd.).

[0085] The reaction conditions were set as follows: constant temperature reaction at 63℃ for 40 minutes.

[0086] After the reaction is complete, remove the reaction tube and immediately place it in an 80°C constant temperature water bath for 2 minutes to inactivate the enzyme and terminate the reaction.

[0087] Technical benefits: Under constant temperature conditions of 63℃, Bst DNA polymerase exhibits strand displacement activity and a high elongation rate, enabling efficient amplification of target genes. A reaction time of 40 minutes is sufficient to produce visible amplification products, achieving rapid detection. The enzyme inactivation step of heating at 80℃ for 2 minutes effectively terminates the reaction, preventing non-specific amplification and ensuring the accuracy of results.

[0088] 4. Result Interpretation (Step S4):

[0089] Visual observation: Under natural light or fluorescent light, directly observe the color change of the solution in the reaction tube. The solution in the positive reaction tube will appear light green, and the solution in the negative reaction tube will appear orange-red (e.g., ...). Figure 7 Experimental results A on the left and Figure 9 The experimental result A on the left is shown.

[0090] UV lamp observation (optional): Place the reaction tube under a UV lamp (wavelength 365nm) and observe the fluorescence changes. Positive reaction tubes show strong green fluorescence, while negative reaction tubes show weak fluorescence or no fluorescence (e.g., ...). Figure 7 Experimental results B on the right and Figure 9 The experimental result on the right is shown in B).

[0091] Result judgment criteria:

[0092] Positive: The solution in the reaction tube shows a light green or green fluorescence, indicating a positive result for equine herpesvirus nucleic acid;

[0093] Negative: The solution in the reaction tube is orange-red or has weak or no fluorescence, indicating that the equine herpesvirus nucleic acid is negative;

[0094] The negative control tube should be orange-red or non-fluorescent, and the positive control tube should be light green or green fluorescent.

[0095] Technical Effects: In the reaction system, when calcein binds to manganese ions, the fluorescence is quenched, and the solution turns orange-red. When the LAMP reaction occurs and a large number of pyrophosphate ions are generated, these pyrophosphate ions preferentially bind to manganese ions, releasing free calcein. This free calcein can then react with divalent metal ions (such as Mg²⁺) in the reaction system. 2+ The combination of these elements produces a strong green fluorescence, which appears light green to the naked eye, thus enabling visual detection. This method requires no instrument assistance; results can be interpreted with the naked eye, making it simple and quick to operate.

[0096] (III) Experimental Results and Analysis

[0097] 1. Visualize LAMP detection results:

[0098] EHV-1 detection ( Figure 6 and Figure 7 LAMP reaction was performed using EHV-1 reference strain DNA as a template, with SYBR Green I dye (added after the reaction) and calcein chromogenic solution (added before the reaction) used as indicators. Figure 6 and Figure 7 As shown, the positive sample reaction tube exhibits green fluorescence or light green / white turbidity, while the negative sample shows no significant color change.

[0099] EHV-4 detection ( Figure 8and Figure 9 LAMP reaction was performed using EHV-4 reference strain DNA as a template, with SYBR Green I dye (added after the reaction) and calcein chromogenic solution (added before the reaction) used as indicators. Figure 8 and Figure 9 As shown, the positive sample reaction tube exhibits green fluorescence or light green / white turbidity, while the negative sample shows no significant color change.

[0100] Results Analysis: Experimental results show that the visual LAMP method established in this invention can effectively detect EHV-1 and EHV-4 using SYBR Green I dye or calcein chromogenic solution as indicators, and the results are intuitive and easy to interpret. The color changes are obvious, making it easy to distinguish between positive and negative results.

[0101] 2. Sensitivity analysis (combined with...) Figure 10 , Figure 11 , Figure 12 and Figure 13 (as shown)

[0102] The DNA of EHV-1 and EHV-4 reference strains were serially diluted 10-fold and used as templates for conventional PCR and LAMP reactions.

[0103] The results are as follows Figure 10 , Figure 11 , Figure 12 and Figure 13 As shown, the limit of detection for EHV-1 LAMP is 9.4 × 10⁻⁶. -5 ng / μL ( Figure 12 The limit of detection for EHV-4LAMP was 1.559 × 10⁻⁶. -4 ng / μL ( Figure 13 ).

[0104] PCR amplification results using primers S and A of EHV-4 PCR: Specific bands appeared in samples 1 to 5, but no target band was observed in sample 6 (see...). Figure 11 The sensitivity of PCR amplification is 1.559 × 10⁻⁶. -3 ng / μL. Furthermore, typical diffuse bands appeared in bands 1 through 6 of the LAMP amplification products (see...). Figure 11 This shows that the sensitivity is 10 times higher than that of PCR, reaching 9.4 × 10⁻⁶. -5 ng / μL.

[0105] Typical diffuse bands were observed in LAMP amplification products from band 1 to band 6. Figure 13 This shows that the sensitivity is 10 times higher than that of PCR, reaching 1.559 × 10⁻⁶. -4 ng / μL.

[0106] The sensitivity results compared to conventional PCR methods (EHV-1 PCR sensitivity is 9.4 × 10⁻⁶) were obtained. -4 ng / μL ( Figure 10 The sensitivity of EHV-4 PCR was 1.559 × 10⁻⁶. -3 ng / μL ( Figure 11 Compared to the previous method, the LAMP method established in this invention has an improved sensitivity of approximately 10 times.

[0107] Technical Benefits: High sensitivity is a key advantage of LAMP technology. The visualization-based LAMP method established in this invention inherits the high sensitivity of LAMP technology, enabling the detection of trace viral nucleic acids. It is suitable for detecting early infections or samples with low viral loads, improving diagnostic accuracy, and is particularly significant for early disease screening of imported racehorses.

[0108] 3. Specificity analysis (combined with...) Figure 14 and Figure 15 (as shown)

[0109] Using EHV-1, EHV-4, EAV, PRV, IBRV, H7N7 EIV, and H3N8 EIV viral nucleic acids as templates, EHV-1 and EHV-4 LAMP detection were performed.

[0110] The results are as follows Figure 14 and Figure 15 As shown, the EHV-1 LAMP method only showed positive results for the EHV-1 template, and the EHV-4 LAMP method only showed positive results for the EHV-4 template (green fluorescence or light green / white turbidity). There was no amplification reaction for other equine viruses and other animal viruses. The results were consistent with the color of the negative control tube.

[0111] Technical Effects: Primer design is crucial for the specificity of LAMP technology. This invention designs LAMP primers targeting the conserved gB gene sequences specific to EHV-1 and EHV-4, ensuring the specificity of detection. Experimental results show that this method has good specificity for EHV-1 and EHV-4, does not cross-react with other common equine viruses or animal viruses, avoids false positive results, and improves the reliability of diagnosis.

[0112] 4. Repeatability analysis (combined with...) Figure 16 and Figure 17 (As shown)

[0113] The same EHV-1 positive sample DNA was subjected to four repeated EHV-1 LAMP tests, and the same EHV-4 positive sample DNA was subjected to four repeated EHV-4 LAMP tests.

[0114] The results are as follows Figure 16 and Figure 17 As shown, the results of the four replicate experiments using the EHV-1 and EHV-4 LAMP methods were all positive, and the color changes were stable and consistent.

[0115] Technical Results: Good repeatability is a crucial indicator for ensuring the stability and reliability of a detection method. The visualization-based LAMP method established in this invention exhibits good repeatability, with consistent results for repeated detection of positive samples, demonstrating that the method is stable, reliable, and suitable for large-scale applications.

[0116] (IV) Conclusion

[0117] This embodiment details the specific implementation process of the visualization LAMP method based on calcein chromogenic solution for detecting equine herpesvirus types 1 and 4, and supplements the description with the detection results of the visualization LAMP method based on SYBR Green I dye. Experimental results fully demonstrate that the method established in this invention has the following significant technical effects:

[0118] Visual interpretation: Using color indicators (calcein chromogenic solution or SYBR Green I dye), the results can be directly interpreted by the naked eye, without the need for complex equipment such as agarose gel electrophoresis and turbidimeter, making the operation extremely simple and intuitive.

[0119] Fast and efficient: LAMP technology itself has the characteristic of rapid amplification. The entire detection process (from reaction to result interpretation) can be completed within 1 hour, which greatly shortens the detection time and improves the detection efficiency.

[0120] High sensitivity: The detection sensitivity is improved compared to the ordinary PCR method, which can detect samples with low viral load and improve the ability of early diagnosis.

[0121] High specificity: The primers are designed specifically for the gB gene of EHV-1 and EHV-4, which has high specificity and can effectively distinguish other equine viruses, avoid false positive results, and ensure the accuracy of the test results.

[0122] Highly applicable to field use: It is easy to operate and requires no special equipment, making it very suitable for grassroots laboratories, port inspection and quarantine departments, or rapid on-site testing applications. It is especially suitable for rapid inspection and quarantine of imported racehorses, providing strong technical support for the prevention and control of equine herpesvirus disease.

[0123] It should also be noted that this embodiment is only a preferred embodiment of the present invention. In practical applications, calcein colorimetric solution or SYBR Green I fluorescent dye can be selected as color indicator according to specific needs, or the reaction system, reaction conditions, etc. can be appropriately optimized within the scope of the claims, all of which fall within the protection scope of the present invention.

[0124] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, 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 method for detecting equine herpesvirus 1 and 4 based on color-change-based visual loop-mediated isothermal amplification, characterized in that, Includes the following steps: S1. Extract nucleic acid from the sample to be tested; S2. Prepare a reaction system containing a LAMP primer set designed for equine herpesvirus, DNA polymerase, and color indicator; S3. The reaction system is subjected to an amplification reaction under isothermal conditions; S4. Determine the test results by observing the color change of the reaction system; Specifically, in step 2, the LAMP primer set binds to the specific gene sequence of equine herpesvirus, and in step 3, isothermal amplification generates a large amount of DNA amplification products. In step 4, a color indicator interacts with the DNA amplification products to produce visible color changes, thereby enabling the visual detection of equine herpesvirus.

2. The method for detecting equine herpesvirus 1 and 4 based on color-change-based visualized loop-mediated isothermal amplification according to claim 1, characterized in that, The LAMP primer set includes primers F3, B3, FIP, BIP, Loop F, and Loop B, which specifically target the gB gene of equine herpesvirus type 1 and / or type 4.

3. The method for detecting equine herpesvirus types 1 and 4 based on color-change-based visual loop-mediated isothermal amplification according to claim 2, characterized in that, The isothermal conditions in step S3 are 60-65℃ for 30-50 minutes, and after the reaction is completed, enzyme inactivation treatment is performed by heating at 80℃ for 2 minutes.

4. The method for detecting equine herpesvirus 1 and 4 based on color-change-based visualized loop-mediated isothermal amplification according to claim 1, characterized in that, The color indicator is SYBR Green I fluorescent dye or calcein colorimetric solution.

5. The method for detecting equine herpesvirus 1 and 4 based on color-change-based visualized loop-mediated isothermal amplification according to claim 4, characterized in that, When the color indicator is SYBR Green I fluorescent dye, the dye is added after the reaction in step S3. The positive reaction shows green fluorescence, while the negative reaction shows no obvious color change; or, by direct visual observation, the solution in the positive reaction tube becomes white and turbid, while the solution in the negative reaction tube remains transparent.

6. The method for detecting equine herpesvirus 1 and 4 based on color-change-based visualized loop-mediated isothermal amplification according to claim 4, characterized in that, When the color indicator is calcein colorimetric solution, the colorimetric solution is added in step S2, wherein the calcein colorimetric solution is a mixture of 0.25 mmol / L calcein and 50 mmol / L manganese chloride; the positive reaction shows a light green color, and the negative reaction shows an orange-red color; or when observed under ultraviolet light, the positive reaction tube shows strong green fluorescence, and the negative reaction tube shows weak fluorescence or no fluorescence.

7. The method for detecting equine herpesvirus 1 and 4 based on color-change-based visual loop-mediated isothermal amplification according to claim 2, characterized in that, The sample to be tested is a nasopharyngeal swab or a blood sample, and DNA extraction is performed in step S1 using a commercial DNA extraction kit or conventional methods.

8. The method for detecting equine herpesvirus 1 and 4 based on color-change-based visual loop-mediated isothermal amplification according to claim 1, characterized in that, The total volume of the reaction system in step S2 is 25 μL, including 2.5 μL of 10×Thermopol buffer, 8 mmol / L MgSO4, 1.4 mmol / L dNTPs, 8 U Bst DNA polymerase, 0.2 μmol / L F3 and B3 primers, 1.6 μmol / L FIP and BIP primers, 0.8 μmol / L Loop F and Loop B primers, and template DNA.

9. The method for detecting equine herpesvirus 1 and 4 based on color-change-based visualized loop-mediated isothermal amplification according to claim 1, characterized in that, The method allows for the determination of test results by directly observing color changes with the naked eye, without the need for agarose gel electrophoresis, turbidimeters, or other special instruments and equipment. It is suitable for rapid on-site testing in grassroots laboratories.

10. The method for detecting equine herpesvirus 1 and 4 based on color-change-based visualized loop-mediated isothermal amplification according to claim 1, characterized in that, The detection limit for equine herpesvirus type 1 nucleic acid is 9.4 × 10⁻⁶. -5 The detection limit for equine herpesvirus 4 nucleic acid is 1.559 × 10 ng / μL. -4 ng / μL.

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