CN114847243A - Method for obtaining BmNPV disease pupae by feeding late-stage 5-year-old virus-inoculated silkworms or puncturing and virus-inoculated silkworms - Google Patents

Abstract

The invention discloses a method for obtaining pure BmNPV diseased pupae free of other virus pathogens by feeding poison-inoculated 5th instar silkworms or piercing and inoculating with poison, comprising: soaking with BmNPV virus liquid with a concentration of 10 ⁵ -10 ⁹ PIB/ml The mulberry leaves were obtained with virus-bearing mulberry leaves; healthy silkworms at the end of the 5th instar of the BmNPV-sensitive strain were selected and fed with virus-bearing mulberry leaves to ensure that among the three feeding mulberry leaves before the silkworms matured, 1 feeding was with virus-bearing mulberry leaves, and when the silkworms matured Cocooning and pupation, diseased pupae are obtained; or silkworms from the 5th instar stage of the BmNPV sensitive strain are selected, and the mulberry leaves with virus are fed, and the blood of live silkworms is taken after the onset, inoculated into the healthy silkworms in the late 5th instar stage of the BmNPV sensitive strain, and the silkworms mature. The upper cocoon and pupation are formed, and the diseased pupa is obtained; or the silkworm from the 5th instar of the BmNPV sensitive strain is selected, and the mulberry leaf with virus is fed. After the silkworm is full, the normal mulberry leaf is fed. 24-48 hours of BmNPV sensitive strain healthy silkworm pupae, diseased pupae were obtained. The invention has the advantages of convenient and simple operation, less impurities, high propagation efficiency and low cost.

Description

Method for obtaining BmNPV diseased pupae by feeding poisoned silkworms at the end of the fifth instar stage or puncturing the poisoning

technical field

The invention relates to the technical field of silkworm disease-resistant breeding, and more particularly, to a method for obtaining pure BmNPV diseased pupae free of other virus pathogens by feeding the silkworms at the end of the fifth instar stage of inoculation or puncture inoculation.

Background technique

Bombyx mori (Bombyx mori) blood pus disease (BmNPV) is a subacute infectious disease of the silkworm, which is one of the main diseases that cause the production of sericulture, especially in the tropical high temperature season of Guangxi. The occurrence of this disease is caused by the infection of silkworms by silkworm nuclear polyhedrosis virus (BmNPV). Both the environment and objects can be a source of reinfection.

The biological characteristics of silkworms and the high-density rearing method in silkworm clusters in a certain space determine that once the disease occurs, it will have the characteristics of large losses and rapid infection, and it also determines that the prevention and treatment of silkworm blood-type pus disease is focused on group control. rather than individual control and treatment. Therefore, silkworm breeding for disease resistance is particularly important. For silkworm disease resistance breeding, BmNPV is generally used to screen moth areas or individuals, and a large amount of BmNPV needs to be used in the entire breeding process.

Chinese patent CN108220250A discloses the method of live piercing and inoculation of silkworm for virus propagation, and the shortcomings are: 1. Silkworm body injection is not easy to operate; When the diseased silkworm is collected and broken, the skin and silk glands of the large silkworm are not easily broken; 4. The silkworm is still in the mulberry-eating stage, and there are many impurities after breaking, which affects the purification of pathogens; 5. The silkworm larvae can be infected with a variety of BmNPV, CPV, FV, etc For a variety of viral diseases, it is difficult to prepare BmNPV polyhedrosis virus without other viral diseases by using BmNPV propagated by larvae.

SUMMARY OF THE INVENTION

It is an object of the present invention to address at least the above-mentioned disadvantages and to provide at least the advantages which will be explained hereinafter.

Another object of the present invention is to provide a method for obtaining pure BmNPV diseased pupae without other viral pathogens by feeding the silkworm at the end of the 5th instar silkworm or by piercing the poison, which is used to feed the silkworm at the end of the 5th instar or the pupa stage. Poison mulberry leaves or inoculate the blood of diseased silkworms, so that after the silkworm disease occurs in the upper cocoon, there will be no manic crawling phenomenon during the onset of the silkworm stage, the pupa body is quieter, and the operation is more convenient and simpler than the lively silkworm body during puncture inoculation, and the pupa body is more convenient and simple than silkworm body. The body is more easily broken, and the broken solution has less impurities than silkworms in the feeding period, which is easy to extract viruses, has high propagation efficiency and low cost, and the obtained pathogens do not contain other virus pathogens.

In order to achieve these objects and other advantages of the present invention, the present invention provides a method for obtaining pure BmNPV diseased pupae that do not contain other viral pathogens by feeding the 5th instar late stage silkworm or puncture inoculation, comprising:

Using the BmNPV virus solution with a concentration of 10 ⁵ -10 ⁹ PIB/ml to impregnate mulberry leaves to obtain virus-carrying mulberry leaves;

A: Select healthy silkworms at the end of the 5th instar of BmNPV-sensitive strains, and feed them with virus-bearing mulberry leaves to ensure that mulberry leaves with virus are fed once within 2 days before the silkworms mature. After, the disease obtains the described BmNPV disease pupae;

Or B: Select silkworms from the 5th instar of the BmNPV-sensitive strain, and feed them with virus-bearing mulberry leaves. After the silkworms are full, feed the normal mulberry leaves until the live silkworms show typical symptoms, and then take the blood of the live silkworms and inoculate them into 5 of the BmNPV-sensitive strains. On the healthy silkworm at the end of the age, after the silkworm matures, the upper cocoon forms and pupates, and the disease obtains the described BmNPV disease pupa;

Or C: Select the 5th instar silkworm of the BmNPV sensitive strain, feed the virus-bearing mulberry leaves, after the silkworm is full, feed the normal mulberry leaf until the live silkworm has typical symptoms, then take the blood of the live silkworm and inoculate it to pupation 24-48 On the healthy silkworm pupae of the BmNPV-sensitive strain of 1 hour, the diseased pupae of the BmNPV were obtained.

According to the characteristics of silkworms, silkworms need to eat enough food before they mature before they can form cocoons. The cocoons are formed in about 2 days after the cocoons, and the cocoons are formed and pupated in about 4 days, that is, the 5th instar silkworm The time from the last stage to cocooning and pupation is 4 to 5 days. According to this characteristic, the above A, B or C schemes designed by the applicant can all ensure that the silkworm will develop a cocoon after the upper cocoon, and the silkworm stage will not appear at this stage. Manic crawling phenomenon at the time of onset, the pupa body is relatively quiet, and the operation is more convenient and simpler than the lively silkworm body during puncture inoculation, the pupa body is more easily broken than the silkworm body, and the broken solution is less impurity than that of the silkworm in the feeding period. By extracting the virus, the propagation efficiency is high and the cost is low, thereby solving the problems existing in the traditional silkworm live puncture inoculation mentioned in the background art.

In addition, during the virus rejuvenation process of B and C, 5-instar silkworms were selected, fed with virus-bearing mulberry leaves, and then the blood of live silkworms was collected. Sufficient, easy to draw blood; second, large food intake, fast growth, can quickly eat mulberry leaves with virus and get sick; second, in the 5th instar, the silkworm in the 5th instar is more sensitive to the virus and has low resistance. Therefore, it is easy to obtain diseased silkworms.

Preferably, in the described method for feeding and inoculating silkworms at the end of the 5th instar stage or piercing and inoculating to obtain pure BmNPV pupae that do not contain other viral pathogens, in described A, the virus concentration using the BmNPV virus solution is 10 ⁵ PIB/ ml, the mulberry leaves were soaked in the virus solution for 10 minutes, and then air-dried to obtain the virus-carrying mulberry leaves;

In the B and C, the virus concentration of the BmNPV virus solution was 10 ⁹ PIB/ml, and the mulberry leaves were soaked in the virus solution for 10 minutes, and then air-dried to obtain the virus-carrying mulberry leaves.

In the above technical scheme, the feeding of A uses a virus solution with a lower virus concentration of 10 ⁵ , and combined with the normal mulberry leaf intake of silkworms, it can ensure that most silkworms ingest an appropriate amount of virus, and avoid excessive intake of virus. , In the later stage, it can form cocoons but cannot pupate, and it can also avoid the situation where the intake of virus is too small and the disease cannot be caused. The feeding of B and C using a higher concentration of virus liquid of 10 ⁹ can make the 5th instar silkworms quickly develop disease, and obtain a large amount of live silkworm blood, which improves the efficiency.

Preferably, in the described method of feeding and inoculating silkworms at the end of the 5th instar stage or piercing and inoculating to obtain pure BmNPV diseased pupae that do not contain other viral pathogens, in the described A, it is specifically the day when the silkworm matures or the day before the feeding with virus On the mulberry leaves, the silkworms mature and grow up, and the immature silkworms continue to feed on the normal mulberry leaves.

In the conventional silkworm raising method, mulberry leaves are fed 1-4 times a day, which is also called 1-4 times a day in the industry. In the present invention, 2 times a day is used. The amount of virus mulberry leaves fed is no different from the amount of conventional feeding, and it is enough to ensure that the silkworms are full. For example, according to the moth area (an average of 400 silkworms in a moth area), the 5th instar silkworms are fed 6kg of mulberry leaves throughout the silkworm period (calculated by 6 days), so that one moth area is fed about 0.5kg of leaves at a time.

Preferably, in the method for the pure BmNPV disease pupae obtained by feeding the poisoned 5th instar silkworm or piercing and inoculating to obtain the pure BmNPV disease pupa that does not contain other virus pathogens, in the B and C, in order to ensure that the silkworm is full of the mulberry leaves with the virus, After feeding the virus-carrying mulberry leaves, wait 8 to 12 hours, and then use normal mulberry leaves to feed after the virus-carrying mulberry leaves have been eaten up or are close to being eaten up.

Preferably, in the described method of feeding and inoculating silkworms at the end of the fifth instar stage or piercing and inoculating to obtain pure BmNPV diseased pupae that do not contain other virus pathogens, the live silkworms with typical symptoms are collected after feeding mulberry leaves with virus for 5 to 6 days. , Use alcohol to sterilize the body surface of the live silkworm. After the body surface is dried, cut the tail to collect blood. The collected blood is immediately used for inoculation, or used within 6 hours of refrigerated storage.

Preferably, in the described method for feeding and inoculating silkworms at the end of the 5th instar stage or piercing and inoculating to obtain pure BmNPV disease pupae that do not contain other viral pathogens, the inoculation method for the healthy silkworms at the end of the 5th instar in B is: removing the poison is good The syringe extracts the blood of the live silkworm, and the silkworm body is counted in the direction of the tail, from any intersegmental membrane of the 6-9th link, pierces the silkworm body wall, and inoculates the blood of the live silkworm;

The inoculation method for the healthy silkworm chrysalis in C is: cancel the poisonous syringe to draw the described diseased silkworm blood, the pupa body is counted toward the tail direction, and the pupa skin is gently pierced from the 2nd or 3rd link on the back of the pupa, and the live Silkworm blood was inoculated into silkworm pupae.

Preferably, in the described method for obtaining pure BmNPV diseased pupae without other virus pathogens by feeding the poisoned silkworms at the end of the fifth instar stage or by puncturing and inoculating with poisoning, in the A and B, after the silkworms mature and grow up, at 25-26 ℃ temperature, normal growth under the condition of 65% air humidity, collect silkworm after 2 days, silkworm pupation after 2 days, cut cocoon to obtain pupa, pupa appears diseased after 3～4 days, collect diseased pupa, store in -20 °C;

In the C, the inoculated silkworm pupae develop for 96 to 120 hours at 25 to 29°C. In the middle and late stages of the disease, the pupae appear diseased, and the diseased pupae are collected and stored at -20°C.

Preferably, in the described method for feeding and inoculating silkworms at the end of the 5th instar stage or piercing and inoculating to obtain pure BmNPV pupae that do not contain other viral pathogens, the method specifically includes:

Take out the BmNPV virus stored at -20°C and thaw it, add sterile water to resuspend, centrifuge at 4000 rpm for 10 minutes to remove the supernatant, repeat 2-3 times, and wash away the drugs added during virus preservation; then add sterile water to resuspend , count the BmNPV suspension with a hemocytometer and an optical microscope, and configure it into a virus solution of 1×10 ⁵ PIB/ml for later use;

The mulberry leaves were soaked in the virus solution for 10 min, fully immersed evenly, and the surface moisture of the mulberry leaves was taken out and dried to obtain virus-bearing mulberry leaves for subsequent use;

Select the late 5th instar silkworms of the BmNPV sensitive strain, on the day or the day before the silkworm matures;

Feed the dried mulberry leaves with virus to silkworms to ensure that 3 times of mulberries are added before ripening, and one of them is virus leaves, and the mature silkworms are swarmed, and the immature silkworms continue to feed normal leaves;

Cooked silkworms are cocooned at 25-26℃ and humidity 65% after cocooning for 2 days, and cocoons are cut after 2 days of pupation. The pupae are laid in a single layer in the silkworm nest and appear after 3-4 days. If the link is relaxed, the pupa is soft, and the body wall is easily broken, the diseased pupae can be collected and stored at -20°C.

Preferably, in the described method for feeding and inoculating silkworms at the end of the 5th instar stage or piercing and inoculating to obtain pure BmNPV pupae that do not contain other viral pathogens, the method specifically includes:

The preserved BmNPV virus was prepared into a virus solution with a concentration of 10 ⁹ PIB/ml, mulberry leaves were soaked in the virus solution for 10 minutes, and the mulberry leaves were picked up and dried to obtain virus-bearing mulberry leaves;

The virus-bearing mulberry leaves were fed to the 5th instar silkworms of BmNPV-sensitive strains to ensure that the silkworms were full. After 8-12 hours, the virus leaves were eaten or close to the light, and then the normal leaves were used for feeding, and the collection appeared after 5-6 days. Live silkworms with typical symptoms of manic crawling, swollen links and intersegmental membranes, oil-soaked trachea, and turbid blood, disinfect the silkworm body with 75% alcohol, and cut the tail angle after drying to collect blood. At this time, the blood contains a lot of virus ;

The collected blood is used for puncture inoculation, and the blood can be used immediately, or the storage time shall not exceed 6 hours;

Select healthy silkworms at the end of the fifth instar of BmNPV sensitive strains; use a 32G syringe, boil for 30 minutes before use, and then use 75% alcohol to sterilize;

The blood is drawn from the syringe with the good detoxification, from the head of the silkworm to any intersegmental membrane of the 6-9th link in the direction of the tail, pierces the silkworm body wall, and inoculates the blood with the BmNPV virus;

After the inoculation, the cooked silkworms are cocooned, carried out at 25-26 ° C, and the normal temperature and humidity protection of 65% air humidity is carried out. After 3-4 days, the link relaxation occurs, the pupae are soft, and the body wall is easily broken. The diseased pupae can be collected and stored at -20°C.

Preferably, in the described method for feeding and inoculating silkworms at the end of the 5th instar stage or piercing and inoculating to obtain pure BmNPV pupae that do not contain other viral pathogens, the method specifically includes:

The preserved BmNPV virus was prepared into a virus solution with a concentration of 10 ⁹ PIB/ml, mulberry leaves were soaked in the virus solution for 10 minutes, and the mulberry leaves were picked up and dried to obtain virus-bearing mulberry leaves;

The virus-bearing mulberry leaves were fed to the 5th instar silkworms of BmNPV-sensitive strains to ensure that the silkworms were full. After 8-12 hours, the virus leaves were eaten or close to the light, and then the normal leaves were used for feeding, and the collection appeared after 5-6 days. Live silkworms with typical symptoms of manic crawling, swollen links and intersegmental membranes, oil-soaked spots on the stoma, and turbid blood were used to disinfect the silkworm body with 75% alcohol by volume. After drying, cut the tail angle to collect blood. a large number of viruses;

The collected blood is used for puncture inoculation, and the blood can be used immediately, or the storage time shall not exceed 6 hours;

Select healthy tender silkworm chrysalis of BmNPV sensitive strain that pupates for 24-48 hours, the pupa skin just hardens now, and the pupa body is soaked and disinfected for 30s with the bleaching powder solution of available chlorine 1%, rinsed, air-dried, and is for subsequent use;

Use a 32G syringe, boil it for 30 minutes before use, and then use 75% alcohol to sterilize it;

The blood is drawn from the syringe that the poison is removed, and the pupa skin is gently pierced from the 2nd or 3rd link of the pupa back of the pupa head to the tail direction, and the blood is inoculated in the silkworm chrysalis;

The silkworm pupa after the inoculation is placed under the condition of 25-29 ℃ and develops for 96-120 hours, to the middle and late stage of the disease, the pupa body appears to be relaxed in the link, and the pupa body begins to soften the symptoms, and the diseased pupa can be collected, and the collected diseased pupa. Store at -20°C.

The present invention includes at least the following beneficial effects:

The method of the invention has low requirements on equipment and tools, and low requirements on the technical level of personnel; low requirements on environmental conditions, no phenomenon of manic crawling during the onset of the silkworm period after the onset of the disease, thereby polluting the environment, and has little impact on the safety and cleanliness of the environment; The pupa body is relatively quiet, and the operation is more convenient and simpler than the lively silkworm body during puncture inoculation; the pupa body is easier to break than the silkworm body, and the solution after breaking is less impurity than the silkworm in the feeding period after breaking, so it is easy to extract the virus; the propagation efficiency High and low cost.

Other advantages, objects, and features of the present invention will appear in part from the description that follows, and in part will be appreciated by those skilled in the art from the study and practice of the invention.

Detailed ways

The present invention will be further described in detail below with reference to the embodiments, so that those skilled in the art can implement according to the description.

It should be understood that terms such as "having", "comprising" and "including" as used herein do not exclude the presence or addition of one or more other elements or combinations thereof.

It should be noted that the experimental methods described in the following embodiments are conventional methods unless otherwise specified, and the reagents and materials can be obtained from commercial sources unless otherwise specified.

Example 1

The method of feeding the poisoned silkworm at the end of the fifth instar stage or puncturing the poisoned pupae to obtain pure BmNPV diseased pupae without other viral pathogens, including:

Take out the BmNPV virus stored at -20°C and thaw it, add sterile water to resuspend, centrifuge at 4000 rpm for 10 minutes to remove the supernatant, repeat 2-3 times, and wash away the drugs added during virus preservation; then add sterile water to resuspend , count the BmNPV suspension with a hemocytometer and an optical microscope, and configure it into a virus solution of 1×10 ⁵ PIB/ml for later use;

The mulberry leaves were soaked in the virus solution for 10 min, fully immersed evenly, and the surface moisture of the mulberry leaves was taken out and dried to obtain virus-bearing mulberry leaves for subsequent use;

Select the late 5th instar silkworms of the BmNPV sensitive strain, on the day or the day before the silkworm matures;

Feed the dried mulberry leaves with virus to silkworms to ensure that 3 times of mulberries are added before ripening, and one of them is virus leaves, and the mature silkworms are swarmed, and the immature silkworms continue to feed normal leaves;

Cooked silkworms are cocooned at 25-26℃ and humidity 65% after cocooning for 2 days, and cocoons are cut after 2 days of pupation. The pupae are laid in a single layer in the silkworm nest and appear after 3-4 days. If the link is slack, the pupae are soft, and the body wall is easily broken, the diseased pupae can be collected and stored at -20°C.

Example 2

The method of feeding the poisoned silkworm at the end of the fifth instar stage or puncturing the poisoned pupae to obtain pure BmNPV diseased pupae without other viral pathogens, including:

The preserved BmNPV virus was prepared into a virus solution with a concentration of 10 ⁹ PIB/ml, mulberry leaves were soaked in the virus solution for 10 minutes, and the mulberry leaves were picked up and dried to obtain virus-bearing mulberry leaves;

The virus-bearing mulberry leaves were fed to the 5th instar silkworms of BmNPV-sensitive strains to ensure that the silkworms were full. After 8-12 hours, the virus leaves were eaten or close to the light, and then the normal leaves were used for feeding, and the collection appeared after 5-6 days. Live silkworms with typical symptoms of manic crawling, swollen links and intersegmental membranes, oil-soaked trachea, and turbid blood, disinfect the silkworm body with 75% alcohol, and cut the tail angle after drying to collect blood. At this time, the blood contains a lot of virus ;

The collected blood is used for puncture inoculation, and the blood can be used immediately, or the storage time shall not exceed 6 hours;

Select healthy silkworms at the end of the fifth instar of BmNPV sensitive strains; use a 32G syringe, boil for 30 minutes before use, and then use 75% alcohol to sterilize;

The blood is drawn from the syringe with the good detoxification, from the head of the silkworm to any intersegmental membrane of the 6-9th link in the direction of the tail, pierces the silkworm body wall, and inoculates the blood with the BmNPV virus;

After the inoculation, the cooked silkworms are cocooned, carried out at 25-26 ° C, and the normal temperature and humidity protection of 65% air humidity is carried out. After 3-4 days, the link relaxation occurs, the pupae are soft, and the body wall is easily broken. The diseased pupae can be collected and stored at -20°C.

Example 3

The method of feeding the poisoned silkworm at the end of the fifth instar stage or puncturing the poisoned pupae to obtain pure BmNPV diseased pupae without other viral pathogens, including:

The preserved BmNPV virus was prepared into a virus solution with a concentration of 10 ⁹ PIB/ml, mulberry leaves were soaked in the virus solution for 10 minutes, and the mulberry leaves were picked up and dried to obtain virus-bearing mulberry leaves;

The virus-bearing mulberry leaves were fed to the 5th instar silkworms of BmNPV-sensitive strains to ensure that the silkworms were full. After 8-12 hours, the virus leaves were eaten or close to the light, and then the normal leaves were used for feeding, and the collection appeared after 5-6 days. Live silkworms with typical symptoms of manic crawling, swollen links and intersegmental membranes, oil-soaked spots on the stoma, and turbid blood were used to disinfect the silkworm body with 75% alcohol by volume. After drying, cut the tail angle to collect blood. a large number of viruses;

The collected blood is used for puncture inoculation, and the blood can be used immediately, or the storage time shall not exceed 6 hours;

Select healthy tender silkworm chrysalis of BmNPV sensitive strain that pupates for 24-48 hours, the pupa skin just hardens now, and the pupa body is soaked and disinfected for 30s with the bleaching powder solution of available chlorine 1%, rinsed, air-dried, and is for subsequent use;

Use a 32G syringe, boil it for 30 minutes before use, and then use 75% alcohol to sterilize it;

The blood is drawn from the syringe that the poison is removed, and the pupa skin is gently pierced from the 2nd or 3rd link of the pupa back of the pupa head to the tail direction, and the blood is inoculated in the silkworm chrysalis;

The silkworm pupa after the inoculation is placed under the condition of 25-29 ℃ and develops for 96-120 hours, to the middle and late stage of the disease, the pupa body appears to be relaxed in the link, and the pupa body begins to soften the symptoms, and the diseased pupa can be collected, and the collected diseased pupa. Store at -20°C.

The method for obtaining Bombyx mori nuclear polyhedrosis virus (BmNPV) through the diseased pupae of the present invention is as follows: after the diseased pupae of Examples 1-3 are broken, extract and collect the extract.

Although embodiments of the present invention have been disclosed above, they are not limited to the applications set forth in the specification and embodiments. It can be fully adapted to various fields suitable for the present invention. Additional modifications can readily be implemented by those skilled in the art.

Claims (5)

1. The method for obtaining pure BmNPV disease pupae without other virus pathogens by feeding virus-inoculated 5-year-old late stage silkworms or puncturing virus inoculation is characterized by comprising the following steps:

the use concentration is 10 ⁵ -10 ⁹ Soaking folium Mori in PIB/ml BmNPV virus solution to obtain virus-containing folium Mori;

a: healthy silkworms of a BmNPV sensitive strain at the late 5 th instar are fed with virus-carrying mulberry leaves, the mulberry leaves are fed within 2 days before the silkworms are mature for 1 time, and after the silkworms are mature, cocooning and pupating are carried out, and the disease of the BmNPV disease pupa is obtained;

or B: selecting silkworms of a BmNPV sensitive strain at the 5 th instar, feeding virus-carrying mulberry leaves, changing the silkworm after the silkworm is fed with virus-carrying mulberry leaves and after the silkworm is saturated with virus-carrying mulberry leaves and feeding the silkworm with virus-carrying mulberry leaves to normal mulberry leaves after the silkworm 2 nd instar until a typical symptom appears in a living silkworm, then taking the blood of the living silkworm, puncturing and inoculating the blood to healthy silkworms of the BmNPV sensitive strain at the 5 th instar later stage, and after the silkworms are mature, mounting cocoons and pupating, and then getting the disease pupas of the BmNPV;

or C: selecting silkworms of BmNPV sensitive strain at the 5 th instar, feeding virus-carrying mulberry leaves, feeding normal mulberry leaves after the silkworms are saturated until the living silkworms have a typical disease state, then taking the blood of the living silkworms, and puncturing and inoculating the blood to healthy silkworm pupas of the BmNPV sensitive strain which pupate for 24-48 hours to obtain the BmNPV diseased pupas.

2. The method for obtaining pure BmNPV disease pupae without other viral pathogens by feeding virus-inoculated 5-year-old silkworm or puncturing virus-inoculation as claimed in claim 1, wherein in B and C, in order to ensure that the silkworm is fed with virus-infected mulberry leaves, waiting for 8-12 hours after feeding the virus-infected mulberry leaves, and feeding the virus-infected mulberry leaves with normal mulberry leaves after the virus-infected mulberry leaves are eaten light or nearly so.

3. The method for obtaining pure BmNPV disease pupae free from other viral pathogens by feeding virus-inoculated 5-year-old silkworm or virus-inoculated by puncture as claimed in claim 4, wherein live silkworms with typical symptoms are collected after 5-6 days of feeding virus-inoculated mulberry leaves, blood is collected by tail cutting, and the collected blood is used for puncture inoculation.

4. The method of claim 1 for obtaining pure BmNPV disease pupae free of other viral pathogens by feeding late 5 th instar vaccinated silkworms or stab vaccination, wherein for late 5 th instar healthy silkworms in B: taking a sterile syringe to extract or an inoculating needle to stick the live silkworm blood, and sticking the live silkworm blood into the body wall of the silkworm to inoculate the live silkworm blood;

for healthy silkworm pupae in C: and (4) taking the sterilized injector to extract the blood of the sick silkworm, and puncturing and inoculating the silkworm pupa skin into the silkworm pupa.

5. The method for obtaining pure BmNPV disease pupae without other virus pathogens by feeding silkworm at the late stage of 5-year virus inoculation or puncturing and inoculation as claimed in claim 1, wherein in A and B, after the silkworm is mature and cocooned, the silkworm is collected after cocooning for 2 days, and then the silkworm is pupated after 2 days, and the silkworm pupae is cut to obtain silkworm pupae, and the disease pupae is collected after the silkworm pupae has symptoms after 3-4 days;

and C, the inoculated silkworm pupas develop for 96-120 hours, and the disease pupas are collected when the pupas have symptoms in the middle and later stages of disease onset.