Loop-mediated isothermal amplification method for rapid inspection and quarantine of imported racer horse herpes viruses 1 and 4

Abstract

The invention relates to a loop-mediated isothermal amplification method for rapid inspection and quarantine of horse herpes viruses 1 and 4 of imported racing horses. The loop-mediated isothermal amplification method comprises the following steps: S1, obtaining a biological sample of the imported racing horses; s2, extracting a nucleic acid template from the biological sample; s3, mixing the nucleic acid template with a reaction system containing nucleic acid polymerase and a specific loop-mediated isothermal amplification primer group aiming at the equine herpes virus type 1 or type 4; s4, performing a nucleic acid amplification reaction under a constant temperature condition; and S5, detecting an amplification product, and judging whether the imported racing horse is infected with the horse herpes virus type 1 or type 4 or not. The loop-mediated isothermal amplification method for rapid inspection and quarantine of the imported racing horse herpes viruses type 1 and type 4 can effectively meet the requirements of rapid customs clearance and epidemic disease prevention and control of imported racing horses.

Description

Technical Field

[0001] This invention relates to the field of molecular biological diagnostics, and more particularly to a loop-mediated isothermal amplification method for rapid detection and quarantine of herpesvirus types 1 and 4 in imported racehorses. Background Technology

[0002] Equine herpesvirus 1 (EHV-1) and equine herpesvirus 4 (EHV-4) are the main pathogens causing equine rhinopneumonitis (ER), which is widespread globally and poses a serious threat to the horse industry, especially the high-value racehorse industry. Equine rhinopneumonitis not only causes respiratory diseases and viral abortions in horses, but can also lead to neurological disorders in severe cases, resulting in significant economic losses. The World Organisation for Animal Health (OIE) has listed equine rhinopneumonitis as a notifiable disease, and my country has also listed it as a Class II animal infectious disease and a key quarantine target for imported and exported equines. Therefore, strict inspection and quarantine of imported racehorses to prevent the introduction of foreign diseases is crucial to ensuring the safety of the domestic horse industry.

[0003] Currently, the commonly used detection methods for equine herpesvirus types 1 and 4 mainly include virus isolation and identification, serological methods, and molecular biological methods. These methods play an important role in disease diagnosis and monitoring, but each has certain limitations, especially in the scenario of rapid inspection and quarantine of imported racehorses, where their shortcomings are particularly prominent.

[0004] Virus isolation and identification is the "gold standard" for diagnosing viral infections. Its principle involves amplifying the virus through cell culture followed by identification. The advantage of this method is that it can obtain live viruses, providing a foundation for virological research. However, virus isolation and identification has significant drawbacks. First, it is time-consuming, typically requiring several days or even weeks to complete, making it difficult to meet the timeliness requirements of rapid quarantine at ports of entry. Second, it is cumbersome, requiring complex cell culture procedures and strict aseptic conditions, resulting in a high technical threshold. Third, it requires specific cell culture conditions and P2 / P3 laboratory facilities, leading to substantial investment in equipment and space. Fourth, there is a risk of virus spread, necessitating high biosafety requirements. These limitations make virus isolation and identification difficult to apply to rapid quarantine scenarios at ports of entry for racing horses.

[0005] Serological methods primarily assist in diagnosis by detecting antibodies against EHV-1 / 4 in equine serum. Commonly used serological methods include neutralization tests and enzyme-linked immunosorbent assays (ELISA). The advantages of serological methods are their relative simplicity and ability to screen large-scale samples. However, serological methods also have significant limitations. First, there is a window period; in the early stages of infection, antibodies may not yet be produced or their titers are low, potentially leading to false negatives and failing to directly reflect the current viral infection status. Second, traditional serological methods may exhibit cross-reactivity; for example, neutralization tests and some early ELISA methods struggle to effectively distinguish between EHV-1 and EHV-4 infections, affecting diagnostic specificity. Third, they are time-consuming; although shorter than virus isolation and identification, neutralization tests typically take several days, and ELISA takes several hours, which remains inefficient in the fast-paced environment of border crossings.

[0006] Molecular biology methods, particularly polymerase chain reaction (PCR)-based techniques, are widely used in virus detection due to their high sensitivity and specificity. Conventional PCR and real-time fluorescence PCR (qPCR) are commonly used methods for EHV-1 / 4 molecular detection. The advantages of PCR technology include high sensitivity and specificity, and shorter detection time compared to virus isolation and serological methods. However, conventional PCR and qPCR still have certain limitations. First, detection still requires several hours to complete, including nucleic acid extraction, PCR amplification, electrophoresis analysis (conventional PCR) or data analysis (qPCR), indicating room for improvement in time efficiency. Second, they rely on sophisticated PCR amplification instruments and professional operators, resulting in high equipment costs and relatively complex operations, limiting rapid detection in resource-constrained environments such as field sites or ports of entry. Third, PCR reactions require temperature cycling, consuming significant energy and hindering the development of portable field testing devices.

[0007] With the increasing frequency of international horse racing exchanges, the timeliness requirements for the inspection and quarantine of imported racehorses are becoming increasingly stringent. Existing technologies are insufficient to fully meet the demands for "fast, accurate, and convenient" inspection and quarantine of imported racehorses. There is an urgent need for a faster, simpler, lower-cost, and equally reliable testing method to shorten the quarantine cycle, improve customs clearance efficiency, and promptly detect and control potential disease risks. Summary of the Invention

[0008] To address the technical problems existing in the prior art, this invention provides a loop-mediated isothermal amplification method for rapid inspection and quarantine of equine herpesvirus types 1 and 4 in imported racehorses, which can effectively meet the needs of rapid customs clearance and disease prevention and control for imported racehorses.

[0009] This invention discloses a loop-mediated isothermal amplification method for rapid detection and quarantine of equine herpesvirus types 1 and 4 in imported racehorses, comprising the following steps:

[0010] Step S1: Obtaining biological samples from imported racehorses: This step is the first step in the testing process and aims to obtain biological samples containing possible equine herpesvirus type 1 or 4 nucleic acid. According to the technical disclosure, the preferred biological sample types are nasopharyngeal swabs or blood, as these two sample types are easy and minimally invasive to collect at ports of entry or quarantine facilities. Nasopharyngeal swabs can directly collect respiratory secretions, while blood can reflect the systemic viral infection status; both are suitable sample sources for detecting equine herpesvirus infection.

[0011] Step S2: Extracting nucleic acid template from the biological sample: This step aims to extract viral nucleic acid from the biological sample collected in step S1, serving as a template for subsequent LAMP amplification. The preparation of the nucleic acid template is a crucial pretreatment step in molecular detection technology. To meet the needs of rapid inspection and quarantine, this step preferably employs a rapid nucleic acid extraction method, such as using a commercially available viral DNA / RNA extraction kit. This method can efficiently extract viral nucleic acid from the biological sample in a short time, obtaining a high-purity nucleic acid template, laying the foundation for subsequent high-sensitivity LAMP detection.

[0012] Step S3: Mix the nucleic acid template with a reaction system containing a nucleic acid polymerase and a specific loop-mediated isothermal amplification primer set targeting equine herpesvirus type 1 or 4. This step constructs the LAMP reaction system, which is the core step in achieving isothermal nucleic acid amplification. The nucleic acid polymerase is the key enzyme driving the LAMP reaction and must possess strand displacement activity, such as BstDNA polymerase. The specific loop-mediated isothermal amplification primer set is a crucial component ensuring the specific amplification of the target viral nucleic acid by the LAMP reaction. This primer set is designed for the specific gene target sequence of equine herpesvirus type 1 or 4, preferably targeting a conserved and specific region of the glycoprotein B gene (gB gene). According to the technical disclosure, this primer set contains outer primers (F3, B3) and inner primers (FIP, BIP), and more preferably, may contain loop primers (LoopF, LoopB) to accelerate the reaction. Primer sequences must be carefully designed and screened to ensure high specificity for EHV-1 and EHV-4, and to work efficiently and synergistically with each other and with reaction system components such as nucleic acid polymerase and reaction buffer. The reaction system must also contain dNTPs, Auxiliary reagents such as betaine are used to optimize enzyme activity and amplification efficiency. The nucleic acid template prepared in step S2 is mixed with the above reaction system to ensure sufficient contact between the template and the reaction components, preparing for the subsequent isothermal amplification reaction.

[0013] Step S4: Nucleic acid amplification reaction under isothermal conditions: This step is the core step in nucleic acid amplification using LAMP technology, demonstrating the isothermal amplification characteristics of this invention. Unlike the thermocyclic temperature-dependent amplification of traditional PCR technology, the LAMP reaction is carried out at a constant temperature, typically around 63°C. This temperature condition is suitable for the efficient catalysis of Bst DNA polymerase and can maintain the specific binding of primers and templates, as well as the smooth progress of strand displacement reactions. According to the optimization results in the technical disclosure, the preferred amplification reaction time is approximately 40 minutes, which is sufficient to achieve efficient amplification of the target viral nucleic acid. Isothermal amplification avoids the need for precision PCR instruments, simplifies the operation process, reduces equipment costs, and is more conducive to rapid application in the field or at ports of entry.

[0014] Step S5: Detect the amplification products to determine whether the imported racehorses are infected with equine herpesvirus type 1 or 4: This step aims to detect and interpret the products of the LAMP amplification reaction in step S4, thereby determining whether the imported racehorses are infected with equine herpesvirus type 1 or 4. Due to the high amplification efficiency and large product quantity of the LAMP reaction, various simple and rapid methods can be used for detection. Preferred detection methods include turbidity observation, fluorescent dye visualization (such as using SYBR Green I or calcein), and real-time fluorescence monitoring. Turbidity observation utilizes the characteristic of magnesium pyrophosphate precipitation produced by LAMP amplification, allowing for visual or turbidimetric judgment of the results; fluorescent dye visualization utilizes the fluorescence signal generated by the binding of dye to double-stranded DNA, enabling rapid interpretation by visual inspection or a fluorescence detector; real-time fluorescence monitoring dynamically monitors changes in fluorescence signals during the amplification process, providing more accurate quantitative or semi-quantitative results. Based on the detection results, it is possible to quickly determine whether the imported racehorses are infected with equine herpesvirus, providing a basis for subsequent quarantine decisions.

[0015] According to the loop-mediated isothermal amplification method for rapid inspection and quarantine of equine herpesvirus types 1 and 4 in imported racehorses, the biological sample is a nasopharyngeal swab or a blood sample.

[0016] The above technical solution limits the biological samples to nasopharyngeal swabs or blood samples, and its technical advantages are: 1. Improved ease of collection and operability: Nasopharyngeal swabs and blood are relatively easy sample types to collect from live racehorses. The collection process causes less stress to the animals, and veterinary procedures are relatively simple. Especially under environmental constraints such as ports of entry or quarantine facilities, the collection of these two types of samples has higher operability, reducing the difficulty and time cost of sample acquisition, and laying the foundation for subsequent rapid inspection and quarantine. 2. Improved viral enrichment and detection accuracy: Equine herpesvirus types 1 and 4 mainly cause respiratory diseases and viral abortions. The virus is distributed in both respiratory secretions (nasopharyngeal swabs) and the circulatory system (blood). Therefore, nasopharyngeal swabs can directly capture the virus at the site of respiratory infection, while blood samples can reflect the status of systemic viral infection. These two sample types improve the enrichment of viral nucleic acid templates, thereby improving the sensitivity and accuracy of the LAMP detection method and reducing the risk of false negative results. 3. Non-invasive or minimally invasive: Compared to invasive sampling methods such as tissue biopsy, nasopharyngeal swabs are non-invasive sampling, and blood collection is also a minimally invasive procedure, which minimizes the impact on the health and welfare of racehorses and meets the requirements of animal ethics and welfare, and is especially suitable for precious imported racehorses.

[0017] According to the loop-mediated isothermal amplification method for rapid detection and quarantine of equine herpesvirus types 1 and 4 in imported racehorses, the reaction system in step S3 comprises: Bst DNA polymerase, deoxyribonucleoside triphosphate, magnesium ions, betaine, and the specific loop-mediated isothermal amplification primer set. Further, in the reaction system, the concentration of magnesium ions is 2.0-4.0 mmol / L, the concentration of betaine is 0.2-0.8 mol / L, and the concentration of deoxyribonucleoside triphosphate is 1.5-3.5 mmol / L.

[0018] The above technical solutions collectively define the key components and their concentration ranges in the LAMP reaction system. This optimized reaction system is the core for achieving rapid, efficient, and sensitive amplification, and its technical effects are reflected in the following aspects: 1. Highly efficient strand displacement ability of Bst DNA polymerase: The present invention specifies the use of "Bst DNA polymerase". Bst DNA polymerase has significant strand displacement activity, which is the key enzymatic basis for the efficient amplification of LAMP technology under isothermal conditions. The strand displacement characteristics of this enzyme can effectively displace the synthesized DNA strand, thereby driving multiple primer binding and cyclic amplification, achieving exponential nucleic acid amplification efficiency, and significantly shortening the reaction time. This is a key technical guarantee for achieving rapid inspection and quarantine. 2. Magnesium ions ( Enzyme activity optimization and product precipitation: The present invention defines the concentration range of "magnesium ions" and "2.0-4.0 mmol / L". Magnesium ions are an essential cofactor for Bst DNA polymerase to exert its enzyme activity. A suitable magnesium ion concentration ensures optimal enzyme activity, improves the catalytic efficiency and fidelity of DNA polymerase, and thus accelerates the LAMP reaction. Furthermore, the LAMP reaction produces a large amount of pyrophosphate byproducts. Magnesium ions can combine with pyrophosphate to form a white magnesium pyrophosphate precipitate. This precipitation phenomenon itself becomes a visual way to interpret positive results, simplifying the result detection steps. The concentration range defined in the present invention is the optimal concentration range for enzyme activity determined through optimization experiments, while also taking into account the visualization effect of magnesium pyrophosphate precipitation. 3. Betaine enhances amplification efficiency and specificity: The present invention defines the concentration range of "betaine" and "0.2-0.8 mol / L". Betaine, as a chemical chaperone, can reduce the stability of DNA's higher-order structure and decrease amplification inhibition caused by high GC content or complex secondary structures, thereby improving the amplification efficiency and stability of LAMP reactions on complex templates. This effect is particularly pronounced when processing complex nucleic acid templates from biological samples. Furthermore, betaine can also improve the specificity of primer binding to target sequences to a certain extent, reducing non-specific amplification and further enhancing the accuracy of detection results. The betaine concentration range specified in this invention has been experimentally optimized to ensure amplification efficiency while avoiding potential enzyme activity inhibition or other negative effects from excessively high concentrations. 4. Deoxynucleoside triphosphates (dNTPs) ensure amplification raw material supply: This invention specifies the concentration range of "deoxynucleoside triphosphates" and their concentration range of "1.5-3.5 mmol / L". dNTPs are fundamental raw materials for DNA synthesis, and a sufficient and appropriate supply of dNTPs is essential for ensuring the continuous and efficient execution of the LAMP reaction. The dNTP concentration range defined by the scheme of this invention is a balance point determined through optimized experiments, which can meet the amplification requirements while avoiding enzyme activity inhibition or mismatch incorporation that may be caused by excessively high concentrations of dNTPs. In summary, the optimized reaction system defined by the above schemes, through the synergistic effect between enzymes, ions, chemical additives, and substrates, constructs a highly efficient, stable, and specific LAMP reaction environment, maximizing nucleic acid amplification efficiency and detection performance, and providing key technical support for achieving rapid inspection and quarantine.

[0019] According to the present invention, a loop-mediated isothermal amplification method for rapid detection and quarantine of equine herpesvirus 1 and 4 in imported racehorses is provided. The specific loop-mediated isothermal amplification primer set is designed based on the glycoprotein B (gB) gene of equine herpesvirus 1 or 4. Each primer set includes an outer primer (F3, B3), an inner primer (FIP, BIP), and a loop primer (LoopF, LoopB), wherein the inner primer FIP contains F1c and F2 sequences, and the inner primer BIP contains B1c and B2 sequences. Further, in the primer set, the concentration of the outer primer is 0.1-0.4 μmol / L, the concentration of the inner primer is 1.2-2.0 μmol / L, the concentration of the loop primer is 0.2-0.8 μmol / L, and the concentration ratio of the inner primer to the outer primer is 6:1 to 10:1. Further, the primer sequence for equine herpesvirus 1 is:

[0020] F3: 5'-ATCAGCACGTACGTTGAACT-3'

[0021] B3: 5'-GCGATCCCCTGCATAATCAC-3'

[0022] FIP: 5'-TCTAGCAGGCCGGTGTCCTC-GGAAGACCGCGAGTTTCTG-3'

[0023] BIP: 5'-CAGCGAAATACAGCGCCGCA-ATTGTCCACGTTGACCACG-3'

[0024] LoopF: 5'-CCGCGTGTACACCTCCA-3'

[0025] LoopB: 5'-GTCTCACGCTCTCAGGT-3';

[0026] And / or,

[0027] The primer sequences for equine herpesvirus type 4 are:

[0028] F3: 5'-GGCAGAGTACCTCCGAGAT-3'

[0029] B3: 5'-CGGTCGATAGGGCAAAGGA-3'

[0030] FIP: 5'-TGGCCCCAGGAGTAGCAAACT-GCTTACCACGACGACGAAG-3'

[0031] BIP: 5'-GTCGGATGGATGCCATGGAGG-CAGACCGCGCTTCTACCT-3'

[0032] LoopF: 5'-AGGTCGAGCTCCACCTCGT-3'

[0033] LoopB: 5'-ACACATCAACCTCTGTCAACTGC-3'。

[0034] The aforementioned technical solutions collectively define the design principles, structural characteristics, concentration parameters, and specific sequences of the specific LAMP primer set. This primer set is the core element of this invention for achieving highly specific detection of equine herpesvirus 1 and 4. Its technical effects are reflected in: 1. Selection of gB gene target and type specificity: The present invention specifies that the primer set is "designed based on the glycoprotein B (gB) gene of equine herpesvirus 1 or 4." Glycoprotein B (gB) is an important membrane protein of equine herpesvirus 1 and 4, playing a crucial role in viral adsorption, invasion, and immune response. The gB gene sequence exhibits specific differences between EHV-1 and EHV-4, while also showing relative conservation within the same type of virus. Selecting the gB gene as the design target of the LAMP primers can maximize type specificity while ensuring detection sensitivity, effectively distinguishing between equine herpesvirus 1 and 4, avoiding cross-reactions with other equine herpesviruses or pathogens, and ensuring the accuracy and reliability of the detection results. This is crucial for the diagnosis and control of diseases in imported racehorses. 2. Multiple Primer Recognition and High Amplification Specificity: The scheme of this invention clearly defines each primer set as including "outer primers (F3, B3), inner primers (FIP, BIP), and loop primers (LoopF, LoopB)". One of the key features that distinguishes LAMP technology from traditional PCR is the use of up to 4-6 primers, which collectively recognize 6-8 different regions on the target gene sequence. The inner primers (FIP, BIP) each contain two sequences (F1c-F2 and B1c-B2) in both directions, forming a hairpin structure to initiate the strand displacement reaction; the outer primers (F3, B3) are responsible for the initial strand displacement; and the loop primers (LoopF, LoopB) are used to accelerate the subsequent circular amplification process. This mechanism of multiple primer synergy greatly improves the specificity of the amplification reaction. Only when all primers precisely match the target sequence can an efficient amplification reaction be initiated. This high specificity stems from the strictness of multi-site recognition, effectively reducing the possibility of false positive results and ensuring the reliability of the detection results. 3. Multiple Primer Recognition and High Amplification Specificity: The scheme of this invention clearly defines each primer set as including "outer primers (F3, B3), inner primers (FIP, BIP), and loop primers (LoopF, LoopB)". One of the key features that distinguishes LAMP technology from traditional PCR is the use of up to 4-6 primers, which collectively recognize 6-8 different regions on the target gene sequence. The inner primers (FIP, BIP) each contain two sequences (F1c-F2 and B1c-B2) in both directions, forming a hairpin structure to initiate the strand displacement reaction; the outer primers (F3, B3) are responsible for the initial strand displacement; and the loop primers (LoopF, LoopB) are used to accelerate the subsequent circular amplification process. This mechanism of synergistic action of multiple primers greatly improves the specificity of the amplification reaction.Efficient amplification can only be achieved when all primers precisely match the target sequence. This high specificity stems from the strictness of multi-site recognition, effectively reducing the possibility of false positives and ensuring the reliability of the detection results. 4. Optimization of primer concentration ratio and efficient amplification: The scheme of this invention limits the concentration of the outer primer to 0.1-0.4 μmol / L, the concentration of the inner primer to 1.2-2.0 μmol / L, the concentration of the loop primer to 0.2-0.8 μmol / L, and the concentration ratio of the inner primer to the outer primer to 6:1 to 10:1. Primer concentration and ratio are important factors affecting the efficiency and specificity of LAMP reaction. Through optimization experiments, it was determined that within the concentration range specified by the scheme of this invention, the inner and outer primers and the loop primer can work synergistically and efficiently, ensuring that the amplification speed and product yield reach the optimal balance. Maintaining an appropriate concentration ratio of inner primer to outer primer (6:1 to 10:1) ensures that the outer primer effectively initiates strand displacement in the initial stage of the reaction, the inner primer drives efficient cyclic amplification in the middle stage, and the loop primer accelerates the reaction process in the later stage, thereby achieving rapid and efficient amplification. 5. Precise Targeting of Specific Primer Sequences: This invention discloses primer sequences for equine herpesvirus type 1 and equine herpesvirus type 4. These specific sequences were obtained through precise screening and design of conserved and specific regions of the gB gene for equine herpesvirus types 1 and 4. These sequences have been experimentally verified to specifically amplify the corresponding viral targets and exhibit good amplification performance. Disclosing these sequences makes the technical solution of this invention more feasible and practical.

[0035] According to the loop-mediated isothermal amplification method for rapid inspection and quarantine of equine herpesvirus types 1 and 4 in imported racehorses of the present invention, the isothermal conditions in step S4 are 60-65°C and the reaction time is 30-60 minutes; preferably, the temperature is 63°C and the reaction time is 40 minutes.

[0036] The above technical solution specifies that in step S4, the "isotropic conditions are 60-65℃, and the reaction time is 30-60 minutes; preferably, the temperature is 63℃, and the reaction time is 40 minutes." Isothermal amplification is one of the core advantages of LAMP technology, and optimized isothermal conditions are directly related to amplification efficiency, specificity, and detection speed. Its technical effects are: 1. Simplified operation and equipment requirements: "Isotropic conditions" are a key feature that distinguishes LAMP technology from traditional PCR technology, which requires variable temperature cycling. Isothermal reactions eliminate the dependence on precision PCR instruments; amplification can be completed with only simple isothermal heating equipment (such as a water bath, isothermal metal bath, or portable isothermal amplification instrument), greatly reducing equipment costs and operational complexity, making the LAMP method easier to promote and apply in resource-limited port sites or grassroots laboratories. 2. Optimal enzyme activity temperature range of 60-65℃: The "60-65℃" specified in this invention is the optimal enzyme activity temperature range for Bst DNA polymerase. Within this temperature range, Bst DNA polymerase exhibits the highest catalytic activity, enabling efficient DNA synthesis and strand displacement, ensuring the rapid and efficient execution of the LAMP reaction. Experimental data show that LAMP amplification results are not significantly different at temperatures of 60.0℃, 61.0℃, 62.0℃, 63.0℃, 64.0℃, and 65.0℃, indicating that this method has a certain temperature tolerance and is easier to operate and control in practical applications. The preferred 63℃ is the midpoint of this temperature range, exhibiting good stability and versatility. 3. Rapid reaction time of 30-60 minutes: The "reaction time of 30-60 minutes; preferably 40 minutes" specified in this invention reflects the rapid detection advantage of LAMP technology. Compared to the amplification time of traditional PCR, which typically requires several hours, the LAMP reaction, under optimized conditions, can be completed in only 30-60 minutes, greatly shortening the detection cycle and meeting the timeliness requirements of rapid inspection and quarantine of imported racehorses. Experimental data show that the EHV-1 / 4 LAMP reaction produces amplification results after 20 minutes, and clear and distinct amplification bands can be observed after 40 minutes. Therefore, 40 minutes is the preferred reaction time, which balances detection speed and sensitivity.

[0037] According to the loop-mediated isothermal amplification method for rapid detection and quarantine of equine herpesvirus types 1 and 4 in imported racehorses of the present invention, the detection method in step S5 is selected from one or more of the following:

[0038] Observe the turbidity and check whether a white magnesium pyrophosphate precipitate is formed in the reaction tube;

[0039] The fluorescent dye method involves adding SYBR Green I or calcein / MnCl2 colorimetric solution to the reaction tube and judging the results by the color change.

[0040] Gel electrophoresis was performed to observe whether characteristic ladder-like bands were produced.

[0041] Real-time fluorescence monitoring determines the results by monitoring changes in fluorescence signals in real time.

[0042] The above technical solution specifies that in step S5, "the detection method is selected from one or more of the following: turbidity observation, fluorescent dye method, gel electrophoresis, and real-time fluorescence monitoring." The diverse result detection methods further enhance the practicality and convenience of the LAMP method, and its technical advantages are:

[0043] 1. Simplified Visualization of Turbidity Observation: "Observing turbidity to see if a white magnesium pyrophosphate precipitate is formed in the reaction tube" is the simplest way to interpret LAMP results. A positive LAMP reaction produces a large amount of magnesium pyrophosphate precipitate, causing the reaction solution to become turbid, which can be directly observed with the naked eye without any additional equipment or reagents. This method is extremely simple, fast, and economical, and is especially suitable for rapid on-site screening or resource-scarce areas.

[0044] 2. Rapid Visualization and High Sensitivity of Fluorescent Dye Methods: The fluorescent dye method, which involves adding SYBR Green I or calcein / MnCl2 chromogenic solution to the reaction tube and interpreting the results by observing color changes, provides a more sensitive and clearer way to interpret the results. It should be noted that SYBR Green I is a universal DNA fluorescent dye that can intercalate into double-stranded DNA and emit green fluorescence. After the reaction, adding SYBR Green I results in positive tubes exhibiting green fluorescence (observable with the naked eye or under UV light), while negative tubes retain the dye's original color (orange), providing a clear color contrast and intuitive interpretation. Regarding the calcein / MnCl2 chromogenic solution, the combination of calcein and MnCl2 represents a more advanced visualization method. The reaction system is pre-added with calcein and MnCl2. In the negative reaction, Mn²⁺ quenches the fluorescence of calcein, turning the solution orange-red. In the positive reaction, pyrophosphate ions are generated, which preferentially bind to Mn²⁺, releasing the fluorescence quenching effect of Mn²⁺ on calcein. The released calcein emits green fluorescence (observable with the naked eye or under UV light). The positive reaction shows a light green color, while the negative reaction shows an orange-red color; the color change is clearly visible. The calcein / MnCl2 method is more sensitive and allows for closed-tube detection, effectively avoiding contamination from opening the tube.

[0045] 3. Validation and Result Confirmation of Gel Electrophoresis: "Gel electrophoresis, observing whether characteristic ladder-like bands are produced," is a classic and reliable nucleic acid detection method. It can be used as a means of validating LAMP results, especially when high accuracy is required or when archiving is necessary. LAMP positive products exhibit a characteristic ladder-like pattern on gel electrophoresis, which is easy to identify and provides more intuitive visual evidence, enhancing the credibility of the results.

[0046] 4. Quantitative and Automated Real-Time Fluorescence Monitoring: Real-time fluorescence monitoring, which judges results by monitoring changes in fluorescence signals in real time, is a more advanced and automated method for interpreting results. It is suitable for laboratories equipped with equipment such as real-time fluorescence PCR instruments. Fluorescent dyes are added to the LAMP reaction system, and the reaction process is monitored using a real-time fluorescence PCR instrument. Positive samples will generate real-time fluorescence amplification curves during the reaction. By analyzing the amplification curves, semi-quantitative or even quantitative analysis can be performed, and the results can be automatically interpreted and recorded, improving the automation level of detection and the objectivity of data analysis.

[0047] Therefore, the diverse result detection methods provided by the above solutions take into account the needs of different application scenarios. They include both simple and quick visual observation methods and sensitive and accurate instrument detection methods. Users can flexibly choose the appropriate detection method according to actual conditions and needs.

[0048] According to the loop-mediated isothermal amplification method for rapid inspection and quarantine of herpesvirus types 1 and 4 in imported racehorses, the total time from sample acquisition to detection completion is no more than 1.5 hours, wherein the nucleic acid extraction time in step S2 is approximately 30 minutes, the amplification reaction time in step S4 is approximately 40 minutes, and the detection time in step S5 is no more than 5 minutes.

[0049] The above technical solution limits the total time from sample acquisition to test completion to no more than 1.5 hours, with nucleic acid extraction in step S2 taking approximately 30 minutes, amplification reaction in step S4 taking approximately 40 minutes, and test in step S5 taking no more than 5 minutes. This limitation on total test time is the core embodiment of the "rapid inspection and quarantine" objective of this invention. Its technical effects are: 1. Meeting the timeliness requirements for rapid customs clearance at ports: The total time from sample acquisition to result output is controlled within 1.5 hours, far shorter than traditional virus isolation, serological testing, and conventional PCR methods. This significantly shortens the inspection and quarantine cycle, meets the timeliness requirements for rapid customs clearance of imported horses, improves port clearance efficiency, and reduces the economic and time costs caused by waiting for test results. 2. Optimization of time allocation and process efficiency in each step: The solution of this invention rationally allocates and optimizes the time for key steps such as nucleic acid extraction, LAMP amplification, and result detection. Nucleic acid extraction takes approximately 30 minutes, LAMP amplification takes approximately 40 minutes, and result detection takes no more than 5 minutes. This time allocation takes into account the operation time and efficiency of each step, making the entire testing process efficient, smooth, and compact, minimizing the total testing time and improving overall testing efficiency. 3. Feasibility of rapid on-site testing: The total testing time of 1.5 hours makes the LAMP method of this invention feasible for rapid on-site testing at ports of entry, quarantine facilities, and other similar locations. Rapid on-site testing can promptly detect and handle racehorses carrying the virus, effectively controlling the spread of the epidemic and providing strong technical support for port quarantine work.

[0050] The loop-mediated isothermal amplification method of the present invention for rapid detection and quarantine of equine herpesvirus types 1 and 4 in imported racehorses can achieve the following technical effects:

[0051] 1. Rapid and Efficient: The isothermal amplification characteristics of LAMP technology, combined with optimized reaction systems and conditions, as well as rapid nucleic acid extraction and simple result interpretation methods, enable the entire testing process to be completed in a shorter time, significantly reducing testing time and improving inspection and quarantine efficiency and customs clearance speed. Compared with traditional virus isolation, serological methods, and conventional PCR methods, this method has a significant time advantage.

[0052] 2. High Sensitivity: LAMP technology utilizes multiple primer pairs to identify multiple regions of the target sequence, improving the specificity and efficiency of amplification. Optimized primer design and reaction system further enhance detection sensitivity, enabling the detection of infections with low viral loads, reducing false negative rates, and identifying potential disease risks earlier.

[0053] 3. High Specificity: The design of the specific LAMP primer set, especially targeting the conserved and specific regions of the gB gene, and the multi-primer synergistic recognition mechanism, ensures the high specificity of this method for equine herpesvirus types 1 and 4. Experiments have shown that this method has no cross-reactivity with other equine pathogens and herpesviruses, avoiding false positive results and improving detection accuracy.

[0054] 4. Simple operation and low equipment requirements: The LAMP reaction is carried out under isothermal conditions, eliminating the need for expensive thermal cyclers and requiring only simple equipment such as a constant temperature water bath or heating module. Results can be interpreted through visualization using turbidity or fluorescent dyes, eliminating the need for complex electrophoresis or real-time fluorescence detection equipment. This lowers the operational threshold and equipment costs, making it easier to promote and apply in ports, grassroots laboratories, or under conditions of limited resources.

[0055] 5. Reduce disease risks and ensure industry safety: Rapid, sensitive, and specific detection methods can promptly identify imported racehorses carrying viruses, buying time for rapid isolation and control measures, and effectively preventing the introduction and spread of overseas diseases. This protects the domestic racehorse industry and the overall horse breeding industry from threats such as EHV-1 / 4, maintaining the healthy development of the industry.

[0056] 6. Applicable to on-site testing: The characteristics of being fast, simple, and requiring low equipment, as well as the selectable sample types and rapid pretreatment methods, enable this invention to perform rapid testing at ports, quarantine sites, or mobile laboratories, meeting the application needs of inbound racehorse inspection and quarantine.

[0057] In summary, the loop-mediated isothermal amplification method of the present invention for rapid detection and quarantine of equine herpesvirus type 1 and 4 in imported racehorses effectively solves the shortcomings of existing technologies in terms of speed, sensitivity, specificity, ease of operation, and equipment dependence. It can efficiently, accurately, and conveniently conduct rapid detection and quarantine of equine herpesvirus type 1 and 4 infection in imported racehorses, and has significant technical effects and application value. Attached Figure Description

[0058] 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.

[0059] Figure 1 This is a flowchart of the method of the present invention;

[0060] Figure 2 This is a diagram showing the optimization results of the ratio of inner and outer primers for equine herpesvirus type 1 in this invention;

[0061] Figure 3 This is a diagram showing the optimization results of the ratio of inner and outer primers for equine herpesvirus type 4 in this invention;

[0062] Figure 4 This is a graph showing the optimization results of Mg²⁺ concentration for equine herpesvirus type 1 in this invention;

[0063] Figure 5 This is a graph showing the optimization results of Mg²⁺ concentration for equine herpesvirus type 4 in this invention;

[0064] Figure 6 This is a graph illustrating the effect of temperature on the LAMP response of equine herpesvirus type 1 in this invention;

[0065] Figure 7 This is a graph illustrating the effect of temperature on the LAMP response of equine herpesvirus type 4 in this invention;

[0066] Figure 8 This is a graph illustrating the effect of time on the LAMP response of equine herpesvirus type 1 in this invention;

[0067] Figure 9 This is a graph illustrating the effect of time on the LAMP response of equine herpesvirus type 4 in this invention;

[0068] Figure 10 This is a visualization of the SYBR Green I LAMP reaction results for equine herpesvirus type 1 in this invention;

[0069] Figure 11 This is a visualization of the SYBR Green I LAMP reaction results for equine herpesvirus type 4 in this invention;

[0070] Figure 12 This is a visualization of the LAMP reaction calcein detection results of equine herpesvirus type 1 in this invention;

[0071] Figure 13 This is a visualization of the LAMP reaction calcein detection results for equine herpesvirus type 4 in this invention;

[0072] Figure 14 This is a graph showing the sensitivity results of conventional PCR amplification of equine herpesvirus type 1 in this invention;

[0073] Figure 15 This is a graph showing the LAMP amplification sensitivity results of equine herpesvirus type 1 in this invention;

[0074] Figure 16 This is a graph showing the sensitivity results of conventional PCR amplification of equine herpesvirus type 4 in this invention;

[0075] Figure 17This is a graph showing the LAMP amplification sensitivity results of equine herpesvirus type 4 in this invention;

[0076] Figure 18 This is a diagram illustrating the specificity verification of the LAMP method for equine herpesvirus type 1 in this invention;

[0077] Figure 19 This is a diagram illustrating the specificity verification of the LAMP method for equine herpesvirus type 4 in this invention;

[0078] Figure 20 This is a real-time fluorescence monitoring image of the LAMP reaction of equine herpesvirus type 1 in this invention;

[0079] Figure 21 This is a real-time fluorescence monitoring image of the LAMP reaction of equine herpesvirus type 4 in this invention. Detailed Implementation

[0080] The loop-mediated isothermal amplification method for rapid detection and quarantine of equine herpesvirus types 1 and 4 in imported racehorses, as described in this invention, will be described in detail below with reference to specific embodiments. It should be noted that the specific embodiments are only for illustrative purposes and should not be construed as limiting the scope of protection of this invention. Those skilled in the art can make adaptive improvements and optimizations to the technical solution of this invention based on the following content without creative effort, but all such improvements and optimizations shall fall within the scope of protection of this invention.

[0081] like Figures 1 to 21 As shown in this embodiment, the purpose of this embodiment is to elaborate in detail a loop-mediated isothermal amplification (LAMP) method for rapid inspection and quarantine of equine herpesvirus type 1 (EHV-1) and equine herpesvirus type 4 (EHV-4) for imported racehorses. This method integrates key steps such as sample collection, nucleic acid extraction, LAMP amplification, result interpretation and process optimization, aiming to achieve rapid, sensitive and specific virus detection of imported racehorses at the port or in a rapid laboratory, effectively meeting the needs of rapid customs clearance and disease prevention and control.

[0082] Step S1: Collection of biological samples from imported racehorses (i.e., obtaining biological samples from imported racehorses)

[0083] Upon arrival at the port of entry or designated quarantine facility, imported racehorses undergo biological sample collection by professionally trained and authorized veterinarians, strictly following animal sampling procedures. To ensure the accuracy and representativeness of the test results, and considering the convenience of on-site operations at the port and animal welfare, nasopharyngeal swabs are preferred as the primary test sample. Blood samples are also accepted as an alternative biological sample type.

[0084] The specific procedures for nasopharyngeal swab collection are as follows: Using a sterile swab, gently insert it into one nostril of the horse, reaching deep into the nasopharynx. Rotate and hold for several seconds to fully aspirate nasal secretions. Remove the swab and immediately place it in a sampling tube containing an appropriate amount of sterile saline or virus preservation solution. Label the sample and proceed with the subsequent nucleic acid extraction steps as soon as possible. If collecting blood samples, use a sterile blood collection needle and vacuum blood collection tube to collect an appropriate amount of venous blood, adding an anticoagulant (such as EDTA) to prevent blood clotting, which could affect the subsequent nucleic acid extraction results. Collected biological samples must be stored and transported at low temperatures (e.g., 4°C) to maintain the integrity of the viral nucleic acid in the sample and the reliability of the test results.

[0085] Step S2: Rapid extraction of nucleic acid template from biological samples (i.e., extraction of nucleic acid template from the biological samples)

[0086] To meet the timeliness requirements of rapid inspection and quarantine, this embodiment uses a commercially available viral DNA extraction kit, such as the viral DNA extraction kit produced by Tiangen Biotech (Beijing) Co., Ltd., to rapidly extract viral nucleic acid from collected nasopharyngeal swabs or blood samples. Compared with traditional phenol-chloroform extraction or boiling methods, the kit method has advantages such as simple operation, short time consumption, high extraction efficiency, and high nucleic acid purity. The entire nucleic acid extraction process can be completed within about 30 minutes, significantly shortening the detection cycle.

[0087] The specific operating procedures are as follows, referring to the kit instructions: Take 200 μL of biological sample (nasopharyngeal swab samples need to be eluted with physiological saline first), add lysis buffer and proteinase K, mix thoroughly, and incubate in a 56°C water bath or constant temperature metal bath for 15 minutes to fully lyse viral particles and release nucleic acid. Then add binding buffer and anhydrous ethanol to allow the nucleic acid to specifically bind to the silica matrix membrane on the adsorption column. Wash twice with washing buffer to remove impurities and proteins. Finally, add elution buffer, incubate at room temperature for 5 minutes, centrifuge, and elute to enrich and purify the viral DNA. The extracted viral DNA can be directly used for subsequent LAMP amplification reactions or stored short-term at -20°C for later use.

[0088] The use of rapid nucleic acid extraction methods can significantly shorten sample processing time, laying the foundation for subsequent rapid LAMP detection and meeting the requirements for nucleic acid extraction time in this invention.

[0089] Step S3: Construction of the LAMP reaction system (i.e., mixing the nucleic acid template with a reaction system containing a nucleic acid polymerase and a specific loop-mediated isothermal amplification primer set for equine herpesvirus type 1 or 4).

[0090] This embodiment constructs independent LAMP reaction systems for equine herpesvirus types 1 and 4, respectively. The core components of the reaction system include: Bst DNA polymerase, a thermophilic DNA polymerase with strand displacement activity, which is the key enzyme in the LAMP reaction; deoxynucleoside triphosphates (dNTPs), which provide the building blocks required for DNA synthesis; and magnesium ions (…). ), as a cofactor of Bst DNA polymerase and involved in the formation of magnesium pyrophosphate precipitate; betaine, a chemical modifier that can reduce the secondary structure of high GC content regions, improving amplification efficiency and specificity; and a specific loop-mediated isothermal amplification primer set for EHV-1 or EHV-4 gB genes.

[0091] The optimized LAMP reaction system (25 μL system) used in this embodiment has the following specific components and final concentrations:

[0092] Components Volume / Concentration Final concentration / volume Bst DNA polymerase (8 U / μL) 1.0 μL 8 U 10× Bst DNA polymerase buffer 2.5 μL 1× dNTP mixture (2.5 mmol / L each) 2.5 μL 2.5 mmol / L <![CDATA[MgSO4 (25 mmol / L)]]> 2.0 μL 3.0 mmol / L Betaine (5 mol / L) 2.5 μL 0.4 mol / L Inner primers FIP / BIP (20 μmol / L) 1.6 μL each 1.6 μmol / L each Outer primer F3 / B3 (20 μmol / L) 0.2 μL each 0.2 μmol / L each Loop primers LoopF / LoopB (20 μmol / L) 0.4 μL each 0.4 μmol / L each Template DNA (extracted viral DNA) 2.0 μL - <![CDATA[Nuclease-free water (ddH2O)]]> Make up to 25 μL -

[0093] The functions and concentrations of each component are selected based on the following criteria:

[0094] Bst DNA polymerase: Provides strand substitution and DNA polymerase activity for the LAMP reaction, ensuring efficient amplification under isothermal conditions. An 8U enzyme dosage is selected to guarantee reaction rate and efficiency.

[0095] 10× Bst DNA polymerase buffer: Provides the appropriate pH and ionic environment required for the LAMP reaction, ensuring maximum enzyme activity;

[0096] dNTP mixture (2.5 mmol / L): Provides sufficient raw materials for DNA synthesis, ensuring the smooth progress of the amplification reaction. The final concentration of 2.5 mmol / L is the optimized optimal concentration, balancing amplification efficiency and cost.

[0097] MgSO4 (3.0 mmol / L): Magnesium ions are an essential cofactor for Bst DNA polymerase and a key ion for the formation of magnesium pyrophosphate precipitate. The final concentration of 3.0 mmol / L represents the optimized optimal concentration, ensuring both enzyme activity and promoting the formation of magnesium pyrophosphate precipitate, facilitating result interpretation. The magnesium ion concentration range in the claims is 2.0-4.0 mmol / L, and the final concentration of 3.0 mmol / L in this embodiment falls within this preferred range.

[0098] Betaine (0.4 mol / L): Betaine can reduce the secondary structure of DNA, especially the secondary structure of GC-rich regions, thereby improving the binding efficiency of primers to the template and the amplification specificity. The final concentration of 0.4 mol / L is the optimized optimal concentration, which can effectively improve amplification efficiency without inhibiting the reaction system. The betaine concentration range defined in the claims is 0.2-0.8 mol / L, and the final concentration of 0.4 mol / L in this embodiment is within this preferred range.

[0099] The specific LAMP primer set includes outer primers (F3, B3), inner primers (FIP, BIP), and loop primers (LoopF, LoopB). The primer sequences are designed from conserved and specific regions of the glycoprotein B (gB) gene of equine herpesvirus types 1 and 4, enabling specific recognition of EHV-1 or EHV-4 nucleic acids, ensuring detection specificity. The primer concentration ratio (outer primer 0.2 μmol / L, inner primer 1.6 μmol / L, loop primer 0.4 μmol / L) and the ratio of inner primer to outer primer concentration are approximately 8:1, representing optimized concentrations and ratios. The primer concentration ranges and ratios defined in the claims cover the preferred concentrations and ratios used in this embodiment.

[0100] The primer sequences for equine herpesvirus type 1 (EHV-1) are as follows:

[0101] Primer name Primer sequence F3 5'-ATCAGCACGCTACGTTGAACT-3' B3 5'-GCGATCCCCTGCATAATCAC-3' FIP 5'-TCTAGCAGGCCGGTGTCCTC-GGAAGACCGCGAGTTTCTG-3' BIP 5'-CAGCGCAAATACAGCGCCGCA-ATTGTCCACGTTGACCACG-3' LoopF 5'-CCGCGTGTACACCTCCA-3' LoopB 5'-GCTCCACGCTCTCAGGT-3'

[0102] The primer sequences for equine herpesvirus type 4 (EHV-4) are as follows:

[0103] Primer name Primer sequence F3 5'-GGCAGAGTACCTCCGAGAT-3' B3 5'-CGGTCGATAGGGCAAAGGA-3' FIP 5'-TGGCCCCAGGAGTAGCAAACT-GCTTACCACGACGACGAAG-3' BIP 5'-GTCGGATGGATGCCATGGAGG-CAGACGCCGCTTCTACT-3' LoopF 5'-AGGTCGAGCTCACCCTCGT-3' LoopB 5'-ACACATCAACCTCTGTCAACTGC-3'

[0104] Template DNA: the viral DNA extracted in step (2).

[0105] Nuclease-free water: Used to replenish the volume of the reaction system and to ensure that there is no nuclease contamination in the system.

[0106] Through systematic optimization of the reaction conditions, the optimal parameter range of the method of this invention was determined. For example... Figure 2 and Figure 3 As shown, the ratio of inner to outer primer concentrations significantly affects the LAMP reaction. When the ratio is 1:8 (0.2 μM outer primer and 1.6 μM inner primer), the resulting ladder-like bands are most prominent, and the amplification efficiency is highest. This phenomenon has been verified in the LAMP reactions of equine herpesvirus type 1 and type 4.

[0107] like Figure 4 and Figure 5As shown, magnesium ion concentration is one of the key factors affecting the efficiency of the LAMP reaction. Experimental results indicate that the LAMP reaction for both equine herpesvirus 1 and 4 achieves optimal amplification at a magnesium ion concentration of 3.0 mmol / L, producing highly specific and clear ladder-shaped bands. When the magnesium ion concentration is below 2.0 mmol / L or above 3.5 mmol / L, the band clarity decreases, indicating that the amplification efficiency is affected.

[0108] Step S4: LAMP isothermal amplification reaction (i.e., nucleic acid amplification reaction performed under isothermal conditions)

[0109] Add 2 μL of extracted viral DNA template to the prepared LAMP reaction solution, mix gently, and then place in a constant temperature heating device for amplification. The preferred constant temperature reaction temperature is 63°C, and the reaction time is 40 minutes. The claims specify a constant temperature of 60-65°C and a reaction time of 30-60 minutes; the 63°C and 40 minutes used in this embodiment are both within this preferred range.

[0110] The LAMP reaction is carried out under isothermal conditions, eliminating the need for expensive thermal cycling equipment such as PCR instruments. Only a simple isothermal water bath, dry incubator, or portable LAMP instrument is required, significantly reducing equipment costs and making it easier to apply at border crossings or in resource-constrained grassroots laboratories. Under a constant temperature of 63℃, Bst DNA polymerase continuously exerts its strand displacement and DNA polymerase activities. The specific primer set efficiently recognizes and binds to the target sequence, initiating the LAMP cycle amplification reaction, exponentially amplifying the target viral nucleic acid in a short time.

[0111] Reaction temperature and time are important parameters for the LAMP reaction. For example... Figure 6 and Figure 7 As shown, the LAMP method of this invention can be effectively carried out in a temperature range of 60℃ to 65℃, indicating that the method has a certain tolerance to temperature changes, which is of great significance for temperature fluctuations that may occur in the field testing environment. Experimental data show that 63℃ is the optimal reaction temperature, at which the reaction efficiency is the highest and the results are the most stable.

[0112] like Figure 8 and Figure 9 As shown, time-kinetic analysis of the LAMP reaction indicates that detectable amplification products begin to form after 20 minutes, but the bands are weak at this time. A clear and distinct trapezoidal band is observed at 40 minutes. Further extending the reaction time to 50-60 minutes does not significantly improve the amplification effect. Therefore, this invention determines the optimal reaction time to be 40 minutes, balancing detection speed and result reliability.

[0113] Step S5: Rapid detection and result interpretation of LAMP reaction products (i.e., detecting the amplification products to determine whether the imported racehorses are infected with equine herpesvirus type 1 or 4).

[0114] After the LAMP reaction is complete, rapid result interpretation can be performed. This embodiment provides multiple result interpretation methods, which can be flexibly selected according to actual conditions and needs, all of which can achieve rapid, simple, and intuitive result interpretation.

[0115] a) Turbidity Observation Method: When the LAMP reaction is positive, a large amount of magnesium pyrophosphate precipitate is produced, causing the solution in the reaction tube to become turbid. The result can be preliminarily judged by directly observing the turbidity change in the reaction tube. Positive reaction tubes show obvious white turbidity, while negative reaction tubes remain clear and transparent. The turbidity observation method is simple to operate, requires no special equipment, and is very suitable for rapid on-site testing.

[0116] b) Visualization method using fluorescent dyes:

[0117] SYBR Green I Dye Method: After the reaction, add 1 μL of SYBR Green I dye to the reaction tube. SYBR Green I is a fluorescent dye that intercalates into double-stranded DNA and emits green fluorescence. Under natural light, the positive reaction tube appears green due to the large amount of amplification product, while the negative reaction tube appears orange due to the dye itself. Under ultraviolet light, the positive reaction tube emits a more pronounced green fluorescence, while the negative reaction tube shows weaker fluorescence or no fluorescence. The SYBR Green I dye method is simple to operate, highly sensitive, and the results can be interpreted visually. Figure 10 and Figure 11 As shown, when using SYBR GREEN I dye for visual detection, positive samples appear bright green, while negative samples retain the original orange color of the dye, with obvious color difference, making it easy to interpret with the naked eye.

[0118] Calcein dye method: Calcein and MnCl2 chromogenic solution are pre-added to the LAMP reaction system. Calcein itself has green fluorescence, but the fluorescence is quenched when it binds to Mn²⁺. In the LAMP positive reaction, the generated pyrophosphate ions preferentially chelate Mn²⁺, releasing free calcein, thus emitting green fluorescence. Under natural light, the positive reaction tube appears bright green, while the negative reaction tube appears dark green. The fluorescence of calcein is quenched, resulting in an orange-red color. The calcein dye method allows for the addition of dye before the reaction, enabling closed-tube detection and effectively avoiding the contamination risks associated with opening the tube. This makes it more suitable for rapid on-site detection scenarios. Figure 12 and Figure 13 As shown, using calcein / When using the colorimetric reagent, positive samples appear light green under sunlight, while negative samples appear orange-red. Under ultraviolet light, positive samples show bright green fluorescence, while negative samples show almost no fluorescence. This visualization method is not only intuitive and easy to identify, but also allows for closed-tube testing, effectively avoiding the risk of contamination caused by opening the tube, making it particularly suitable for rapid on-site testing applications.

[0119] c) Gel electrophoresis verification method (optional): For result verification or interpretation of complex situations, a small amount of LAMP reaction products can be analyzed by agarose gel electrophoresis. Positive LAMP reaction products will exhibit a characteristic ladder-like pattern on the gel, with the bands diffusely distributed; this is a typical characteristic of LAMP amplification. Although the gel electrophoresis verification method is slightly more complex, the results are intuitive and reliable, and it can be used as the gold standard or auxiliary method for result interpretation.

[0120] d) Real-time fluorescence monitoring (optional): A fluorescent dye (such as SYBR Green I or EvaGreen) is added to the LAMP reaction system, and real-time monitoring is performed using a real-time fluorescence detector with isothermal incubation function (such as the isothermal mode of a quantitative PCR instrument). Positive samples will rapidly generate fluorescence signals during the reaction and show a clear fluorescence amplification curve within 10-20 minutes, while negative samples will not show obvious changes in fluorescence signals. Real-time fluorescence monitoring can achieve quantitative or semi-quantitative detection and provides more objective and accurate results interpretation.

[0121] In this embodiment, turbidity observation or fluorescent dye visualization is preferably used for rapid result interpretation, while gel electrophoresis and real-time fluorescence monitoring can be used as verification methods. The method of this invention incorporates real-time fluorescence monitoring technology, enabling more accurate detection and analysis, such as... Figure 20 As shown, in real-time fluorescence monitoring of the equine herpesvirus type 1 LAMP reaction, positive samples showed significant fluorescence signal enhancement 13-20 minutes after the start of the reaction, with fluorescence intensity increasing rapidly with increasing reaction time, forming a typical exponential growth curve, while negative samples remained at baseline. Similarly, as... Figure 21 As shown, real-time fluorescence monitoring of the LAMP reaction for equine herpesvirus type 4 revealed a significant enhancement of fluorescence signal in positive samples within 10-16 minutes after the reaction began. Real-time fluorescence monitoring not only enables more accurate result interpretation but also allows for semi-quantitative analysis of viral load, providing more information for clinical diagnosis and epidemiological studies. Furthermore, real-time monitoring technology can be used to optimize LAMP reaction conditions, providing a basis for further methodological improvements.

[0122] Step S6: Inspection process optimization and application effect

[0123] By integrating the aforementioned steps of sample collection, nucleic acid extraction, LAMP amplification, and result interpretation, this embodiment constructs a rapid inspection and quarantine process for equine herpesvirus types 1 and 4 in imported racehorses. The entire testing process, from sample collection to result interpretation, can be completed within 1-1.5 hours, significantly outperforming traditional methods such as virus isolation and identification, serological methods, and conventional PCR, greatly improving the efficiency of inspection and quarantine and customs clearance speed for imported racehorses.

[0124] The beneficial effects achieved by the method described above include: 1. Rapid and efficient: The entire detection process can be completed within 1.5 hours, and the LAMP amplification reaction itself only requires 40 minutes, greatly shortening the quarantine cycle and improving customs clearance efficiency. 2. High sensitivity: such as Figure 14 and Figure 15 As shown, the detection limit for equine herpesvirus type 1 using conventional PCR methods is [insert value here]. The detection limit of the LAMP method of this invention can reach [value missing]. Sensitivity is increased by about 10 times, similarly, such as Figure 16 and Figure 17 As shown, the detection limit for equine herpesvirus type 4 using conventional PCR methods is [insert value here]. The detection limit of the LAMP method of this invention can reach [value missing]. The sensitivity is also increased by about 10 times, enabling the detection of infections with low viral loads and the early detection of potential disease risks. In other words, this significantly improved detection sensitivity allows the method of this invention to detect samples in the early stages of infection or carrying low viral loads, which is of great significance for the early detection and control of diseases in imported racehorses and can effectively reduce the risk of missed detections due to insufficient detection sensitivity. 3. High specificity: The specific primer set designed based on the gB gene can specifically recognize EHV-1 and EHV-4, and has no cross-reactivity with other equine pathogens and herpesviruses, ensuring the accuracy of the detection results. Figure 18 As shown, when the LAMP method of this invention was used to detect samples of equine herpesvirus type 1, equine herpesvirus type 4, 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), only the equine herpesvirus type 1 sample showed a positive result, while the other virus samples were negative. Similarly, as... Figure 19As shown, the LAMP method for equine herpesvirus 4 also exhibits similar high specificity, with only equine herpesvirus 4 samples showing positive results, while other virus samples were negative. This high specificity stems from the primer set designed in this invention specifically recognizing multiple sites on the gB genes of equine herpesvirus 1 and 4, ensuring the accuracy and reliability of the detection results and effectively avoiding false positives, which is crucial for the accurate quarantine of imported racehorses. 4. Simple operation and low equipment requirements: The LAMP reaction is carried out under isothermal conditions, eliminating the need for expensive thermal cyclers. The results are intuitively interpreted, requiring low technical skills from operators, and are easy to promote and apply at port sites or grassroots laboratories. 5. Effectively reduces disease risk and ensures industry safety: Rapid and accurate detection can promptly identify horses carrying the virus, allowing for isolation and other control measures, effectively preventing the introduction and spread of imported diseases such as EHV-1 / 4, protecting domestic racehorses and the entire horse breeding industry from disease threats, and maintaining the healthy development of the industry. 6. Suitable for on-site testing: Its rapid, simple, and low-equipment-requirement features make it suitable for on-site or mobile laboratory testing at ports of entry and quarantine facilities, providing strong technical support for the rapid quarantine of imported racehorses.

[0125] In summary, this embodiment details the implementation process of a rapid inspection and quarantine method for equine herpesvirus types 1 and 4 in imported racehorses based on LAMP technology. It fully demonstrates the technical solution, operation steps, key parameters, and achievable technical effects of the present invention, proving the speed, sensitivity, specificity, and practicality of the method. It fully meets the needs of rapid inspection and quarantine of imported racehorses and has significant application value and promotion prospects.

Claims

1. A loop-mediated isothermal amplification method for rapid quarantine inspection of Equid Herpesvirus 1 and 4 in imported racehorses, characterized by, The method comprises the following steps: S1, obtaining a biological sample of an imported racehorse; S2, extracting a nucleic acid template from the biological sample; S3, mixing the nucleic acid template with a reaction system comprising a nucleic acid polymerase and a specific loop-mediated isothermal amplification primer set for equine herpesvirus type 1 or type 4; S4, performing a nucleic acid amplification reaction under constant temperature conditions; S5, detecting the amplification product to determine whether the imported racehorse is infected with equine herpesvirus type 1 or type 4.

2. The loop-mediated isothermal amplification method for rapid quarantine of equine herpesviruses type 1 and 4 in imported racehorses according to claim 1, characterized in that, The biological sample is a nasopharyngeal swab or a blood sample.

3. The loop-mediated isothermal amplification method for rapid quarantine of equine herpesvirus type 1 and 4 of racehorses according to claim 1, wherein, The reaction system in step S3 comprises Bst DNA polymerase, deoxynucleotide triphosphate, magnesium ions, betaine and the specific loop-mediated isothermal amplification primer set.

4. The loop-mediated isothermal amplification method for rapid quarantine of equine herpesviruses type 1 and 4 in imported racehorses according to claim 3, characterized in that, In the reaction system, the concentration of magnesium ions is 2.0-4.0 mmol / L, the concentration of betaine is 0.2-0.8 mol / L, and the concentration of deoxynucleotide triphosphate is 1.5-3.5 mmol / L.

5. The loop-mediated isothermal amplification method for rapid quarantine of equine herpesviruses type 1 and 4 in imported racehorses according to claim 1, characterized in that, The specific loop-mediated isothermal amplification primer set is designed based on the glycoprotein B (gB) gene of equine herpesvirus type 1 or type 4, and each primer set comprises outer primers (F3, B3), inner primers (FIP, BIP) and loop primers (LoopF, LoopB), wherein the inner primer FIP comprises F1c and F2 sequences, and the inner primer BIP comprises B1c and B2 sequences.

6. The loop-mediated isothermal amplification method for rapid quarantine of equine herpesviruses type 1 and 4 in imported racehorses according to claim 5, wherein, In the primer set, the concentration of outer primers is 0.1-0.4 μmol / L, the concentration of inner primers is 1.2-2.0 μmol / L, the concentration of loop primers is 0.2-0.8 μmol / L, and the concentration ratio of inner primers to outer primers is 6:1 to 10:

1.

7. The loop-mediated isothermal amplification method for rapid quarantine of equine herpesviruses type 1 and 4 in imported racehorses according to claim 5, characterized in that ； The primer sequences of equine herpesvirus type 1 are as follows: F3: 5'-ATCAGCACGTACGTTGAACT-3' B3: 5'-GCGATCCCCTGCATAATCAC-3' FIP: 5'-TCTAGCAGGCCGGTGTCCTC-GGAAGACCGCGAGTTTCTG-3' BIP: 5'-CAGCGAAATACAGCGCCGCA-ATTGTCCACGTTGACCACG-3' LoopF: 5'-CCGCGTGTACACCTCCA-3' LoopB: 5'-GCTCCACGCTCTCAGGT-3'; and / or, The primer sequences of equine herpesvirus type 4 are as follows: F3: 5'-GGCAGAGTACCTCCGAGAT-3' B3: 5'-CGGTCGATAGGGCAAAGGA-3' FIP: 5'-TGGCCCCAGGAGTAGCAAACT-GCTTACCACGACGACGAAG-3' BIP: 5'-GTCGGATGGATGCCATGGAGG-CAGACCGCGCTTCTACCT-3' LoopF: 5'-AGGTCGAGCTCCACCTCGT-3' LoopB: 5'-ACACATCAACCTCTGTCAACTGC-3'.

8. The loop-mediated isothermal amplification method for rapid quarantine of equine herpesviruses type 1 and 4 in imported racehorses according to claim 1, characterized in that, The constant temperature condition in the step S4 is 60-65℃, and the reaction time is 30-60 minutes.

9. The loop-mediated isothermal amplification method for rapid quarantine of equine herpesviruses type 1 and 4 in imported racehorses according to claim 1, characterized in that, The detection method in the step S5 is selected from one or more of the following: Turbidity observation, observing whether white magnesium pyrophosphate precipitate is generated in the reaction tube; Fluorescent dye method, adding SYBR Green I or adding calcein / MnCl2 color developing solution to the reaction tube, and judging the result by color change; Gel electrophoresis, observing whether characteristic ladder bands are generated; Real-time fluorescence monitoring, judging the result by real-time monitoring of fluorescence signal change.

10. The loop-mediated isothermal amplification method for rapid quarantine of equine herpesviruses type 1 and 4 in imported racehorses according to claim 1, characterized in that, The total time of the method from sample acquisition to completion of detection is not more than 1.5 hours, wherein the nucleic acid extraction time of the step S2 is about 30 minutes, the amplification reaction time of the step S4 is about 40 minutes, and the detection time of the step S5 is not more than 5 minutes.

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