A detection method based on molecular biology fluorescence assay

Abstract

This invention relates to a detection method based on molecular biological fluorescence assay. Detection points are uniformly set within a test area. Samples are taken at these points, and several test samples are processed to obtain a detection template. This template is then added to a molecular fluorescence quantitative PCR reaction system to obtain detection results. Based on the results, positive and negative points are identified, a test area map is plotted, and positive points are marked. A preliminary positive concentration area is delineated based on the marked positive points in the test area map. This area is then magnified according to specified rules to obtain a detection amplification area. Detection points are rearranged within the detection amplification area, and the detection process is repeated. The final positive concentration area within the detection amplification area is obtained. Based on this final positive concentration area, the source region of the silkworm disease pathogen is analyzed. Furthermore, the pathogen's accumulation and transmission routes are analyzed. Based on the pathogen's accumulation and transmission routes, corresponding control areas are delineated, and matching control measures are implemented.

Description

Technical Field

[0001] This invention belongs to the field of molecular fluorescence measurement technology, and particularly relates to a detection method based on molecular biological fluorescence measurement. Background Technology

[0002] Silkworm diseases are one of the major causes of economic losses in silkworm farming. At the same time, silkworm diseases directly affect the yield of cocoons and the quality of silk. Accurate detection and effective prevention and control of silkworm diseases are of great significance to industrial development, industry stability, and the income of silkworm farmers.

[0003] Silkworms, raised in dense groups, are highly susceptible to various diseases caused by pathogenic microorganisms, parasites, or other external factors. Originally, midgut septicemia and hollow-head softening were the main diseases. However, the specific diseases caused vary depending on the rearing time and climatic conditions. Currently, blood septicemia, mushy disease, and pesticide poisoning pose the most serious threats to sericulture. Silkworm diseases are one of the main factors causing losses during silkworm rearing. Traditional diagnostic techniques for silkworm diseases mainly rely on visual inspection combined with microscopic examination. Often, by the time a disease is diagnosed, irreparable economic losses have already occurred. Therefore, traditional silkworm disease detection is reactive and has a certain time lag.

[0004] Testing silkworms before they become infected and analyzing the accumulation of pathogens is crucial for sericulture. Predicting disease progression based on pathogen accumulation information allows for early intervention and preventative measures, avoiding the need for measures to be taken only after infection. Once silkworms are infected, the disease can spread over a large area, making control difficult and potentially requiring expanded treatment, resulting in significant losses.

[0005] Therefore, it is urgent to conduct environmental and silkworm body testing before silkworms become infected, analyze the accumulation and transmission routes of pathogens, and delineate corresponding prevention and control areas based on the accumulation and transmission routes of pathogens, and implement matching prevention and control measures in advance to prevent and control diseases from causing significant economic losses. Summary of the Invention

[0006] The purpose of this invention is to provide a detection method based on molecular biology fluorescence measurement, which can be used to detect the environment and silkworms before silkworms become diseased, analyze the accumulation of pathogens and their transmission routes, delineate corresponding prevention and control areas based on the accumulation of pathogens and their transmission routes, and deploy matching prevention and control measures.

[0007] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is as follows:

[0008] A detection method based on molecular biology fluorescence assay includes the following steps:

[0009] S1: Detection points are evenly set up within the test area, and samples are taken at the detection points to obtain a number of test samples;

[0010] S2: Process the sample to be tested to obtain a detection template, and add the detection template to the molecular fluorescence quantitative PCR reaction system;

[0011] S3: Obtain the detection results of each sample to be tested, and based on the detection results, obtain the positive and negative points in the detection points, draw the test range map, and mark the positive points in the test range map;

[0012] S4: Based on the markings of positive points in the test range diagram, delineate the initial positive concentration area, enlarge the initial positive concentration area according to the specified rules to obtain the detection amplification area, rearrange the detection points in the detection amplification area, and execute steps S1-S3 again;

[0013] S5: Obtain the final positive concentration area within the detection amplification area, and analyze the source region of the silkworm pathogen based on the final positive concentration area;

[0014] S6: Delineate control areas based on the source region of the silkworm disease pathogen and implement corresponding control measures.

[0015] Preferably, the specific process of step S1 is as follows:

[0016] S11: Prepare sampling consumables and testing consumables. The sampling consumables include medical cotton swabs, labels, self-sealing bags, and markers. The testing consumables include sterile water, EP tubes, 96-well quantitative plates, and sealing films.

[0017] S12: Set the test range, evenly set the test points within the test range, and use sampling consumables to take samples at the test points to obtain several test samples.

[0018] Preferably, the specific process of step S2 is as follows:

[0019] S21: Use testing consumables to process the sample to be tested. Add a quantitative amount of sterile water to the EP tube, put the collected medical cotton swab environmental sample into the EP tube for dilution and mixing, and use it as a test template to store in a 4°C refrigerator for later use.

[0020] S22: Prepare a molecular fluorescence quantitative PCR reaction system and add the detection template to the molecular fluorescence quantitative PCR reaction system.

[0021] Preferably, the molecular fluorescence quantitative PCR reaction system is configured as follows: 12.5 μl Mix, 0.5 μl upstream primer, 0.5 μl downstream primer, 0.5 μl probe, 8.5 μl ddH2O, 2.5 μl template, with three replicate sample wells for each sample, and two positive control wells and two negative control wells per plate.

[0022] Preferably, the specific process in step S3 is as follows:

[0023] S31: Number all test samples, obtain the test results of each sample, and enter the location information and test results of each test sample into a preset template table according to the number to obtain the test table;

[0024] S32: Draw a test range diagram based on the test range, and mark the location information corresponding to the positive test results recorded in the test table on the test range diagram.

[0025] Preferably, in step S32, the area map to be tested is drawn using a preset GIS drawing software, a new point feature layer is created, and the location information corresponding to the positive detection results is converted into coordinate information and then imported into the layer.

[0026] Preferably, the specific process of step S4 is as follows:

[0027] S41: Set a density threshold for positive points, and use GIS drawing software to draw a bounding box around the area where the density of positive points is greater than the density threshold;

[0028] S42: Establish density contour lines in the region where the density of the positive points is greater than the density threshold, and establish a mapping table between density contour lines and magnification ratio based on the density contour lines;

[0029] S43: According to the mapping relationship between the density contour lines and the magnification ratio, the area where the density of the positive point is greater than the density threshold is magnified to obtain the detection magnification area, and the detection magnification area is framed in the test range map;

[0030] S44: Rearrange the detection points in the detection amplification area and execute steps S1-S3 again.

[0031] Preferably, the specific process of step S44 is as follows:

[0032] S441: The detection points in the detection amplification area are rearranged according to the density contour lines, and the detection point density is set to match the density contour lines.

[0033] S442: Execute steps S1-S3 based on the density of the detection points that match the density contour lines.

[0034] Preferably, the specific process of step S5 is as follows:

[0035] S51: Obtain the final positive concentration area within the detection amplification area, and collect environmental data and personnel flow data of the final positive concentration area;

[0036] S52: Starting from the final positive concentration area, and combining silkworm breeding activities and personnel flow, trace the transmission path of the pathogen. By investigating the daily work routes of breeders, the use and circulation of silkworm equipment, and the treatment of silkworm excrement, determine the transmission path of the pathogen in the area to be tested, and analyze the source of silkworm disease pathogens based on the transmission path.

[0037] Preferably, the specific process of step S6 is as follows:

[0038] S61: Delineate key prevention and control areas based on the analyzed transmission routes of pathogens within the area to be tested and the source of silkworm pathogens;

[0039] S62: Divide the key prevention and control areas into different areas, and deploy corresponding prevention and control measures according to the designated areas;

[0040] S63: Implement appropriate prevention and control measures for silkworm diseases according to the different regions.

[0041] The beneficial effects of this invention include:

[0042] This invention provides a detection method based on molecular biology fluorescence assay. Detection points are uniformly set within the target area. Samples are taken at these points, processed, and a detection template is obtained. This template is then added to a molecular fluorescence quantitative PCR system to obtain detection results. Based on these results, positive and negative points are identified, and a target area map is plotted with positive points marked. A preliminary positive concentration area is delineated based on the marked positive points in the target area map. This area is then magnified according to specified rules to obtain a detection amplification area. Detection points are rearranged within this amplification area, and the detection process is repeated. The final positive concentration area within the amplification area is obtained, and the source region of the silkworm disease pathogen is analyzed based on this final positive concentration area. Furthermore, the pathogen's accumulation and transmission pathways are analyzed. Based on the pathogen's accumulation and transmission pathways, corresponding control areas are delineated, and matching control measures are implemented.

[0043] First, a preliminary positive concentration area is delineated based on positive spot markers, and then the area is magnified according to specified rules to obtain a detection magnification area. Within this magnification area, the detection points are then rearranged. By setting a positive spot density threshold, establishing density contour lines, and creating a mapping table between density contour lines and magnification ratios, the detection magnification area can be scientifically and rationally determined based on the distribution density of positive spots, and the detection points can be rearranged according to the density contour lines. This dynamic optimization of the detection area and points allows resources to be concentrated on more detailed detection of high-risk areas, improving detection efficiency while reducing detection costs, achieving efficient resource utilization.

[0044] Secondly, the test results of each sample were numbered and recorded, and a map of the testing area was drawn. Using GIS drawing software, the location information corresponding to the positive test results was converted into coordinate information and imported into a layer, achieving precise location and visualization of the positive points. This helps to intuitively understand the distribution of silkworm disease within the testing area, facilitates the analysis of transmission paths, and provides a clear and accurate basis for subsequent prevention and control work.

[0045] Furthermore, by acquiring the preliminary and final positive concentration areas within the detection amplification zone, it is possible to accurately analyze the source area of ​​silkworm disease pathogens, and further analyze the pathogen's accumulation and transmission routes. Based on the pathogen's accumulation and transmission routes, corresponding prevention and control areas can be delineated, and matching prevention and control measures can be implemented in advance to prevent the occurrence and spread of silkworm diseases.

[0046] Finally, by collecting environmental and population movement data from the final positive concentration areas and tracing the pathogen transmission path in conjunction with silkworm farming activities, the source of silkworm disease pathogens was analyzed in depth from multiple dimensions. This not only accurately identifies the source of the pathogen but also clearly grasps its transmission path, providing strong support for the formulation of targeted prevention and control measures. Compared with traditional detection methods, the tracing of pathogens is more comprehensive and accurate. Attached Figure Description

[0047] Figure 1 This is a schematic flowchart of the detection method based on molecular biology fluorescence measurement according to the present invention.

[0048] Figure 2 This is a schematic diagram showing the distribution of NPV and DNV positive samples in this invention.

[0049] Figure 3 This is a statistical diagram illustrating the ingestion-related environmental RT-PCR detection results of the present invention. Detailed Implementation

[0050] The following is in conjunction with the appendix Figure 1-3 The present invention will be further described in detail below:

[0051] Example 1

[0052] See appendix Figure 1 As shown, a detection method based on molecular biology fluorescence assay includes the following steps:

[0053] S1: Detection points are evenly distributed within the testing area. Samples are taken from these detection points to obtain several test samples. The testing area includes various areas of the silkworm rearing area and the silkworm house. Detection points are initially evenly distributed within these areas, with a relatively sparse distribution. Samples are then taken from these detection points using sampling consumables. The sampling process is as follows: Using clean, sterile medical cotton swabs, 3-5 sites are selected on the surface of the sampling site. Each site is smeared 2-3 times, with a single smear length greater than 10cm. The cotton swab is then rotated with the fingers. After smearing each site, the cotton swab should be placed in a self-sealing sterile bag, labeled, and then smeared at the next site.

[0054] S2: The sample to be tested is processed using testing consumables to obtain a testing template, and the testing template is added to the molecular fluorescence quantitative PCR reaction system for detection;

[0055] S3: Obtain the detection results of each sample to be tested, and based on the detection results, obtain the positive and negative points in the detection points, draw the test range map, and mark the positive points in the test range map;

[0056] S4: Based on the markings of positive points in the test range diagram, delineate the initial positive concentration area, enlarge the initial positive concentration area according to the specified rules to obtain the detection amplification area, rearrange the detection points in the detection amplification area, and execute steps S1-S3 again;

[0057] S5: Obtain the final positive concentration area within the detection amplification area, and analyze the source region of the silkworm pathogen based on the final positive concentration area;

[0058] S6: Delineate control areas based on the source region of the silkworm disease pathogen and implement corresponding control measures.

[0059] The principle of the detection method based on molecular biology fluorescence assay of the present invention is as follows: Utilizing the principle of fluorescence signal enhancement accompanying the increase of the target PCR product, fluorescence signal values ​​are collected during the PCR amplification process, and the Ct value is used to determine whether the test sample contains silkworm concentrated nucleovirus. Specific PCR primers are designed based on the conserved sequence of the BmDNV orf gene to perform the PCR amplification reaction.

[0060] The specific process of step S1 is as follows:

[0061] S11: Prepare sampling consumables and testing consumables. The sampling consumables include medical cotton swabs, labels, self-sealing bags, and markers. The testing consumables include sterile water, EP tubes, 96-well quantitative plates, and sealing films.

[0062] S12: Set the test range, evenly set the test points within the test range, and use sampling consumables to take samples at the test points to obtain several test samples.

[0063] The specific process of step S2 is as follows:

[0064] S21: Use testing consumables to process the sample to be tested. Add 400μl of sterile water to a 1.5ml EP tube, put the collected medical cotton swab environmental sample into the EP tube for dilution and mixing, and store it in a 4℃ refrigerator as a test template for later use.

[0065] S22: Prepare a molecular fluorescence quantitative PCR reaction system, add the detection template to the molecular fluorescence quantitative PCR reaction system, and perform PCR reaction quality control.

[0066] The molecular fluorescence quantitative PCR reaction system was configured as follows: 12.5 μl Mix, 0.5 μl upstream primer, 0.5 μl downstream primer, 0.5 μl probe, 8.5 μl ddH2O, 2.5 μl template, with three replicate sample wells for each sample, and two positive control wells and two negative control wells per plate.

[0067] During the testing process, after the amplification reaction is complete, the positive control shows a typical amplification curve, while the fluorescence curves of the blank control and negative control are flat or less than 15% lower than the fluorescence value of the positive control, indicating that the reaction system is working normally. Otherwise, it indicates that the PCR reaction system is abnormal and needs to be tested again.

[0068] The result determination process is as follows: Assuming the PCR reaction system is functioning normally, the result is determined based on the collected fluorescence curve and Ct value. The determination rules are as follows:

[0069] a. If the Ct value of the sample to be tested is less than or equal to the Ct value of the positive control, the sample is determined to have been found to contain silkworm nucleovirus;

[0070] b. If the Ct value of the sample to be tested is greater than or equal to the Ct value of the negative control, it is determined that the sample does not contain the silkworm nucleovirus.

[0071] c. If the Ct value of the sample to be tested is greater than the Ct value of the positive control and less than the Ct value of the negative control, PCR amplification should be repeated. If the repeated experiment shows a typical amplification curve and the Ct value is still within this range, the sample is suspected of containing silkworm nucleovirus.

[0072] Example 2

[0073] Based on Example 1, the specific process in step S3 is as follows:

[0074] S31: Number all test samples, obtain the test results of each sample, and enter the location information and test results of each test sample into a preset template table according to the number to obtain the test table;

[0075] S32: Draw a test range diagram based on the test range, and mark the location information corresponding to the positive test results recorded in the test table on the test range diagram.

[0076] In step S32, the area to be tested is drawn using a preset GIS drawing software, a new point feature layer is created, and the location information corresponding to the positive detection results is converted into coordinate information and then imported into this layer.

[0077] The specific process of step S4 is as follows:

[0078] S41: Set a density threshold for positive points, and use GIS drawing software to draw a bounding box around the area where the density of positive points is greater than the density threshold;

[0079] S42: Establish density contour lines in the region where the density of the positive points is greater than the density threshold, and establish a mapping table between density contour lines and magnification ratio based on the density contour lines;

[0080] S43: According to the mapping relationship between the density contour lines and the magnification ratio, the area where the density of the positive point is greater than the density threshold is magnified to obtain the detection magnification area, and the detection magnification area is framed in the test range map;

[0081] S44: Rearrange the detection points in the detection amplification area and execute steps S1-S3 again.

[0082] The specific process of step S44 is as follows:

[0083] S441: The detection points in the detection amplification area are rearranged according to the density contour lines, and the detection point density is set to match the density contour lines.

[0084] S442: Execute steps S1-S3 based on the density of the detection points that match the density contour lines.

[0085] The specific process of step S5 is as follows:

[0086] S51: Obtain the final positive concentration area within the detection amplification area, and collect environmental data and personnel flow data of the final positive concentration area;

[0087] S52: Starting from the final positive concentration area, and combining silkworm breeding activities and personnel flow, trace the transmission path of the pathogen. By investigating the daily work routes of breeders, the use and circulation of silkworm equipment, and the treatment of silkworm excrement, determine the transmission path of the pathogen in the area to be tested, and analyze the source of silkworm disease pathogens based on the transmission path.

[0088] The specific process of step S6 is as follows:

[0089] S61: Delineate key prevention and control areas based on the analyzed transmission routes of pathogens within the area to be tested and the source of silkworm pathogens;

[0090] S62: Divide the key prevention and control areas into different areas, and deploy corresponding prevention and control measures according to the designated areas;

[0091] S63: Implement appropriate prevention and control measures for silkworm diseases according to the different regions.

[0092] In another embodiment, the samples were divided into four categories: ground samples, which represent the main route of pathogen circulation and widespread transmission, and the testing sites included the floors of the co-breeding room and the large silkworm rearing room; mulberry leaf-related samples, which were samples consumed by silkworms, including mulberry leaves, mulberry storage rooms, silkworm loading and unloading equipment, silkworm feeding equipment, and mulberry cutting machines; surface samples, which were surfaces in direct contact with silkworms, including silkworm beds, silkworm boxes, and silkworm seats; and silkworm rearing equipment. A total of 682 samples were collected for this pathogen testing: 214 ground samples, 227 mulberry leaf-related samples, 223 surface samples (including silkworm beds, silkworm boxes, and silkworm seats), and 18 silkworm rearing equipment samples.

[0093] The collected samples were tested for NPV and DNV, and the results are shown in Figure 1. Based on the test results, the positive rate was calculated with any pathogen detection as the positive rate. It was found that the overall positive rate of this survey reached 23.02%, and the NPV positive rate was higher than the DNV positive rate, which was 1.41 times that of the DNV positive rate. Among them, the positive rate of silkworm rearing trays was the highest at 61.11%, which was 3.32 times that of the surface samples of silkworm rearing.

[0094] Table 1. Statistical analysis of RT-PCR results in silkworm rearing environment samples

[0095]

[0096] See Figure 2As shown, based on the sample distribution, the main source of positive samples was ground samples. Whether it was NPV positive samples or DNV positive samples, ground samples were the largest source of positive samples. The second largest source of NPV positive samples was surface samples, and the second largest source of DNV positive samples was mulberry leaf-related samples. Although the proportion of positive samples from clumps was relatively small, among the 18 clump samples collected, the NPV positive rate was 61.1% and the DNV positive rate was 50.00%, indicating that the sample quantity affected the distribution results.

[0097] Table 2. Statistical analysis of RT-PCR detection results in food-related environments.

[0098]

[0099] Mulberry leaf-related samples were categorized into three types: mulberry storage room environment, mulberry loading equipment, and mulberry cutting machine. Their distribution in positive samples of mulberry leaf-related samples was obtained. (See attached image). Figure 3 As shown in Table 2, the test results revealed that the overall positive rate of the mulberry storage room was significantly higher than that of the mulberry loading equipment and the mulberry cutting machine. However, the positive rates of NPV and DNV were significantly higher in the mulberry cutting machine than in the storage room and the loading equipment. This result indicates that the number of samples that were simultaneously positive for NPV and DNV in the mulberry cutting machine was greater, and the pathogen residues showed a certain degree of diversity.

[0100] According to the results of this invention, 23.02% of the detection sites in the silkworm rearing environment carried pathogens. These pathogens remained on the ground, in mulberry storage rooms, mulberry cutting machines, silkworm rearing platforms, and silkworm boxes. Ground pathogens are easily spread through the movement of people. Pathogens in mulberry leaf samples have a very high probability of being ingested by silkworms, thus infecting them with silkworm diseases. Surface samples come into direct contact with silkworms, and the pathogens contained in these places will directly affect the health of silkworm rearing. Through the accumulation of pathogens in these places, a silkworm disease outbreak can gradually form. In particular, the high positive rate of 28.50% for ground pathogens should be given attention. Ground pathogens can circulate and spread with the movement of people, thus causing large-scale silkworm disease outbreaks. Proper ground disinfection can effectively prevent the spread of pathogens.

[0101] The circulation of mulberry leaves may be one of the main channels through which pathogens enter the silkworm rearing environment.

[0102] In the mulberry leaf-related samples of this invention, the positive rate was 19.38%. After further analysis of the sample types, the positive rate was found to be: mulberry storage room > mulberry cutting machine > mulberry loading equipment. Because mulberry storage rooms store mulberry leaves for extended periods, pathogens are more likely to accumulate in the leaves. The mulberry loading equipment, having less contact time with the leaves, had a significantly lower positive rate than the storage room and the mulberry cutting machine. On the other hand, due to farmers' feeding habits, the mulberry cutting machine is disinfected less frequently. This may explain why there were more samples from the mulberry cutting machine that tested positive for both NPV and DNV. Different pathogens enter the rearing environment and the silkworms through the mulberry cutting machine.

[0103] Meanwhile, during the circulation of mulberry leaves, ground pathogens are also one of the main pathways for pathogen accumulation in the mulberry storage room. The mulberry storage room is one of the most frequently used places in the silkworm rearing process. The movement of personnel brings ground pathogens into the mulberry storage room, and then from the ground into the mulberry leaves. The pathogens on the mulberry leaves then enter the mulberry cutting machine and flow to the silkworm rearing platform and silkworm boxes. Therefore, in the silkworm rearing process, effective disinfection of each key link in the circulation of mulberry leaves is necessary to control the accumulation of pathogens and the outbreak of silkworm diseases.

[0104] The patterns of pathogen residue and accumulation exhibit regional characteristics. Although no data analysis has been performed in this invention, it was found during the detection process that pathogen residue and accumulation show distinct regional characteristics. Different regional climate conditions, mulberry garden management methods, and silkworm rearing habits may all affect the distribution characteristics of pathogens. This invention will further explore the impact of regional conditions and silkworm rearing habits on pathogen accumulation and distribution, thereby proposing regional targeted prevention and control measures to achieve a precise disinfection and control plan for the silkworm rearing environment.

[0105] In summary, the detection method based on molecular biological fluorescence assay provided by this invention involves uniformly setting detection points within the test area, sampling at these points to obtain and process several test samples, obtaining a detection template, and then adding it to a molecular fluorescence quantitative PCR reaction system to obtain detection results. Based on the detection results, positive and negative points are identified, a test area map is plotted, and positive points are marked. A preliminary positive concentration area is delineated based on the marked positive points in the test area map, and this area is magnified according to specified rules to obtain a detection amplification area. Detection points are rearranged within the detection amplification area, and the detection process is repeated. The final positive concentration area within the detection amplification area is obtained, and the source region of the silkworm disease pathogen is analyzed based on this final positive concentration area. Furthermore, the pathogen's accumulation and transmission routes are analyzed, and corresponding prevention and control areas are delineated based on the pathogen's accumulation and transmission routes, and matching prevention and control measures are implemented.

[0106] Based on the marking of positive points, a preliminary positive concentration area is delineated, and the detection amplification area is obtained by enlarging the area according to specified rules. Within this amplified area, the detection points are then rearranged. By setting a positive point density threshold, establishing density contour lines, and creating a mapping table between density contour lines and amplification ratios, the detection amplification area can be scientifically and rationally determined based on the distribution density of positive points, and the detection points can be rearranged according to the density contour lines. This dynamic optimization of the detection area and points allows for more detailed detection of high-risk areas by concentrating resources, improving detection efficiency while reducing costs, and achieving efficient resource utilization. The detection results of each sample are numbered and recorded, and a detection range map is drawn. Using GIS drawing software, the location information corresponding to the positive detection results is converted into coordinate information and imported into a layer, achieving precise positioning and visualization of positive points. This helps to intuitively understand the distribution of silkworm disease within the detection range, facilitates the analysis of transmission paths, and provides a clear and accurate basis for subsequent prevention and control work.

[0107] By acquiring preliminary and final positive concentration areas within the detection amplification zone, the source area of ​​silkworm disease pathogens can be accurately analyzed. This allows for analysis of pathogen accumulation and transmission routes. Based on these factors, corresponding prevention and control areas can be delineated, and matching prevention and control measures can be implemented proactively to prevent the occurrence and spread of silkworm diseases. By collecting environmental and personnel movement data from the final positive concentration areas and tracing pathogen transmission routes in conjunction with silkworm farming activities, the source of silkworm disease pathogens can be analyzed in depth from multiple dimensions. This not only accurately identifies the source of the pathogen but also clearly understands its transmission route, providing strong support for developing targeted prevention and control measures. Compared to traditional detection methods, this approach offers a more comprehensive and accurate tracing of pathogen origins.

Claims

1. A detection method based on molecular biological fluorescence assay, characterized in that, Includes the following steps: S1: Detection points are evenly set up within the test area, and samples are taken at the detection points to obtain a number of test samples; S2: Process the sample to be tested to obtain a detection template, and add the detection template to the molecular fluorescence quantitative PCR reaction system; S3: Obtain the detection results of each sample to be tested, and based on the detection results, obtain the positive and negative points in the detection points, draw the test range map, and mark the positive points in the test range map; S4: Based on the markings of positive points in the test range diagram, delineate the initial positive concentration area, enlarge the initial positive concentration area according to the specified rules to obtain the detection amplification area, rearrange the detection points in the detection amplification area, and execute steps S1-S3 again; S5: Obtain the final positive concentration area within the detection amplification area, and analyze the source region of the silkworm pathogen based on the final positive concentration area; S6: Delineate control areas based on the source areas of the silkworm disease pathogens and implement corresponding control measures; The specific process in step S3 is as follows: S31: Number all test samples, obtain the test results of each sample, and enter the location information and test results of each test sample into a preset template table according to the number to obtain the test table; S32: Draw a test range map based on the test range, and mark the location information corresponding to the positive test results recorded in the test table on the test range map; In step S32, the area to be tested is drawn using a preset GIS drawing software, a new point feature layer is created, and the location information corresponding to the positive detection results is converted into coordinate information and then imported into the layer. The specific process of step S4 is as follows: S41: Set a density threshold for positive points, and use GIS drawing software to draw a bounding box around the area where the density of positive points is greater than the density threshold; S42: Establish density contour lines in the region where the density of the positive points is greater than the density threshold, and establish a mapping table between density contour lines and magnification ratio based on the density contour lines; S43: According to the mapping relationship between the density contour lines and the magnification ratio, the area where the density of the positive point is greater than the density threshold is magnified to obtain the detection magnification area, and the detection magnification area is framed in the test range map; S44: Rearrange the detection points in the detection amplification area and execute steps S1-S3 again; The specific process of step S5 is as follows: S51: Obtain the final positive concentration area within the detection amplification area, and collect environmental data and personnel flow data of the final positive concentration area; S52: Starting from the final positive concentration area, and combining silkworm breeding activities and personnel flow, trace the transmission path of the pathogen. By investigating the daily work routes of breeders, the use and circulation of silkworm equipment, and the treatment of silkworm excrement, determine the transmission path of the pathogen in the area to be tested, and analyze the source of silkworm disease pathogens based on the transmission path.

2. The detection method based on molecular biology fluorescence assay according to claim 1, characterized in that, The specific process of step S1 is as follows: S11: Prepare sampling consumables and testing consumables. The sampling consumables include medical cotton swabs, labels, self-sealing bags, and markers. The testing consumables include sterile water, EP tubes, 96-well quantitative plates, and sealing films. S12: Set the test range, evenly set the test points within the test range, and use sampling consumables to take samples at the test points to obtain several test samples.

3. The detection method based on molecular biology fluorescence assay according to claim 2, characterized in that, The specific process of step S2 is as follows: S21: Use testing consumables to process the sample to be tested. Add a quantitative amount of sterile water to the EP tube, put the collected medical cotton swab environmental sample into the EP tube for dilution and mixing, and use it as a test template to store in a 4°C refrigerator for later use. S22: Prepare a molecular fluorescence quantitative PCR reaction system and add the detection template to the molecular fluorescence quantitative PCR reaction system.

4. The detection method based on molecular biology fluorescence assay according to claim 2, characterized in that, The molecular fluorescence quantitative PCR reaction system was configured as follows: 12.5 μl Mix, 0.5 μl upstream primer, 0.5 μl downstream primer, 0.5 μl probe, 8.5 μl ddH2O, 2.5 μl template, with three replicate sample wells for each sample, and two positive control wells and two negative control wells per plate.

5. The detection method based on molecular biology fluorescence assay according to claim 1, characterized in that, The specific process of step S44 is as follows: S441: The detection points in the detection amplification area are rearranged according to the density contour lines, and the detection point density is set to match the density contour lines. S442: Execute steps S1-S3 based on the density of the detection points that match the density contour lines.

6. The detection method based on molecular biology fluorescence assay according to claim 5, characterized in that, The specific process of step S6 is as follows: S61: Delineate key prevention and control areas based on the analyzed transmission routes of pathogens within the area to be tested and the source of silkworm pathogens; S62: Divide the key prevention and control areas into different areas, and deploy corresponding prevention and control measures according to the designated areas; S63: Implement appropriate prevention and control measures for silkworm diseases according to the different regions.

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