A method for detecting silkworm microparticle viruses

Abstract

This invention discloses a method for detecting silkworm microparticle viruses. The invention relates to the field of detection technology. Using a controlled variable method, three experimental groups were conducted. By controlling the pH value and ion concentration under different environments, the final number of microparticles was detected, verifying the influence of different pH values ​​and ion concentrations on the accuracy of the experiment. Simultaneously, after sample purification, the impurity content was significantly reduced. The appearance characteristics of the microparticles were extracted and identified using a microscope. Color marking enabled the microparticles to be counted in a short time, resulting in high efficiency.

Description

Technical Field

[0001] This invention relates to the field of detection technology, and specifically to a method for detecting silkworm microparticle viruses. Background Technology

[0002] Microsporidiosis of silkworms is a contagious disease caused by microsporidia parasites in silkworms, which once dealt a huge blow to the sericulture industry. Through research on microsporidiosis, the French microbiologist Louis Pasteur proposed the microscopic selection method for mother moths more than 100 years ago. Since then, this microscopic selection technique has been consistently used for the examination of silkworm microsporidiosis.

[0003] In the prior art, such as Chinese Patent No. CN104945049A, a method for killing silkworm nucleopolyhedrovirus and microspores in silkworm yarn includes the following steps: A. Material preparation: Prepare the following components in parts by weight: 1000-1125 parts fresh silkworm excrement, 100-166 parts sugarcane filter mud, 2.5 parts fermentation agent, and 7.5 parts urea; B. Pile preparation: First, mix the fermentation agent, urea, and sugarcane filter mud evenly to form a mixture. Spread a layer of fresh silkworm excrement 28-32 cm thick on one-third of the mixture, then evenly sprinkle one-third of the mixture on the spread fresh silkworm excrement. Repeat the above method to lay two more layers; spray water on the silkworm excrement pile to maintain the dryness of the fresh silkworm excrement at 50%-55%; C. Turning the pile: Turn the silkworm excrement pile obtained in step B; D. Fermentation: Ferment the silkworm excrement pile obtained in step C at high temperature for 19-22 days. This invention can solve the problem of diseases in silkworm excrement affecting the yield and quality of silkworm cocoons.

[0004] However, existing technologies, such as the traditional method of selecting mother moths using a microscope, suffer from drawbacks such as high labor intensity, low work efficiency, and inability to reproduce test results. Furthermore, human eyes are prone to fatigue after prolonged operation, which can also adversely affect the test results. At the same time, due to the small size of microparticle viruses, they are easily mixed with external impurities, making it difficult to observe the distribution of microparticle viruses directly using traditional optical microscopes. Summary of the Invention

[0005] To address the shortcomings of existing technologies, the technical solution adopted by this invention is: a method for detecting silkworm microparticle viruses, comprising the following steps:

[0006] Step 1: Take three samples of the same type of silkworm eggs and label them A, B, and C respectively. Place the three samples A, B, and C in a constant temperature incubator at a temperature of 26 degrees Celsius for a 20-day incubation period. Select the hatched larvae and remove the unhatched eggs, and label them A1, A2, and A3 respectively.

[0007] Step 2: Take three glass culture chambers, sterilize them, and place the three samples in a sealed aerobic environment, continuously feeding them mulberry leaves;

[0008] Step 3: One and a half weeks before cocoon formation, inoculate the virus spores at a concentration of 10... 5 Add a dose of / to mulberry leaves and administer to A1 samples; inoculate viral spores at 10 3 / dose added to mulberry leaves; A2 sample was given to consume, and virus spores were added to mulberry leaves at a dose of 0; A3 sample was given to consume.

[0009] Step 4: Continue to culture the three samples until they form cocoons, and then purify the microparticles;

[0010] Step 5: Microsporidia are activated to germinate and then fluorescently labeled.

[0011] Step 6: Observe the images using an electron microscope and calculate the number of microsporin viruses.

[0012] In a preferred embodiment, the purification method for the microparticles in step four is as follows:

[0013] Step a: Disinfect the surface of the three samples of silkworm pupae (A1, A2, and A3) with 75% ethanol, grind them thoroughly in a mortar, and collect the grinding liquid in a 10mL centrifuge tube;

[0014] Step b: After diluting the grinding solution, set the centrifuge to 500 r / min, centrifuge for 5 min, and collect the supernatant;

[0015] Step c: Filter the supernatant through four layers of gauze, collect the filtrate, and centrifuge the filtrate at 3000 r / min for 5 min. Keep the precipitate and discard the supernatant.

[0016] Step d: Resuspend the precipitate with sterile hydrogen peroxide;

[0017] Step e: Soak sterile absorbent cotton in water and press it into a 10mL syringe. Then filter the spore suspension through the syringe, centrifuge at 3000r / min for 5min, discard the supernatant, and repeat steps d and e three times.

[0018] Step f: Add an appropriate amount of hydrogen peroxide to resuspend the precipitate, which is the purified spore suspension.

[0019] In a preferred embodiment, step five, the activation and budding of microsporidia are crucial steps in the host infection process. During host infection, purified spore polar filaments are ejected, infecting host cells. The conditions required for activation mainly include environmental pH and calcium. 2+ Concentration, intraspore osmotic pressure, and other important aspects;

[0020] Microsporidia require a specific pH environment for activation. Microsporidia that parasitize lepidopteran insects need to be treated with an ion buffer solution of a certain pH value in order to germinate.

[0021] In a preferred embodiment, the sporofilament ejection and host cell infection include the following steps:

[0022] (1) Adhesion of spores to host cells;

[0023] (2) Spore activation;

[0024] (3) The osmotic pressure inside the spores increases;

[0025] (4) The wires pop outwards;

[0026] (5) The sporogenous material enters the cytoplasm of the host cell through the polar tube.

[0027] In a preferred embodiment, in step five, the fluorescent imaging labeling uses KMnO4-methyl violet to stain the silkworm microparticles purple, while the green spores and Aspergillus spores appear purplish-brown, and the pollen grains appear as pale yellow lumps.

[0028] In a preferred embodiment, the microparticle image in step six has the following characteristics:

[0029] (1) The microparticles are oval in shape, with a size of (3.6~3.8)μm×(2.0~2.3)μm. They appear pale green under a microscope, have a smooth surface, and have strong refractive properties.

[0030] (2) The image contains a large number of silkworm moth fragments and moth hairs of various shapes, as well as spores that are similar in appearance to microparticles, such as green spores.

[0031] (3) There is overlap between spores;

[0032] (4) Since the specimen is a water-immersed specimen, the microparticles move, which causes changes in the shape and size of the microparticles and blurs the overall image.

[0033] In a preferred embodiment, the silkworm microparticle image needs to undergo chain code tracking. After chain code tracking, seven feature parameters of the microparticle virus need to be extracted: perimeter, width, height, area, elongation, complexity, and rectangularity. The calculation method for these feature parameters is as follows:

[0034] (1) Circumference

[0035] The perimeter of a silkworm microparticle, i.e., the number of pixels occupied by the microspore's edge outline, can be calculated using the 8-connected chain code method:

[0036] L=sqrt(2)*N0+N e

[0037] In the formula: N0 is the number of pixels in the odd chain code, N e The number of pixels in an even-numbered chain code;

[0038] (2) Long axis

[0039] The major axis of a silkworm microparticle is the length occupied by the pixels of the major axis of the ellipse containing the microparticle;

[0040] (3) Minor axis

[0041] The minor axis of a silkworm microparticle is the length of the line segment that intersects the outer edge of the silkworm microparticle with a straight line that passes through the center point of the ellipse containing the silkworm microparticle and is perpendicular to the major axis.

[0042] (4) Area

[0043] The area of ​​a silkworm microparticle is the sum of the number of all pixels contained in the microparticle; in a binary image, if 1 is used to represent a microparticle and 0 is used to represent the background, then the area is the sum of the number of pixels in the image with a value of 1.

[0044] (5) Elongation

[0045] Elongation represents the ratio of the width to the length of the smallest bounding rectangle of a microparticle, indicating the elongation of the microparticle. As the shape of silkworm microparticles becomes more elongated, their elongation will decrease. When E is 1, the shape is square, and the elongation reflects how close the microparticle is to an ellipse. Its calculation formula is:

[0046] E = min(W,H) / max(W,H);

[0047] (6) Complexity

[0048] Complexity is calculated from the area and perimeter, reflecting how closely the silkworm microparticles resemble a circle. When the identified image portion is circular, the complexity reaches its maximum of 1, calculated using the following formula:

[0049] Z = L 2 / A, where A is the area of ​​the region and L is the perimeter of the region;

[0050] (7) Rectangularity

[0051] Rectangularity is the ratio of the area of ​​a silkworm microparticle to the area of ​​its smallest bounding rectangle, reflecting how closely the silkworm microparticle resembles its bounding rectangle. The closer the shape of the silkworm microparticle is to a rectangle, the larger the rectangularity value. Its mathematical expression is as follows:

[0052] R = S / S mer In the formula, S is the area of ​​the microparticle. mer It is the area of ​​the smallest bounding rectangle of the microparticle.

[0053] The beneficial effects of this invention are as follows:

[0054] Using the controlled variable method, three experimental groups were conducted. By controlling the pH value and ion concentration under different environments, the final number of microparticles was detected, verifying the influence of different pH values ​​and ion concentrations on the accuracy of the experiment. At the same time, after the samples were purified, the impurity content was significantly reduced. The appearance characteristics of microparticles were extracted and identified using a microscope. By using color marking, microparticles could be counted in a short time with high efficiency. Attached Figure Description

[0055] Figure 1 This is the accuracy rate of the present invention under the condition that the ion concentration and pH value remain constant;

[0056] Figure 2 This is the accuracy of the invention under conditions where the ion concentration increases;

[0057] Figure 3 This refers to the accuracy of the invention when the pH value increases. Detailed Implementation

[0058] The present invention will now be described in further detail with reference to the accompanying drawings and specific embodiments. The embodiments of the present invention are given for illustrative and descriptive purposes only, and are not intended to be exhaustive or to limit the invention to the forms disclosed. Many modifications and variations will be apparent to those skilled in the art. The embodiments were chosen and described to better illustrate the principles and practical application of the invention, and to enable those skilled in the art to understand the invention and design various embodiments with various modifications suitable for a particular purpose.

[0059] Example 1:

[0060] Please see Figure 1 - Figure 3 This invention provides a technical solution: a method for detecting silkworm microparticle viruses, comprising the following steps:

[0061] Step 1: Take three samples of the same type of silkworm eggs and label them A, B, and C respectively. Place the three samples A, B, and C in a constant temperature incubator at a temperature of 26 degrees Celsius for a 20-day incubation period. Select the hatched larvae and remove the unhatched eggs, and label them A1, A2, and A3 respectively.

[0062] Step 2: Take three glass culture chambers, sterilize them, and place the three samples in a sealed aerobic environment, continuously feeding them mulberry leaves;

[0063] Step 3: One and a half weeks before cocoon formation, inoculate the virus spores at a concentration of 10... 5 Add a dose of / to mulberry leaves and administer to A1 samples; inoculate viral spores at 10 3 / dose added to mulberry leaves; A2 sample was given to consume, and virus spores were added to mulberry leaves at a dose of 0; A3 sample was given to consume.

[0064] Step 4: Continue to culture the three samples until they form cocoons, and then purify the microparticles;

[0065] Step 5: Microsporidia are activated to germinate and then fluorescently labeled.

[0066] Step 6: Observe the images using an electron microscope and calculate the number of microsporin viruses.

[0067] In step four, the purification method for the microparticles is as follows:

[0068] Step a: Disinfect the surface of the three samples of silkworm pupae (A1, A2, and A3) with 75% ethanol, grind them thoroughly in a mortar, and collect the grinding liquid in a 10mL centrifuge tube;

[0069] Step b: After diluting the grinding solution, set the centrifuge to 500 r / min, centrifuge for 5 min, and collect the supernatant;

[0070] Step c: Filter the supernatant through four layers of gauze, collect the filtrate, and centrifuge the filtrate at 3000 r / min for 5 min. Keep the precipitate and discard the supernatant.

[0071] Step d: Resuspend the precipitate with sterile hydrogen peroxide;

[0072] Step e: Soak sterile absorbent cotton in water and press it into a 10mL syringe. Then filter the spore suspension through the syringe, centrifuge at 3000r / min for 5min, discard the supernatant, and repeat steps d and e three times.

[0073] Step f: Add an appropriate amount of hydrogen peroxide to resuspend the precipitate, which is the purified spore suspension.

[0074] Table 1

[0075] Group <![CDATA[Ca 2+ Concentration pH value temperature Number of tests Accuracy (%) Experimental group A1 0.1 9 20℃ 2500 100 Experimental group A2 0.1 9 20℃ 1000 100 Experimental group A3 0.1 9 20℃ 0 100

[0076] Example 2:

[0077] Please see Figure 1 - Figure 3 This invention provides a technical solution: a method for detecting silkworm microparticle viruses, comprising the following steps:

[0078] Step 1: Take three samples of the same type of silkworm eggs and label them A, B, and C respectively. Place the three samples A, B, and C in a constant temperature incubator at a temperature of 26 degrees Celsius for a 20-day incubation period. Select the hatched larvae and remove the unhatched eggs, and label them A1, A2, and A3 respectively.

[0079] Step 2: Take three glass culture chambers, sterilize them, and place the three samples in a sealed aerobic environment, continuously feeding them mulberry leaves;

[0080] Step 3: One and a half weeks before cocoon formation, inoculate the virus spores at a concentration of 10... 5 Add a dose of / to mulberry leaves and administer to A1 samples; inoculate viral spores at 10 3 / dose added to mulberry leaves; A2 sample was given to consume, and virus spores were added to mulberry leaves at a dose of 0; A3 sample was given to consume.

[0081] Step 4: Continue to culture the three samples until they form cocoons, and then purify the microparticles;

[0082] Step 5: Microsporidia are activated to germinate and then fluorescently labeled.

[0083] Step 6: Observe the images using an electron microscope and calculate the number of microsporin viruses.

[0084] In step four, the purification method for the microparticles is as follows:

[0085] Step a: Disinfect the surface of the three samples of silkworm pupae (A1, A2, and A3) with 75% ethanol, grind them thoroughly in a mortar, and collect the grinding liquid in a 10mL centrifuge tube;

[0086] Step b: After diluting the grinding solution, set the centrifuge to 500 r / min, centrifuge for 5 min, and collect the supernatant;

[0087] Step c: Filter the supernatant through four layers of gauze, collect the filtrate, and centrifuge the filtrate at 3000 r / min for 5 min. Keep the precipitate and discard the supernatant.

[0088] Step d: Resuspend the precipitate with sterile hydrogen peroxide;

[0089] Step e: Soak sterile absorbent cotton in water and press it into a 10mL syringe. Then filter the spore suspension through the syringe, centrifuge at 3000r / min for 5min, discard the supernatant, and repeat steps d and e three times.

[0090] Step f: Add an appropriate amount of hydrogen peroxide to resuspend the precipitate, which is the purified spore suspension.

[0091] Table 2

[0092] Group <![CDATA[Ca 2+ Concentration pH value temperature Number of tests Accuracy (%) Experimental group A1 0.2 9 20℃ 2500 100 Experimental group A2 0.2 9 20℃ 1000 100 Experimental group A3 0.2 9 20℃ 0 100

[0093] As can be seen from Tables 1 and 2, compared with Table 1, the accuracy rates of the experimental groups in Table 1 and Table 2 are equal, while the differences in ion concentration, pH value, and temperature among the experimental groups are not significant (p>0.05).

[0094] Example 3:

[0095] Please see Figure 1 - Figure 3 This invention provides a technical solution: a method for detecting silkworm microparticle viruses, comprising the following steps:

[0096] Step 1: Take three samples of the same type of silkworm eggs and label them A, B, and C respectively. Place the three samples A, B, and C in a constant temperature incubator at a temperature of 26 degrees Celsius for a 20-day incubation period. Select the hatched larvae and remove the unhatched eggs, and label them A1, A2, and A3 respectively.

[0097] Step 2: Take three glass culture chambers, sterilize them, and place the three samples in a sealed aerobic environment, continuously feeding them mulberry leaves;

[0098] Step 3: One and a half weeks before cocoon formation, inoculate the virus spores at a concentration of 10... 5 Add a dose of / to mulberry leaves and administer to A1 samples; inoculate viral spores at 10 3 / dose added to mulberry leaves; A2 sample was given to consume, and virus spores were added to mulberry leaves at a dose of 0; A3 sample was given to consume.

[0099] Step 4: Continue to culture the three samples until they form cocoons, and then purify the microparticles;

[0100] Step 5: Microsporidia are activated to germinate and then fluorescently labeled.

[0101] Step 6: Observe the images using an electron microscope and calculate the number of microsporin viruses.

[0102] In step four, the purification method for the microparticles is as follows:

[0103] Step a: Disinfect the surface of the three samples of silkworm pupae (A1, A2, and A3) with 75% ethanol, grind them thoroughly in a mortar, and collect the grinding liquid in a 10mL centrifuge tube;

[0104] Step b: After diluting the grinding solution, set the centrifuge to 500 r / min, centrifuge for 5 min, and collect the supernatant;

[0105] Step c: Filter the supernatant through four layers of gauze, collect the filtrate, and centrifuge the filtrate at 3000 r / min for 5 min. Keep the precipitate and discard the supernatant.

[0106] Step d: Resuspend the precipitate with sterile hydrogen peroxide;

[0107] Step e: Soak sterile absorbent cotton in water and press it into a 10mL syringe. Then filter the spore suspension through the syringe, centrifuge at 3000r / min for 5min, discard the supernatant, and repeat steps d and e three times.

[0108] Step f: Add an appropriate amount of hydrogen peroxide to resuspend the precipitate, which is the purified spore suspension.

[0109] Table 3

[0110] Group <![CDATA[Ca 2+ Concentration pH value temperature Number of tests Accuracy (%) Experimental group A1 0.2 10 20℃ 2500 100 Experimental group A2 0.2 12 20℃ 500 50 Experimental group A3 0.2 11 20℃ 0 100

[0111] As can be seen from Tables 2 and 3, the accuracy of Table 2 is much higher than that of Table 3 (p<0.05).

[0112] In step five, the activation and budding of microsporidia are crucial steps in the host infection process. During host infection, purified spore polar filaments are ejected, infecting host cells. The conditions required for activation mainly include environmental pH and calcium. 2+ Concentration, intraspore osmotic pressure, and other important aspects;

[0113] Microsporidia require a specific pH environment for activation. Microsporidia parasitizing lepidopteran insects need to be treated with an ion buffer solution of a certain pH value to germinate. The germination effect of silkworm microsporidia is most significant in an external environment of 20℃ and pH value of 9.0-11.0.

[0114] Ca 2+ It is a second messenger essential for many cell activation processes, and plays a crucial role in the activation and invasion of host cells by microsporidia spores. During the invasion of host cells, as the sporotubes are ejected, calcium bound to the spore wall and plasma membrane... 2+ Gradually decreasing, microspore germination depends on Ca. 2+ Experiments showed that when the external CaCl2 concentration was 0.1 mol / L or 0.2 mol / L and the pH was 9.0, it facilitated the infection of microsporidia. After the integranins and calmodulin in the intermediate lamellar assemblage of the microspores were removed, their germination activation ability was inhibited, resulting in permanent inactivation of the spores. 2+ It also has side effects, increasing the amount of calcium in the environment. 2+ The concentration of P ≤ 0.5 will inhibit the germination of microsporidia and thus hinder the infection of microsporidia.

[0115] The sporofilament ejection and host cell infection process includes the following steps:

[0116] (1) Adhesion of spores to host cells;

[0117] (2) Spore activation;

[0118] (3) The osmotic pressure inside the spores increases;

[0119] (4) The wires pop outwards;

[0120] (5) The sporogenous material enters the cytoplasm of the host cell through the polar tube.

[0121] In step five, the fluorescent developing label uses KMnO4-methyl violet to stain the silkworm microparticles purple, while the green spores and Aspergillus spores appear purplish-brown, and the pollen grains appear as pale yellow lumps.

[0122] In step six, the microparticle image has the following characteristics:

[0123] (1) The microparticles are oval in shape, with a size of (3.6~3.8)μm×(2.0~2.3)μm. They appear pale green under a microscope, have a smooth surface, and have strong refractive properties.

[0124] (2) The image contains a large number of silkworm moth fragments and moth hairs of various shapes, as well as spores that are similar in appearance to microparticles, such as green spores.

[0125] (3) There is overlap between spores;

[0126] (4) Since the specimen is a water-immersed specimen, the microparticles move, which causes changes in the shape and size of the microparticles and blurs the overall image.

[0127] Silkworm microparticle images require chain code tracking. After chain code tracking, seven feature parameters of the microparticle virus need to be extracted: perimeter, width, height, area, elongation, complexity, and rectangularity. The calculation methods for these feature parameters are as follows:

[0128] (1) Circumference

[0129] The perimeter of a silkworm microparticle, i.e., the number of pixels occupied by the microspore's edge outline, can be calculated using the 8-connected chain code method:

[0130] L=sqrt(2)*N0+N e

[0131] In the formula: N0 is the number of pixels in the odd chain code, N e The number of pixels in an even-numbered chain code;

[0132] (2) Long axis

[0133] The major axis of a silkworm microparticle is the length occupied by the pixels of the major axis of the ellipse containing the microparticle;

[0134] (3) Minor axis

[0135] The minor axis of a silkworm microparticle is the length of the line segment that intersects the outer edge of the silkworm microparticle with a straight line that passes through the center point of the ellipse containing the silkworm microparticle and is perpendicular to the major axis.

[0136] (4) Area

[0137] The area of ​​a silkworm microparticle is the sum of the number of all pixels contained in the microparticle; in a binary image, if 1 is used to represent a microparticle and 0 is used to represent the background, then the area is the sum of the number of pixels in the image with a value of 1.

[0138] (5) Elongation

[0139] Elongation represents the ratio of the width to the length of the smallest bounding rectangle of a microparticle, indicating the elongation of the microparticle. As the shape of silkworm microparticles becomes more elongated, their elongation will decrease. When E is 1, the shape is square, and the elongation reflects how close the microparticle is to an ellipse. Its calculation formula is:

[0140] E = min(W,H) / max(W,H);

[0141] (6) Complexity

[0142] Complexity is calculated from the area and perimeter, reflecting how closely the silkworm microparticles resemble a circle. When the identified image portion is circular, the complexity reaches its maximum of 1, calculated using the following formula:

[0143] Z = L 2 / A, where A is the area of ​​the region and L is the perimeter of the region;

[0144] (7) Rectangularity

[0145] Rectangularity is the ratio of the area of ​​a silkworm microparticle to the area of ​​its smallest bounding rectangle, reflecting how closely the silkworm microparticle resembles its bounding rectangle. The closer the shape of the silkworm microparticle is to a rectangle, the larger the rectangularity value. Its mathematical expression is as follows:

[0146] R = S / S mer In the formula, S is the area of ​​the microparticle. mer It is the area of ​​the smallest bounding rectangle of the microparticle.

[0147] Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of them. All other embodiments obtained by those skilled in the art and related fields based on the embodiments of the present invention without inventive effort should fall within the scope of protection of the present invention. Structures, devices, and operating methods not specifically described and explained in the present invention, unless otherwise specified or limited, shall be implemented according to conventional means in the art.

Claims

1. A method for detecting silkworm microparticle viruses, characterized in that, Includes the following steps: Step 1: Take three samples of the same type of silkworm eggs and label them A, B, and C respectively. Place the three samples A, B, and C in a constant temperature incubator at a temperature of 26 degrees Celsius for a 20-day incubation period. Select the hatched larvae and remove the unhatched eggs, and label them A1, A2, and A3 respectively. Step 2: Take three glass culture chambers, sterilize them, and place the three samples in a sealed aerobic environment, continuously feeding them mulberry leaves; Step 3: One and a half weeks before cocoon formation, inoculate the virus spores at a concentration of 10... 5 Add a dose of / to mulberry leaves and administer to A1 samples; inoculate viral spores at 10 3 / dose added to mulberry leaves; A2 sample was given to consume, and virus spores were added to mulberry leaves at a dose of 0; A3 sample was given to consume. Step 4: Continue to culture the three samples until they form cocoons, and then purify the microparticles; Step 5: Microsporidia are activated to germinate and then fluorescently labeled. Step Six: Observe the images using an electron microscope and calculate the number of microsporin viruses; In step four, the purification method for the microparticles is as follows: Step a: Disinfect the surface of the three samples of silkworm pupae (A1, A2, and A3) with 75% ethanol, grind them thoroughly in a mortar, and collect the grinding liquid in a 10mL centrifuge tube; Step b: After diluting the grinding slurry, set the centrifuge to 500 r / min, centrifuge for 5 min, and collect the supernatant; Step c: Filter the supernatant through four layers of gauze, collect the filtrate, and centrifuge the filtrate at 3000 r / min for 5 min. Keep the precipitate and discard the supernatant. Step d: Resuspend the precipitate with sterile hydrogen peroxide; Step e: Soak sterile absorbent cotton in water and press it into a 10mL syringe. Then filter the spore suspension through the syringe, centrifuge at 3000r / min for 5min, discard the supernatant, and repeat steps d and e three times. Step f: Add an appropriate amount of hydrogen peroxide to resuspend the precipitate, which is the purified spore suspension; In step five, the activation and budding of microsporidia are crucial steps in the host infection process. During host infection, purified spore polar filaments are ejected, infecting host cells. The conditions required for activation mainly include environmental pH and calcium. 2+ Concentration, intraspore osmotic pressure, and other important aspects; Microsporidia require a specific pH range of 9.0-11.0 for activation. Microsporidia that parasitize lepidopteran insects need to be treated with an ion buffer solution with a pH range of 9.0-11.0 to germinate.

2. The method for detecting silkworm microparticle virus according to claim 1, characterized in that: The sporofilament ejection and host cell infection include the following steps: (1) Adhesion of spores to host cells; (2) Spore activation; (3) The osmotic pressure inside the spores increases; (4) The wires pop outwards; (5) The sporogenous material enters the cytoplasm of the host cell through the polar tube.

3. The method for detecting silkworm microparticle virus according to claim 1, characterized in that: In step five, the fluorescent imaging labeling uses KMnO4-methyl violet to stain the silkworm microparticles purple, while the green spores and Aspergillus spores appear purplish-brown, and the pollen grains appear as pale yellow lumps.

4. The method for detecting silkworm microparticle viruses according to claim 1, characterized in that: In step six, the microparticle image has the following characteristics: (1) The microparticles are oval in shape, with a size of (3.6~3.8)μm×(2.0~2.3)μm. They appear pale green under a microscope, have a smooth surface, and have strong refractive properties. (2) The image contains a large number of silkworm moth fragments and moth hair impurities of various shapes, as well as green spores and microparticles that are similar in appearance. (3) There is overlap between spores; (4) Since the specimen is a water-immersed specimen, the microparticles move, which causes changes in the shape and size of the microparticles and blurs the overall image.

Images