A method for collecting pig classification samples
A standardized method for pig sample collection using disposable tubes and detailed record-keeping addresses the inadequacies of existing animal sample collection methods, ensuring contamination prevention and efficient storage.
Patent Information
- Application Number
- CN202210278358.5
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2022-03-21
- Publication Date
- 2024-12-31
- Estimated Expiration
- 2042-03-21
AI Technical Summary
Existing methods for collecting animal samples, such as bovine serum, are inadequate for collecting pig samples and require manual addition of sodium chloride, which is complex and not standardized, lacking comprehensive protocols for pig classification samples.
A method for collecting pig classification samples involving blood, tissue, organ, intestinal, fecal, and swab samples using disposable vacuum tubes, with specific protocols for each type, including the use of anticoagulants and PBS buffer, and detailed record-keeping to prevent contamination and ensure sample integrity.
The method provides a standardized, comprehensive, and contamination-free collection process that reduces direct contact with sick pigs and humans, facilitating efficient sample storage and tracing.
Smart Images
Figure CN114831668B_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the technical field of sample collection, and in particular relates to a method for collecting pig classification samples. Background Art
[0002] Reasonable collection of sick pig samples can reduce direct contact between sick pigs and the environment and humans. Therefore, there is an urgent need for a more complete and standardized method of collecting pig samples.
[0003] A Chinese patent application disclosed a method for collecting animal serum, more specifically, bovine serum. The method includes the following steps: blood collection, blood collection, centrifugation, and aliquoting. The key is to add 0.9% sodium chloride injection to the blood collection bottle at a ratio of 1% of the total volume. Before centrifugation, the blood collection bottle is placed in a 38°C water bath, and centrifugation is not performed until the serum begins to precipitate. The advantage of this invention is that the addition of sodium chloride injection to the blood collection bottle creates an isotonic environment, which does not change the osmotic pressure of the blood and prevents hemolysis due to red blood cell rupture. Placing the blood collection bottle in a 38°C water bath ensures uniform heating of the blood, which facilitates the precipitation of unstable proteins and facilitates the precipitation, centrifugation, and separation of serum. The collected serum is yellow and translucent. However, this method only describes the collection of animal serum and does not address how to collect samples from other animals. Furthermore, the method requires manual addition of 0.9% sodium chloride injection to the blood collection bottle to prevent red blood cell rupture, a complex process. Summary of the Invention
[0004] In response to the lack of methods for collecting pig samples, the present invention aims to provide a method for collecting pig samples by classification. The method proposed in this invention involves the collection of various pig samples, which is more comprehensive. Furthermore, when collecting blood and serum, disposable vacuum blood collection tubes are directly used, which is more convenient and protects the samples from contamination, thus preventing them from polluting the environment and threatening human health.
[0005] The present invention proposes a method for collecting pig classification samples, comprising the following steps:
[0006] A method for collecting pig classification samples comprises the following steps:
[0007] (1) Blood collection: First, paste accurate sample background information on the surface of the collection tube, then shave the pig hair at the blood collection site, then disinfect it, wait for the blood collection site to dry, puncture it with a needle, collect the blood into the blood collection tube and mix it;
[0008] (2) Collection of various tissues and organs: After the pig dies, the body cavity is opened, the tissues and organs are stripped, and then the tissue blocks are cut and packaged. The remaining tissues are stored separately, and the sample background information record sheet is recorded;
[0009] (3) Collection of intestinal contents: After the pig dies, the entire intestine is removed under sterile conditions, and then the large intestine and small intestine are cut out. The intestinal contents are squeezed out and packaged, and the remaining intestine is stored separately, or the intestine is cut open from both ends with tissue scissors, flushed with buffer, and the flushed contents are packaged, and the background information of the sample is recorded;
[0010] (4) Fecal collection: collect feces from outside the body, prepare feces containers to collect pig feces, and then divide the pig feces samples into smaller pieces and record the background information of the samples;
[0011] (5) Collection of swab samples:
[0012] 1) Anal swab
[0013] Clean the area around the anus, insert a sterile cotton swab into the anal crypt, rotate it, and remove it. Cut the head of the cotton swab and immerse it in buffer solution. Record the background information of the sample.
[0014] 2) Respiratory swab
[0015] Use a sterile cotton swab to collect secretions from the pig's nasal cavity, throat, or trachea, then remove them. Cut off the head of the cotton swab and immerse it in buffer. Record the background information of the sample.
[0016] 3) Eye swab
[0017] Open the eyelids and then use a sterile cotton swab to collect secretions, avoiding contact with eyelashes and the edge of the face. After sampling, cut the head of the cotton swab and immerse it in buffer, and record the background information of the sample;
[0018] 4) Skin swab
[0019] Place a sterile cotton swab on the skin surface to be sampled and wipe it. After wiping, cut off the head of the cotton swab at each sampling point and immerse it in buffer solution. Record the background information of the sample.
[0020] 5) Vaginal swab
[0021] Insert a sterile cotton swab into the sow's vagina, rotate it, and remove it. Cut off the head of the cotton swab and immerse it in buffer solution. Record the background information of the sample.
[0022] Furthermore, in step (2), the blood is collected using a disposable vacuum blood collection tube containing an anticoagulant when collecting whole blood; a disposable vacuum blood collection tube or a disposable blood collector without any additives is used to prepare serum; and at least three tubes of blood need to be collected from each sick pig.
[0023] Furthermore, in step (2), the tissue organs are heart, liver, spleen, lung, kidney and lymph node; and the tissue blocks are 3 to 5 tissue blocks.
[0024] Furthermore, in step (3), the contents are divided into at least 3 centrifuge tubes; and the buffer is PBS buffer (PH 7.2 to PH 7.4).
[0025] Furthermore, in step (4), the stool sample is divided into at least 3 centrifuge tubes.
[0026] Furthermore, the buffer solution in step (5) is PBS buffer solution (PH 7.2 to PH 7.4).
[0027] Furthermore, in step (5), at least three anal swabs, respiratory swabs, eye swabs, skin swabs and vaginal swabs need to be collected from each pig.
[0028] Furthermore, the blood collection in step (1) should be carried out in the morning from pigs that have not been fed or have stopped eating for 6 hours but have sufficient drinking water to prevent incomplete blood coagulation and the formation of a jelly-like sample; in step (1), the blood collection tube should be tilted during blood collection, and the action should be gentle when inverting and mixing to avoid hemolysis; in step (1), for whole blood samples, the collected whole blood should be stored in a refrigerator at 2 to 6°C, and the separated serum sample should be stored in a refrigerator at -15 to -20°C.
[0029] Furthermore, in step (4), the stool sampler for collecting stool should sample from the inside of the stool, because the outside of the stool is easily contaminated after contact with air, and the DNA of some bacteria will begin to degrade.
[0030] Furthermore, in step (2), step (3), step (4) and step (5), the collected samples are stored in a refrigerator at -20 to -80°C.
[0031] Furthermore, a method for collecting pig classification samples is specifically as follows:
[0032] (1) Preparation before sample collection; (2) Collection of blood; (3) Collection of various tissues and organs; (4) Collection of intestinal contents; (5) Collection of feces; (6) Collection of swab samples.
[0033] Furthermore, the specific steps of preparing for sample collection in step (1) are as follows:
[0034] 1) The type of sample to be collected should be determined based on the different disease symptoms or test purposes, such as blood, tissues, organs, contents, feces, or various swabs, so that sterile operating instruments and consumables can be prepared in advance. When the cause of the disease cannot be estimated, comprehensive collection can be performed;
[0035] 2) Before collecting samples, staff must wear lab coats, masks, gloves, and goggles if necessary to ensure their safety;
[0036] 3) To record detailed sample information, this method proposes a sample background information record sheet, which is printed out and recorded before sampling;
[0037] Furthermore, in step (2), blood is collected using appropriate collection tubes according to the specific test items. For example, when collecting whole blood, a disposable vacuum blood collection tube (EDTA-purple cap) containing an anticoagulant is used; when preparing serum, a disposable vacuum blood collection tube (red cap) without any additives is used. First, accurate sample background information is affixed to the surface of the collection tube. Then, the pig hair at the blood collection site is shaved clean, and then disinfected with 75% (volume percentage concentration) alcohol. After the blood collection site is dry, it is punctured with a disposable needle. The blood is collected into the blood collection tube and gently inverted to mix. At least three tubes are required for each sick pig.
[0038] Furthermore, the various tissues and organs described in step (3) are collected by opening the body cavity with sterile dissecting instruments after the pig dies, and removing the tissues and organs (heart, liver, spleen, lungs, kidneys, lymph nodes, etc.) with sterile tissue scissors, a scalpel, and forceps. Then, 3 to 5 tissue blocks of 1 cm × 1 cm × 1 cm are cut with a scalpel and placed separately in 10 mL centrifuge tubes. The remaining tissue is placed in the corresponding homogenization bag. The background information of the sample is recorded. After collecting one tissue and organ, new sterile tissue scissors, a scalpel, and forceps should be replaced in time when collecting another.
[0039] Furthermore, the intestinal contents of step (4) are collected after the pig dies by using sterile tissue scissors and a scalpel to remove the entire intestine under sterile conditions. Then, after cutting the required part of the intestine, the intestinal contents are squeezed out and divided into multiple tubes (at least 3 tubes) of 10mL centrifuge tubes, and the remaining part is placed in the corresponding homogenization bag. If the contents are small, the intestine can be cut open from both ends with tissue scissors, rinsed with PBS buffer, and the rinsed contents are divided into 10mL centrifuge tubes. Record the background information of the sample.
[0040] Furthermore, the feces collected in step (5) is collected from outside the body and should be fresh. A sterile, dry and clean feces container is prepared to collect pig feces. The feces samples of the tested pigs are then divided into multiple (at least 3) 10 mL centrifuge tubes using a sterile feces sampler, and the background information of the samples is recorded.
[0041] Furthermore, the collection of the swab sample in step (6) includes:
[0042] 1) Anal swab
[0043] Clean the area around the anus. Insert a sterile cotton swab into the anal crypt, gently rotate, and remove. Immediately cut the tip of the cotton swab and immerse it in a 2-mL sterile centrifuge tube containing 1 mL of PBS buffer. Seal the tube with parafilm and place the collected sample in an ice box. Record the background information of the sample. At least three anal swabs should be collected from each sick pig.
[0044] 2) Respiratory swab
[0045] Use a sterile cotton swab to collect secretions from the pig's nasal cavity, throat, or trachea. Immediately cut the tip of the swab and immerse it in a 2-mL sterile centrifuge tube containing 1 mL of PBS buffer. Seal the tube with parafilm and place the collected sample in an ice box. Record the background information of the sample. At least three respiratory swabs should be collected from each sick pig.
[0046] 3) Eye swab
[0047] To collect eyelids, first use an eyelid speculum to open the eyelids. Then, use a sterile cotton swab to collect a sufficient amount of secretions, avoiding contact with eyelashes and the edge of the face. After sampling, cut the tip of the cotton swab and immerse it in a 2mL sterile centrifuge tube filled with 1mL of PBS buffer. Seal the tube with sealing film and place the collected sample in an ice box. Record the background information of the sample. At least three eye swabs should be collected from each sick pig.
[0048] 4) Skin swab
[0049] Press a sterile cotton swab onto the skin surface to be sampled. Bend it firmly at a 45-degree angle to the surface being wiped. Slowly and steadily wipe the surface. Flip the swab over and wipe the other side perpendicular to the direction of the previous swab. After wiping, cut the tip of the cotton swab from each sampling point and immerse it in a 2-mL sterile centrifuge tube containing 1 mL of PBS buffer. Seal the tube with parafilm and place the collected sample in a centrifuge tube ice box. Record the sample's background information. At least three skin swabs should be collected from each sampling point.
[0050] 5) Vaginal swab
[0051] Insert a sterile cotton swab into the sow's vagina and gently rotate it before removing it. Immediately cut the tip of the swab and place it in a 2-mL sterile centrifuge tube containing 1 mL of PBS buffer. Seal the tube with parafilm and place the collected sample in an ice box. Record the background information of the sample. At least three vaginal swabs should be collected from each sick pig.
[0052] Preferably, the sample background information is recorded in detail according to the sample background information record table, and the test results can be associated with the sample information later.
[0053] Preferably, the blood in step (2) should be collected in the morning from pigs that have not been fed (on an empty stomach) or have not been fed for 6 hours but have sufficient drinking water, to prevent incomplete blood coagulation and the formation of a jelly-like sample.
[0054] Preferably, in step (2), the blood collection tube should be slightly tilted when collecting blood, and the inversion and mixing should be done gently to avoid hemolysis.
[0055] Preferably, in step (2), for whole blood samples, if nucleic acid extraction cannot be performed as soon as possible, the collected whole blood needs to be stored in a refrigerator at 2 to 6°C; and the separated serum sample should be stored in a refrigerator at -15 to -20°C.
[0056] Preferably, the tissues and organs are cut into pieces and packaged, so as to avoid repeated freezing and thawing of the tissues during subsequent sampling.
[0057] Preferably, replacing new sterilized tissue scissors, scalpels and tweezers can reduce cross-contamination between tissues.
[0058] Preferably, PBS buffer is used for the collection of intestinal contents and swab samples, so that cells can survive within a suitable pH range and maintain cell integrity.
[0059] Preferably, in step (5), the stool sampler used for collecting the stool should sample from the inside of the stool as much as possible, because the outside of the stool is easily contaminated after contact with air, and the DNA of some bacteria will begin to degrade.
[0060] Preferably, when collecting swab samples, sealing film is used to seal the mouth of the centrifuge tube to prevent leakage of PBS buffer and mutual contamination between samples.
[0061] Preferably, in step (3), step (4), step (5) and step (6), the collected samples are stored in a refrigerator at -20 to -80°C.
[0062] Preferably, in steps (2), (3), (4), (5) and (6), the collected samples should be backed up for future information tracing.
[0063] The method of the present invention has the following beneficial effects: (1) Different sampling methods are used for samples of different disease types, which is beneficial for staff to standardize operations and avoid directly killing sick pigs. (2) The proposed sample background information record table can be used to improve sample background information and facilitate information tracing. (3) Based on the sampling method of the present invention, direct contact between sick pigs and the environment and humans can be reduced, avoiding environmental pollution and threats to human health. BRIEF DESCRIPTION OF THE DRAWINGS
[0064] Figure 1This is a detailed sample background information record table of the sick pig eye swab in Example 1 of the present invention.
[0065] Figure 2 This is a detailed sample background information record table of the sick pig skin swab in Example 2 of the present invention.
[0066] Figure 3 This is a detailed sample background information record table of the sick pig heart in Example 3 of the present invention.
[0067] Figure 4 This is a flow chart of the pig classification sample collection method proposed in the present invention. DETAILED DESCRIPTION
[0068] The following will be combined with the accompanying drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. If the process or parameters are not particularly described in detail below, they can be understood or implemented by those skilled in the art with reference to the existing technology.
[0069] Figure 4 This is a flow chart of the pig classification sample collection method proposed in the present invention.
[0070] Example 1
[0071] Reference Figure 4 , a pig classification sample collection method, comprising the following steps:
[0072] (1) Preparation before sample collection
[0073] 1) In March 2017, boars at a large-scale pig farm in Guangzhou, Guangdong Province, developed symptoms such as emaciation, cloudy eyes, and difficulty walking. The cause of the disease could not be directly estimated, so a comprehensive collection of samples was conducted on a sick boar, including blood, tissues, organs, contents, feces, and various swabs (excluding vaginal swabs), and sterile operating instruments and consumables were prepared in advance.
[0074] 2) Before collecting samples, staff should wear lab coats, masks, gloves, and goggles to ensure their safety.
[0075] 3) Before sampling, print out a sample background information record sheet proposed by this method and record detailed sample background information.
[0076] (2) Blood collection
[0077] For whole blood collection, use disposable vacuum blood collection tubes (EDTA - purple caps) containing an anticoagulant; for serum preparation, use disposable vacuum blood collection tubes (red caps) without any additives. First, accurately label the sample background information on the surface of the collection tube. Next, shave the pig hair at the blood collection site and disinfect it with 75% (volume percentage concentration) alcohol. Once the blood collection site is dry, puncture it with a disposable needle. Collect blood into the tube and gently invert to mix. Three each of purple-capped and red-capped vacuum blood collection tubes are used. Whole blood samples are stored in a refrigerator at 2°C; separated serum samples are stored in a refrigerator at -15°C.
[0078] (3) Collection of various tissues and organs
[0079] After the pigs died, the body cavity was opened with sterile dissecting instruments. Sterile tissue scissors, scalpels, and forceps were used to remove the tissues and organs—the heart, liver, spleen, lungs, kidneys, and lymph nodes. Three 1 cm × 1 cm × 1 cm pieces of tissue were cut from each of the heart, liver, spleen, lungs, kidneys, and lymph nodes using a scalpel. These were placed separately in 10 mL centrifuge tubes. The remaining tissue was placed in a homogenization bag, and a sample background information sheet was affixed to the surface of the homogenization bag. After collecting one tissue organ, new sterile tissue scissors, scalpels, and forceps were used to collect another. Tissue samples were stored in a -20°C freezer.
[0080] (4) Collection of intestinal contents
[0081] Using sterile tissue scissors and a scalpel, aseptically remove the entire intestine. After dissecting the large and small intestines, squeeze the intestinal contents and divide them into three 10 mL centrifuge tubes. Separately transfer the remaining intestine into a homogenizing bag. Record the background information of the sample. Store the intestinal content samples in a -20°C freezer.
[0082] (5) Collection of feces
[0083] Feces collected from outside the body should be fresh. Prepare a sterile, dry and clean feces container to collect pig feces. Then use a sterile feces sampler to divide the feces samples of the tested pigs into 3 10mL centrifuge tubes. Record the background information of the samples. The feces samples should be stored in a -20℃ refrigerator.
[0084] (6) Collection of swab samples
[0085] 1) Anal swab
[0086] Clean the area around the anus. Insert a sterile cotton swab into the anal crypt, gently rotate, and remove. Cut the tip of the cotton swab and immerse it in a 2-mL sterile centrifuge tube containing 1 mL of PBS buffer (pH 7.2). Seal the tube with parafilm and place the collected sample in a centrifuge tube ice box. Record the sample background information. Collect three anal swabs and store them in a -20°C freezer.
[0087] 2) Respiratory swab
[0088] Use a sterile cotton swab to collect secretions from the pig's nasal cavity, remove the swab, cut the tip, and immerse it in a 2-mL sterile centrifuge tube containing 1 mL of PBS buffer (pH 7.2). Seal the tube with sealing film and place the collected sample in a centrifuge tube ice box. Record the sample background information. Collect three respiratory swabs and store them in a -20°C freezer.
[0089] 3) Eye swab
[0090] When sampling, first use an eyelid speculum to open the eyelids, then use a sterile cotton swab to collect a sufficient amount of secretions, avoiding contact with eyelashes and the edge of the face. After sampling, cut the head of the cotton swab and immerse it in a 2 mL sterile centrifuge tube containing 1 mL PBS buffer (pH 7.2). Seal the mouth of the centrifuge tube with sealing film, place the collected sample in a centrifuge tube ice box, and record the background information of the sample ( Figure 1 ). Three eye swabs were collected and stored in a -20°C refrigerator.
[0091] 4) Skin swab
[0092] Press a sterile cotton swab onto the skin surface to be sampled, bend it firmly at a 45-degree angle to the surface being wiped, and wipe the sample surface steadily and slowly. Flip the swab over and wipe the other side of the swab, but perpendicular to the direction of the previous wiping movement. After wiping, cut the tip of the cotton swab from each sampling point and immerse it in a 2-mL sterile centrifuge tube containing 1 mL of PBS buffer (pH 7.2). Seal the tube with sealing film and place the collected sample in a centrifuge tube ice box. Record the background information of the sample. Select three sampling points on the sick pigs and collect three skin swabs at each sampling point, for a total of nine skin swabs. The skin swab samples were stored in a -20°C refrigerator.
[0093] Example 2
[0094] Reference Figure 4 , a pig classification sample collection method, comprising the following steps:
[0095] (1) Preparation before sample collection
[0096] 1) In October 2018, sows at a large-scale pig farm in Shaoguan City, Guangdong Province, developed symptoms of skin ulceration and peeling. Based on these symptoms, it was determined that the sample collection type was skin swabs. Therefore, skin swabs were collected from four sows, and sterile operating instruments and consumables were prepared in advance;
[0097] 2) Before collecting samples, staff should wear lab coats, masks, gloves, and goggles to ensure their safety;
[0098] 3) Before sampling, print out a sample background information record sheet proposed by this method and record detailed sample background information.
[0099] (2) Collection of skin swabs
[0100] Press the sterile cotton swab on the skin surface to be sampled, bend it with force to form a 45-degree angle with the wiping surface, wipe the sampling surface steadily and slowly, turn the cotton swab over, and wipe the other side of the cotton swab, but perpendicular to the direction of the previous wiping movement. After wiping, cut the head of the cotton swab at each sampling point and immerse it in a 2 mL sterile centrifuge tube containing 1 mL PBS buffer (PH 7.3). Seal the mouth of the centrifuge tube with sealing film, place the collected sample in a centrifuge tube ice box, and record the background information of the sample ( Figure 2 Three sampling sites were selected on each sow, and four skin swabs were collected from each site, for a total of 48 skin swabs. The skin swab samples were stored in a -50°C freezer.
[0101] Example 3
[0102] Reference Figure 4 , a pig classification sample collection method, comprising the following steps:
[0103] (1) Preparation before sample collection
[0104] 1) In March 2018, piglets at a large-scale pig farm in Yangjiang City, Guangdong Province, developed symptoms of tremors, difficulty walking, and an inability to suck colostrum. Unable to directly assess the cause of the illness, a comprehensive collection of blood, tissues, organs, contents, feces, and various swabs was performed on one affected pig (mother), with sterile instruments and consumables prepared in advance.
[0105] 2) Before collecting samples, staff should wear lab coats, masks, gloves, and goggles to ensure their safety;
[0106] 3) Before sampling, print out a sample background information record sheet proposed by this method and record detailed sample background information.
[0107] (2) Blood collection
[0108] For whole blood collection, use disposable vacuum blood collection tubes containing an anticoagulant (EDTA - purple cap); for serum preparation, use disposable vacuum blood collection tubes without any additives (red cap). First, accurately label the sample background information on the surface of the collection tube. Next, shave the pig hair at the blood collection site and disinfect it with 75% (volume percentage concentration) alcohol. Once the blood collection site is dry, puncture it with a disposable needle. Collect blood into the tube and gently invert to mix. Five each of purple and red vacuum blood collection tubes are used. Whole blood samples are stored at 6°C; separated serum samples are stored at -20°C.
[0109] (3) Collection of various tissues and organs
[0110] After the pig died, the body cavity was opened with sterile dissecting instruments, and the tissue organs (heart, liver, spleen, lung, kidney, lymph node) were removed with sterile tissue scissors, scalpels and forceps. Then, 5 tissue blocks of 1 cm × 1 cm × 1 cm were cut from the heart, liver, spleen, lung, kidney and lymph node respectively with a scalpel and placed separately in 10 mL centrifuge tubes. The remaining tissue was placed in a homogenizing bag and the recorded sample background information record sheet ( Figure 3 ) on the surface of the homogenizing bag. After collecting one tissue or organ, replace the sterilized tissue scissors, scalpel, and forceps promptly when collecting another. Tissue and organ samples should be stored in a -80°C freezer.
[0111] (4) Collection of intestinal contents
[0112] Using sterile tissue scissors and a scalpel, aseptically remove the entire intestine. After dissecting the large and small intestines, squeeze the intestinal contents and distribute them into five 10 mL centrifuge tubes. Separately transfer the remaining intestine into a homogenizing bag. Record the background information of the sample. Store the intestinal content samples in a -80°C freezer.
[0113] (5) Collection of feces
[0114] When collecting feces from outside the body, ensure that they are fresh. Prepare sterile, dry, and clean fecal containers to collect the pig feces. Then, use a sterile fecal sampler to divide the fecal samples into five 10 mL centrifuge tubes. Record the background information of the samples. Store the fecal samples in a -80°C freezer.
[0115] (6) Collection of swab samples
[0116] 1) Anal swab
[0117] Clean the area around the anus. Insert a sterile cotton swab into the anal crypt, gently rotate, and remove. Cut the tip of the cotton swab and immerse it in a 2-mL sterile centrifuge tube containing 1 mL of PBS buffer (pH 7.4). Seal the tube with parafilm and place the collected sample in an ice box. Record the background information of the sample. Collect five anal swabs. Store the anal swab samples in a -80°C freezer.
[0118] 2) Respiratory swab
[0119] Use a sterile cotton swab to collect throat secretions and remove them. Cut the tip of the swab and immerse it in a 2-mL sterile centrifuge tube containing 1 mL of PBS buffer (pH 7.4). Seal the tube with parafilm and place the collected sample in a centrifuge tube ice box. Record the sample background information. Collect five respiratory swabs. Store respiratory swab samples in a -80°C freezer.
[0120] 3) Eye swab
[0121] To collect eyelid samples, first use an eyelid speculum to open the eyelids. Next, use a sterile cotton swab to collect a sufficient amount of secretions, avoiding contact with eyelashes and the edge of the face. After collection, cut the tip of the cotton swab and immerse it in a 2-mL sterile centrifuge tube containing 1 mL of PBS buffer (pH 7.4). Seal the tube with sealing film and place the collected sample in a centrifuge tube ice box. Record the background information of the sample. Collect five eye swabs. Store the eye swab samples in a -80°C freezer.
[0122] 4) Skin swab
[0123] Press a sterile cotton swab onto the skin surface to be sampled. Bend it firmly at a 45-degree angle to the surface being wiped. Smoothly and slowly wipe the surface. Flip the swab over and wipe the other side perpendicular to the direction of the previous swab. After wiping, cut the tip of the cotton swab from each sampling point and immerse it in a 2-mL sterile centrifuge tube containing 1 mL of PBS buffer (pH 7.4). Seal the tube with parafilm and place the collected sample in a centrifuge tube ice box. Record the sample's background information. Three sampling sites were selected on the sick pigs, with five skin swabs collected from each site, for a total of 15 skin swabs. Store the skin swab samples at -80°C.
[0124] 5) Vaginal swab
[0125] Insert a sterile cotton swab into the pig's vagina, gently rotate, and remove. Cut the tip of the swab and place it in a 2-mL sterile centrifuge tube containing 1 mL of PBS buffer (pH 7.4). Seal the tube with parafilm and place the collected sample in an ice box. Record the background information of the sample. Collect five vaginal swabs. Store the vaginal swab samples in a -80°C freezer.
[0126] The above description is only a preferred embodiment of the present invention, which is used to explain the present invention, not to limit the present invention. Changes, substitutions, modifications, etc. made by those skilled in the art without departing from the spirit of the present invention should fall within the scope of protection of the present invention.
Claims
1. A method for collecting pig classification samples, characterized in that: Confirm the type of sample to be collected according to different disease symptoms or test purposes, including blood, tissues and organs, contents, feces or various swabs. When the cause cannot be estimated, conduct comprehensive collection; specifically, the following steps are included: (1) Blood collection: First, paste accurate sample background information on the surface of the collection tube, then shave the pig hair at the blood collection site, then disinfect it, wait for the blood collection site to dry, puncture it with a needle, collect the blood into the blood collection tube and mix it evenly; (2) Collection of various tissues and organs: After the pig dies, the body cavity is opened, the tissues and organs are stripped, and then the tissue blocks are cut and packaged. The remaining tissues are stored separately, and the sample background information record sheet is recorded; the blood is collected by using a disposable vacuum blood collection tube containing an anticoagulant when collecting whole blood; a disposable vacuum blood collection tube or a disposable blood collection device without any additives is used to prepare serum; at least 3 tubes of blood are required for each sick pig; the tissues and organs are the heart, liver, spleen, lung, kidney and lymph node; the tissue blocks cut are 3 to 5 tissue blocks; (3) Collection of intestinal contents: After the pig dies, remove the entire intestine under sterile conditions, then cut the large intestine and small intestine, squeeze out the intestinal contents and package them, and store the remaining intestine separately, or use tissue scissors to cut the intestine from both ends, flush it with buffer, and package the flushed contents, and record the background information of the sample; (4) Feces collection: collect feces from outside the body, prepare feces containers to collect pig feces, then divide the pig feces samples into smaller pieces and record the background information of the samples; (5) Collection of swab samples: 1) Anal swab Clean the area around the anus, insert a sterile cotton swab into the anal crypt, rotate it, and remove it. Cut off the head of the cotton swab and immerse it in buffer solution. Record the background information of the sample. 2) Respiratory swab Use a sterile cotton swab to dip the secretions from the pig's nose, throat or trachea and remove them. Cut off the head of the cotton swab and immerse it in buffer. Record the background information of the sample. 3) Eye swab Open the eyelids, then use a sterile cotton swab to collect secretions, avoiding contact with eyelashes and the edge of the face. After sampling, cut off the head of the cotton swab and immerse it in buffer, and record the background information of the sample; 4) Skin swab Place a sterile cotton swab on the skin surface to be sampled and wipe it. After wiping, cut off the head of the cotton swab at each sampling point and immerse it in the buffer solution, and record the background information of the sample; 5) Vaginal swab Insert a sterile cotton swab into the sow's vagina, rotate it, and then remove it. Cut off the head of the cotton swab and immerse it in buffer solution. Record the background information of the sample.
2. A method for collecting pig classification samples according to claim 1, characterized in that: In step (3), the contents are packaged into at least 3 centrifuge tubes; and the buffer is PBS buffer.
3. The method for collecting pig classification samples according to claim 1, characterized in that: In step (4), the stool sample is packaged into at least 3 centrifuge tubes.
4. The method for collecting pig classification samples according to claim 1, characterized in that: The buffer in step (5) is PBS buffer.
5. The method for collecting pig classification samples according to claim 1, characterized in that: In step (5), at least three anal swabs, respiratory swabs, eye swabs, skin swabs and vaginal swabs must be collected from each pig.
6. The method for collecting pig classification samples according to claim 1, characterized in that: The blood collection in step (1) should be carried out in the morning from pigs that have not been fed or have stopped eating for 6 hours but have sufficient drinking water to prevent incomplete blood coagulation and the formation of a jelly-like sample; in step (1), the blood collection tube should be tilted during blood collection, and the action should be gentle when inverting and mixing to avoid hemolysis; in step (1), for whole blood samples, the collected whole blood needs to be stored in a refrigerator at 2~6℃, and the separated serum samples should be stored in a refrigerator at -15~-20℃.
7. The method for collecting pig classification samples according to claim 1, characterized in that: In step (4), the stool sampler for collecting stool should sample from the inside of the stool, because the outside of the stool is easily contaminated after contact with air, and the DNA of some bacteria will begin to degrade.
8. A method for collecting pig classification samples according to any one of claims 1 to 7, characterized in that: In step (2), step (3), step (4) and step (5), the collected samples are stored in a refrigerator at -20 to -80 °C.