Separation method for separating duck circovirus by using SPF duck embryo
Through the SPF duck embryo yolk sac inoculation and PCR amplification electrophoresis analysis, the problem of difficulty in isolation of duck circovirus is solved, and a simple, economical and safe method of virus isolation is provided, which promotes the study of the pathogenic mechanism of duck circovirus.
Patent Information
- Application Number
- CN202510430961.4
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-04-08
- Publication Date
- 2025-07-04
AI Technical Summary
In the prior art, duck circovirus is difficult to reproduce successfully in cell lines and other systems, resulting in limited research on the pathogenic mechanism of duck circovirus and lacks simple, economical and less risky isolation methods.
The SPF duck embryo was used for yolk sac inoculation, combined with PCR amplification and gel electrophoresis analysis, and the 8-day-old SPF duck embryo was inoculated through the yolk sac, and cultured to 27 days old. The duck cyclovirus nucleic acid was extracted and PCR amplified. After electrophoresis analysis, the passage was performed until the stable passage of virus was obtained.
A simple, economical and low-risk infection isolation method is achieved, supporting the isolation, identification and antigen preparation of duck cyclovirus, and promoting the study of the pathogenic mechanism of duck cyclovirus.
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Figure CN120249225A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the technical field of biological vaccine prevention and control, and specifically to a method for isolating duck circovirus using SPF duck embryos. Background Art
[0002] Virus isolation is the "gold standard" for pathogen detection. So far, except for porcine circovirus (PCV) which can reproduce in cells such as PK-15, no other circoviruses have been successfully obtained methods for virus reproduction in cell lines and other systems. Among them, for duck farming, the isolation of duck circovirus is relatively difficult, and the inability to isolate the virus directly affects the research progress of veterinary workers on the pathogenic mechanism of duck circovirus.
[0003] Duck circovirus belongs to the genus Circovirus of the family Circoviridae. According to the genetic characterization of the virus, DuCV can be divided into three genotypes: DuCV-1, DuCV-2, and DuCV-3, which contain three main open reading frames (ORFs), namely ORF1, ORF2, and ORF3. ORF1 encodes the Rep protein, which is a protein necessary for virus replication; ORF2 encodes the Cap protein, which is the main structural protein of the virus and constitutes the nucleocapsid of the virus; ORF3 is located on the complementary strand of ORF1, and the related protein is involved in apoptosis.
[0004] Therefore, for the duck farming field at present, there is an urgent need for a method for isolating duck circovirus that is simple to operate, economically suitable, and has relatively low potential risks. Summary of the Invention
[0005] Aiming at the disadvantages and deficiencies existing in the prior art, the present invention provides a method for isolating duck circovirus using SPF duck embryos, which includes the duck circovirus inoculation method, inoculation method, and method for obtaining virus cultures. This virus isolation method is simple to operate, economically suitable, and has relatively low potential infection risks, and can be used as a common research method for isolating, identifying, and preparing antigens of duck circovirus.
[0006] To achieve the above objectives, the present invention is realized through the following technical solutions: The method for isolating duck circovirus using SPF duck embryos provided by the present invention includes the following steps:
[0007] (1) Preparation of the sample of the virus culture to be tested: Select healthy SPF fertilized duck embryos for cultivation, inoculate SPF duck embryos at 8-day-old, the inoculation method is yolk sac inoculation, and the virus culture obtained is the duck embryo body.
[0008] (2) Extracting nucleic acid of duck circovirus: Extracting the nucleic acid of duck circovirus from each inoculated SPF duck embryo.
[0009] (3) PCR amplification: After extracting the nucleic acid target gene of duck circovirus in step (2), perform PCR amplification to obtain an amplification product;
[0010] (4) Electrophoresis analysis: Then use gel electrophoresis to detect the amplification product. When a duck circovirus-positive embryo is obtained, continue to passage the obtained material from the positive embryo using the culture method of repeating steps (1)-(2) until a duck circovirus that can be stably passaged on duck embryos is obtained.
[0011] Preferably, the selection method for collecting virus cultures is as follows:
[0012] Discard the duck embryos that die within 48 hours after inoculating SPF duck embryos. Extend the culture time of duck embryos until the duck embryos reach 27 days of age. If there are dead duck embryos during this period, collect each embryo separately and mark the death time and number;
[0013] If the 27-day-old duck embryos do not die, place the duck embryos in a refrigerator at -20°C for 1 hour, then collect the embryos, collect each embryo separately, and make good marks.
[0014] Preferably, extract the nucleic acid of duck circovirus from each SPF duck embryo after inoculation. Refer to the specific primers designed based on the published target gene ORF1 of duck circovirus, and design the amplified target fragment length to be 619 bp; Design primers: For the target gene ORF1, its upstream primer Ducv-F and downstream primer Ducv-R sequences are respectively:
[0015] Ducv-F: SEQ ID NO.1; (5’-CGGCGCTTGTACTCCGTACTC-3’);
[0016] Ducv-R: SEQ ID NO.2; (5’-CCCGCGTGGTTTGTAATACTTG-3’);
[0017] Perform PCR amplification. After designing the primers, amplify the target gene to obtain an amplification product.
[0018] Preferably, the specific parameters of PCR amplification are as follows:
[0019] The expected length of the amplified target fragment is 619 bp; The PCR reaction system is 25 μl, including 2 μl of template DNA, 1 μl each of the upstream and downstream primers at 20 mmol / L, and make up the system with dd H2O;
[0020] The PCR reaction conditions are: pre-denaturation at 95°C for 3 min, denaturation at 95°C for 15 s, annealing at 50°C for 15 s, extension at 72°C for 1 min, for 32 cycles; Finally, extend at 72°C for 5 min.
[0021] Preferably, in the electrophoresis analysis step, a duck circovirus positive sample and dd H2O are used as positive and negative controls respectively, and the PCR amplification product is taken for gel electrophoresis detection.
[0022] Preferably, the sterile syringe used for embryo inoculation is a 1 ml syringe barrel equipped with a 5 ml syringe needle.
[0023] The present invention provides a method for isolating duck circovirus using SPF duck embryos. It has the following beneficial effects:
[0024] (1) The method for isolating duck circovirus using SPF duck embryos in the present invention includes the duck circovirus inoculation method, inoculation method, and method for obtaining virus cultures. Among them, the inoculation age of SPF duck embryos is 8 days old, the inoculation method is yolk sac inoculation, the culture time is extended to 27 days old after inoculation, and the virus collection is the duck embryo body.
[0025] (2) Virus isolation is the "gold standard" for pathogen detection. This virus isolation method is simple to operate, economically suitable, and has a relatively low potential infection risk. It can be used as a common research method for duck circovirus isolation, identification, antigen preparation, etc. It successfully solves the current dilemma of duck circovirus isolation and is of great significance for studying the pathogenic mode and mechanism of the immunosuppressive disease caused by duck circovirus. BRIEF DESCRIPTION OF THE DRAWINGS
[0026] Figure 1 It is a schematic diagram of the inoculation method of duck circovirus for this patent application;
[0027] Figure 2 It is a schematic diagram of the embryo body for collecting duck circovirus for this patent application;
[0028] Figure 3 It is a schematic diagram of a duck embryo in which duck circovirus is stably passed to the 28th generation in this patent application;
[0029] Figure 4 It is a PCR identification result diagram of duck circovirus in this patent application. DETAILED DESCRIPTION OF THE INVENTION
[0030] To make the objectives, technical solutions, and advantages of this invention patent clearer, the spirit of what is disclosed in this invention will be described in detail below. After any person skilled in the relevant technical field understands the embodiments of this content, they can make changes and modifications based on the techniques taught by this content, which do not depart from the spirit and scope of this content.
[0031] The method for isolating duck circovirus using SPF duck embryos provided by the present invention includes the duck circovirus inoculation method, inoculation method, and method for obtaining virus cultures.
[0032] Specifically select the age of the SPF duck embryos for embryo inoculation, the method of embryo inoculation, and the collection of virus cultures: Select healthy SPF fertilized duck embryos, with the inoculation age being 8-day-old duck embryos, the method of embryo inoculation being yolk sac inoculation, and the collection of virus cultures being the embryo bodies of duck embryos.
[0033] Materials to be prepared: Incubator, egg candler, laminar flow hood, positive duck circovirus disease materials, 1 ml sterile syringe, 5 ml sterile syringe, surgical forceps, alcohol lamp, egg tray, aqueous filters (0.22 μm, 0.45 μm), sterilized 1.5 ml EP tubes, paraffin, refrigerator, centrifuge, sterilized normal saline, gloves, masks, etc.
[0034] The sterile syringe used for embryo inoculation is a 1 ml syringe barrel equipped with a 5 ml syringe needle.
[0035] Cultivation of SPF duck embryos after inoculation: Discard the duck embryos that die within 48 hours after inoculating SPF duck embryos, extend the cultivation time of duck embryos until the duck embryos reach 27 days of age. If there are dead duck embryos during this period, collect the embryo bodies of each embryo separately and mark the death time and number. If the 27-day-old duck embryos do not die, place the duck embryos in the refrigerator at -20 °C for 1 hour, and then collect the embryo bodies. Collect each embryo separately and make good marks.
[0036] Extract the duck circovirus nucleic acid of each SPF duck embryo collected after inoculation, and refer to the specific primers designed according to the published ORF1 of the target gene of duck circovirus. The specific primer sequences are shown in Table 1.
[0037] Table 1 Ducv PCR primer sequences
[0038]
[0039] The expected amplification length is 619 bp; the PCR reaction system is 25 μl, including 2 μl of template DNA, 1 μl of each of the upstream and downstream primers at 20 mmol / L, and dd H2O is added to make up the system.
[0040] The PCR reaction conditions are: pre-denaturation at 95 °C for 3 min, denaturation at 95 °C for 15 s, annealing at 50 °C for 15 s, extension at 72 °C for 1 min, for 32 cycles; finally, extension at 72 °C for 5 min.
[0041] Electrophoresis analysis: Use positive duck circovirus disease materials and dd H2O as positive and negative controls respectively, and take the PCR products for gel electrophoresis detection. After detecting positive embryo bodies of duck circovirus, continue to passage the obtained materials of positive embryo bodies according to the above-mentioned duck embryo inoculation and cultivation method until a duck circovirus that can be stably passaged on duck embryos is obtained.
[0042] The isolation method for isolating duck circovirus using SPF duck embryos is as follows:
[0043] Preparation of the sample of the virus culture to be tested - inoculation and culture of the duck embryo body:
[0044] As Figure 1 shown in the schematic diagram of yolk sac inoculation, the whole SPF duck embryo includes an air chamber 1, a yolk sac 2, and egg white 3.
[0045] 1. Mark the air chamber: Select healthy and viable 8-day-old SPF duck embryos and place them vertically on the egg tray. Use an egg candler to candler the eggs, and use a pencil to mark the positions of the air chamber and the embryo on the other side of the embryo at the highest point of the eggshell, avoiding the area with dense blood vessels. Make a mark with a pencil, which is the inoculation needle insertion point. The arrow direction in
[0046] is the needle insertion direction. Figure 1 The arrow direction in is the needle insertion direction.
[0047] 2. Treatment of the diseased material: Use it after irradiating the ultra-clean workbench with ultraviolet light for 30 min. Repeatedly freeze-thaw the positive diseased material of duck circovirus 3 times, centrifuge it at 12,000 r / min for 5 min with a high-speed centrifuge, and perform aseptic operation in the ultra-clean bench. Take the supernatant of the diseased material and filter it first with a 0.45 μm aqueous filter and then with a 0.22 μm aqueous filter to remove impurities and bacteria.
[0048] 3. Punch and inoculate: Mark the inoculation information on the eggshell, including the number, date, and passage number. First, disinfect it with iodine tincture, and then use a 75% alcohol cotton ball to deiodize and disinfect the eggshell. After sterilizing and cooling the punching tool by burning, punch a hole at the marked place, requiring to punch through the eggshell without damaging the shell membrane. When inoculating, use a 1 ml medical syringe needle tube with a 5 ml syringe needle, vertically insert it from the needle insertion point, and insert the 5 ml syringe needle completely. Pay attention not to insert the needle towards the eggshell direction to avoid puncturing the blood vessels. After inoculation, use paraffin to seal the needle insertion point.
[0049] 4. Incubate and observe: Put the sealed SPF duck embryos into the incubator at 37 °C for incubation, and discard the duck embryos that die within 48 h. Candler the embryos once in the morning and once in the afternoon every day.
[0050] 5. Harvest the embryos and perform PCR identification: Incubate the inoculated SPF duck embryos until they reach 27 days of age. If there are dead duck embryos during this period, separately harvest the embryo bodies of each embryo and mark the death time and number. If the 27-day-old duck embryos do not die, place the duck embryos in the refrigerator at -20 °C for 1 h, and wait for the blood vessels to contract and fall off before harvesting the embryo bodies. Separate and harvest each SPF duck embryo and make good marks. As Figure 2 shown, take a photo of the embryo body.
[0051] Extract the nucleic acid of duck circovirus from the embryo body of each duck embryo and perform gel electrophoresis detection. Discard the negative ones, keep the positive ones and continue to passage according to the above method until a duck circovirus that can be stably passaged on duck embryos is obtained.
[0052] As Figure 3The impact of the 28th passage of duck circovirus on duck embryos is shown as follows. Diseased materials that were identified as positive by PCR after collecting embryo bodies from the 27th passage of circovirus were centrifuged at 12,000 r / min for 5 min using a high-speed centrifuge. On October 26, 2024, 5 8-day-old SPF duck embryos were inoculated via the yolk sac, 0.2 ml per embryo, and the embryos were candled once in the morning and once in the afternoon every day. On November 7, 2024, one embryo died during candling (20 days old), and on November 8, 2024, one embryo died during candling (21 days old). On November 11, 2024, one embryo died during candling, and two were frozen to death (24 days old). Each embryo body was collected separately and subjected to PCR identification. The dead duck embryos showed bleeding on the surface of the embryo bodies and the embryo bodies were dwarfed.
[0053] As Figure 4 Shown in the PCR identification result diagram of duck circovirus as follows. Among them, the PCR identification results showed that the DUCV detected in the embryo bodies obtained from the 28th passage of circovirus were all positive. Among them, the red label 1 represents: collected from the natural death of DUCV 28th passage on November 7, 2024; the red label 2 represents: collected from the natural death of DUCV 28th passage on November 8; the red label 3 represents: collected from the natural death of DUCV 28th passage on November 11; the red label 4 represents: collected from the frozen death of DUCV 28th passage on November 11 ①; the red label 5 represents: collected from the frozen death of DUCV 28th passage on November 11 ②; the red label Y represents: negative control.
[0054] In summary, the isolation method of duck circovirus using SPF duck embryos in the present invention is simple to operate, economically suitable, and has a relatively small potential infection risk, and can be used as a common research method for the isolation, identification, and antigen preparation of duck circovirus.
[0055] The above is only the preferred specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any person skilled in the art within the technical scope disclosed by the present invention, according to the technical solution and inventive concept of the present invention, making equivalent substitutions or changes, should be covered by the protection scope of the present invention.
Claims
1. A method for isolating duck circovirus using SPF duck embryos, characterized in that, The steps are as follows: (1) Preparation of the sample to be tested: Select healthy SPF fertilized duck embryos for cultivation. The inoculation age is 8-day-old SPF duck embryos, and the embryo inoculation method is yolk sac inoculation. The virus culture obtained is the duck embryo body. (2) Extraction of duck circovirus nucleic acid: Extract the duck circovirus nucleic acid from each SPF duck embryo after inoculation. (3) PCR amplification: After amplifying the target gene of the duck circovirus nucleic acid extracted in step (2), perform PCR amplification to obtain an amplification product. (4) Electrophoresis analysis: Then use gel electrophoresis to detect the amplification product. When a duck circovirus positive embryo body is obtained, the obtained material of the positive embryo body is continuously passaged by repeating the culture method of steps (1)-(2) until a duck circovirus that can be stably passaged on duck embryos is obtained.
2. The isolation method of isolating duck circovirus using SPF duck embryos according to claim 1, characterized in that, The selection method for the obtained virus culture is as follows: Discard the duck embryos that die within 48 hours after inoculating SPF duck embryos. Extend the culture time of the duck embryos until they reach 27 days old. If there are dead duck embryos during this period, collect the embryo body of each embryo separately and mark the death time and number. If the 27-day-old duck embryos do not die, place the duck embryos in a refrigerator at -20°C and freeze them for 1 hour, then collect the embryo bodies, collect each embryo separately, and make good marks.
3. The isolation method of isolating duck circovirus using SPF duck embryos according to claim 1, characterized in that, Extract the duck circovirus nucleic acid from each SPF duck embryo after inoculation. Refer to the specific primers designed according to the published ORF1 of the duck circovirus target gene, and design the amplification target fragment length to be 619bp. Design primers: for the target gene ORF1, the upstream primer Ducv-F and the downstream primer Ducv-R sequences are respectively: Ducv-F: SEQ ID NO.1; (5’-CGGCGCTTGTACTCCGTACTC-3’); Ducv-R: SEQ ID NO.2; (5’-CCCGCGTGGTTTGTAATACTTG-3’); Perform PCR amplification. After designing the primers, amplify the target gene to obtain an amplification product.
4. The isolation method for isolating duck circovirus using SPF duck embryos according to claim 3, wherein, The specific parameters of the PCR amplification are as follows: The expected amplification target fragment length is 619bp; the PCR reaction system is 25ul, including 2ul of template DNA, 1ul of each of the upstream and downstream primers at 20mmol / L, and dd H2O is added to make up the system. The PCR reaction conditions are: pre-denaturation at 95°C for 3 minutes, denaturation at 95°C for 15 seconds, annealing at 50°C for 15 seconds, extension at 72°C for 1 minute, for 32 cycles; finally, extension at 72°C for 5 minutes.
5. The isolation method of isolating duck circovirus using SPF duck embryos according to claim 4, characterized in that, In the electrophoresis analysis step, use duck circovirus positive sample and dd H2O as the positive control and negative control respectively, and take the PCR amplification product for gel electrophoresis detection.
6. The isolation method for isolating duck circovirus using SPF duck embryos according to claim 1, characterized in that, The sterile syringe used for embryo inoculation is a 1ml syringe barrel equipped with a 5ml syringe needle.