Poultry dried blood spot swab and application

Abstract

The invention relates to a dried poultry blood spot swab and application, the dried poultry blood spot swab comprises a blood sampling area, a transition area and a handheld area, the blood sampling area comprises a spherical blood sampler and a circular truncated cone, and the surface of the spherical blood sampler is completely covered with flocking. According to the dried poultry blood spot swab, the dried poultry blood spot swab is divided into the blood taking area, the transition area and the handheld area, the surface of a spherical blood taking device in the blood taking area is completely covered with the flocking, the flocking is adopted for blood suction, the handheld area and the blood taking area can be isolated through the transition area, the environmental pollution probability is reduced, and when the dried poultry blood spot swab is adopted for blood taking, the sampling wound surface is small, and the blood taking amount is small; the growth of poultry is not influenced, and rapid and large-scale extraction of poultry blood nucleic acid can be realized.

Description

[0001] This application claims priority to patent application number 202511284609.0 (the earlier application was filed on September 9, 2025, and is entitled "A Dried Blood Spot Swab for Birds and Its Application"). Technical Field

[0002] This invention relates to the field of blood collection technology, and in particular to a dried avian blood spot swab and its application. Background Technology

[0003] Dried blood spots (DBS) are a novel sample collection technique and morphology, also known as dried blood dots or dried blood patches. They are formed by placing a drop of blood from the subject's fingertip or heel onto a sample collection card and allowing it to dry at room temperature. This technique offers advantages such as convenient collection, minimal invasiveness, and wide coverage. It has wide applications not only in traditional newborn screening but also in medical, public health, and scientific research fields. Nucleic acid extraction from dried blood spot samples is a core step in upstream experiments. In the poultry farming industry, dried blood spots are the preferred sampling method due to the small size of young birds and the difficulty of blood collection.

[0004] The current conventional method for dried blood spot sampling involves collecting blood from the subject's fingertip or heel, dropping it onto a sample collection card, drying it at room temperature to form a blood spot, and then collecting the collection card for subsequent testing. However, this traditional dried blood spot sampling method is extremely inconvenient. It suffers from a lack of standardization and normalization, with significant variations depending on the procedure, blood volume, drying time, and elution method, leading to inconsistent results between laboratories. Furthermore, differences in the hydrophilicity and blood cell adsorption capacity of different filter paper materials result in large fluctuations in nucleic acid yield. The pretreatment method for traditional dried blood spot sampling still relies on manual perforation, and automated equipment has not yet fully addressed the issue of parallel processing of high-throughput samples. Moreover, poultry farming differs from human medicine, requiring the collection of large batches of samples in a short time, necessitating further optimization of the convenience of traditional dried blood spot sampling methods.

[0005] Therefore, there is an urgent need for a simple, fast, and efficient method for blood collection. Summary of the Invention

[0006] To address the aforementioned technical problems, this invention provides a dried poultry blood spot swab and its application. The dried poultry blood spot swab of this invention can link blood aspiration with nucleic acid extraction, enabling rapid and large-scale extraction of poultry blood nucleic acid.

[0007] To achieve this objective, the present invention adopts the following technical solution: In a first aspect, the present invention provides a dried blood spot swab for poultry, the dried blood spot swab for poultry includes a blood collection area, a transition area and a handheld area, the blood collection area includes a spherical blood collector and a frustum, the surface of the spherical blood collector is completely covered with flocking.

[0008] In this invention, the dried blood spot swab of poultry is divided into a blood collection area, a transition area, and a handheld area. The blood collection area is used to collect blood, and the surface of the spherical blood collector in the blood collection area is completely covered with flocking to collect blood. The transition area can isolate the handheld area from the blood collection area, reducing the probability of environmental pollution. This method results in a small sampling wound, a small amount of blood collected, no impact on poultry growth, and enables rapid and large-scale extraction of poultry blood nucleic acid.

[0009] Preferably, the flocking material includes polyester fiber.

[0010] In this invention, polyester fiber is used as flocking material to absorb blood. First, polyester fiber has a high liquid absorption capacity, which can ensure sufficient extraction volume and increase the concentration of nucleic acid. Second, unlike cotton and other materials, polyester fiber does not have problems such as lint shedding that affects the extraction effect.

[0011] Preferably, the diameter of the spherical blood collection device is 1-3 mm. For example, it can be 1 mm, 1.5 mm, 2 mm, 2.5 mm or 3 mm, etc.

[0012] In this invention, the aforementioned diameter ensures a blood absorption volume of 5-10 μL, which guarantees an appropriate range of extracted nucleic acid concentrations to support subsequent detection.

[0013] Preferably, the diameter of the connection between the frustum and the spherical blood collection device is 0.5-1.5 mm. For example, it can be 0.5 mm, 0.6 mm, 0.7 mm, 0.8 mm, 0.9 mm, 1.0 mm, 1.1 mm, 1.2 mm, 1.3 mm, 1.4 mm, or 1.5 mm, etc.

[0014] Preferably, the height of the frustum (5) is 6-10 mm. For example, it can be 6 mm, 6.5 mm, 7 mm, 7.5 mm, 8 mm, 8.5 mm, 9 mm, 9.5 mm or 10 mm, etc.

[0015] Preferably, the diameter of the connection between the frustum and the transition zone is 2-4 mm. For example, it can be 2 mm, 2.5 mm, 3 mm, 3.5 mm, or 4 mm.

[0016] Preferably, the transition region has a length of 4-5 mm, a width of 2-4 mm, and a thickness of 2-3 mm. The 4-5 mm can be, for example, 4 mm, 4.2 mm, 4.4 mm, 4.6 mm, 4.8 mm, or 5 mm. The 2-4 mm can be, for example, 2 mm, 2.5 mm, 3 mm, 3.5 mm, or 4 mm. The 2-3 mm can be, for example, 2 mm, 2.2 mm, 2.4 mm, 2.5 mm, 2.6 mm, 2.8 mm, or 3 mm.

[0017] Preferably, the transition zone is recessed on both sides.

[0018] The transition zone in this invention can firstly prevent the overall structure from being vertical, thus preventing the swab from slipping from the sampler's hand during sampling. Secondly, it can visually distinguish the sampling area from the handheld area, preventing the sampling area from being contaminated.

[0019] Preferably, the depth of the recess is 0.5-0.6 mm. For example, it can be 0.5 mm, 0.52 mm, 0.54 mm, 0.56 mm, 0.58 mm or 0.6 mm, etc.

[0020] Preferably, the handheld area has a length of 10-20 mm, a width of 2-4 mm, and a thickness of 1-2 mm. The 10-20 mm can be, for example, 10 mm, 11 mm, 12 mm, 13 mm, 14 mm, 15 mm, 16 mm, 17 mm, 18 mm, 19 mm, or 20 mm. The 2-4 mm can be, for example, 2 mm, 2.5 mm, 3 mm, 3.5 mm, or 4 mm. The 1-2 mm can be, for example, 1 mm, 1.2 mm, 1.4 mm, 1.5 mm, 1.6 mm, 1.8 mm, or 2.0 mm.

[0021] In a second aspect, the present invention provides an application of the dried avian blood spot swabs described in the first aspect in the extraction of avian blood nucleic acid.

[0022] Thirdly, the present invention provides a method for extracting avian blood nucleic acid, the method comprising collecting blood using the avian dried blood spot swabs described in the first aspect and then performing detection.

[0023] Preferably, the method includes drawing blood using a spherical blood collection device made from dried poultry blood spot swabs, placing the spherical blood collection device completely inside a deep-well plate after blood collection, drying the blood, and then extracting nucleic acid.

[0024] Preferably, the amount of blood drawn is 5-10 μL. For example, it can be 5 μL, 6 μL, 7 μL, 8 μL, 9 μL, or 10 μL.

[0025] Preferably, the drying time is 2-4 hours and the temperature is 20-30°C. The 2-4 hours can be, for example, 2 hours, 2.5 hours, 3 hours, 3.5 hours, or 4 hours.

[0026] Compared with the prior art, the present invention has at least the following beneficial effects: 1. In this invention, the dried blood spot swab of poultry is divided into a blood collection area, a transition area, and a handheld area. The blood collection area is used to collect blood, and the surface of the spherical blood collector in the blood collection area is completely covered with flocking to collect blood. The transition area can isolate the handheld area from the blood collection area, reducing the probability of environmental pollution. This method results in a small sampling wound, a small amount of blood collected, no impact on poultry growth, and enables rapid and large-scale extraction of poultry blood nucleic acid.

[0027] 2. Based on the dried blood spot swabs of poultry, a method for extracting nucleic acid from poultry blood was developed. This method can connect blood aspiration with nucleic acid extraction, realizing the continuity of aspiration and nucleic acid extraction. Compared with conventional dried blood spot extraction methods, this method has a more convenient blood collection process, is less likely to cause sample contamination, causes less damage to poultry, requires less blood, and does not affect poultry growth. Attached Figure Description

[0028] Figure 1 This is an image of the appearance of a dried blood spot swab from poultry. In the image, 1 is the blood collection area, 2 is the transition area, 3 is the handheld area, 4 is the blood collection device, and 5 is a truncated cone.

[0029] Figure 2 The structure of a dried blood spot swab from birds.

[0030] Figure 3 This is the electrophoresis image of test example 4.

[0031] Figure 4 This is the electrophoresis image of test example 5.

[0032] Figure 5 This is an external view of a 96-hole deep hole plate. Detailed Implementation

[0033] The technical solution of the present invention will be further described below with reference to the accompanying drawings and specific embodiments. However, the following examples are merely simplified examples of the present invention and do not represent or limit the scope of protection of the present invention. The scope of protection of the present invention is determined by the claims.

[0034] The reagents used in the following examples were sourced from the following sources: DNA Extraction Kit: GenoPrep® Avian Dried Blood Spot DNA Extraction Kit (Magnetic Bead Method).

[0035] Example 1 This embodiment prepares a dried avian blood spot swab. like Figure 1 and Figure 2 As shown, the blood collection area, transition area, and handheld area are connected sequentially. The blood collection area consists of a spherical blood collection device and a frustum. The entire surface of the spherical blood collection device is covered with polyester fiber flocking. The diameter of the spherical blood collection device is 2 mm. The diameter of the connection between the frustum and the spherical blood collection device is 1 mm, and the height of the frustum is 8.4 mm. The diameter of the connection between the frustum and the transition area is 3 mm. The transition area is 4.6 mm long, 3 mm wide, and 3 mm thick, with recesses on both sides at a depth of 0.5 mm. The handheld area is 15 mm long, 3 mm wide, and 1 mm thick.

[0036] Example 2 This embodiment prepares a dried avian blood spot swab. like Figure 1 and Figure 2 As shown, the blood collection area, transition area, and handheld area are connected sequentially. The blood collection area consists of a spherical blood collection device and a frustum. The entire surface of the spherical blood collection device is covered with polyester fiber flocking. The diameter of the spherical blood collection device is 1 mm. The diameter of the connection between the frustum and the spherical blood collection device is 0.5 mm, and the height of the frustum is 10 mm. The diameter of the connection between the frustum and the transition area is 2 mm. The transition area is 5 mm long, 2 mm wide, and 2 mm thick, with recesses on both sides at a depth of 0.55 mm. The handheld area is 20 mm long, 2 mm wide, and 2 mm thick.

[0037] Example 3 This embodiment prepares a dried avian blood spot swab. like Figure 1 and Figure 2 As shown, the blood collection area, transition area, and handheld area are connected sequentially. The blood collection area consists of a spherical blood collection device and a frustum. The entire surface of the spherical blood collection device is covered with polyester fiber flocking. The diameter of the spherical blood collection device is 3 mm. The diameter of the connection between the frustum and the spherical blood collection device is 1.5 mm, and the height of the frustum is 6 mm. The diameter of the connection between the frustum and the transition area is 4 mm. The transition area is 4 mm long, 4 mm wide, and 2 mm thick, with recesses on both sides at a depth of 0.6 mm. The handheld area is 10 mm long, 4 mm wide, and 2 mm thick.

[0038] Example 4 This embodiment prepares a dried avian blood spot swab. like Figure 1 and Figure 2As shown, the blood collection area, transition area, and handheld area are connected sequentially. The blood collection area consists of a spherical blood collection device and a frustum. The entire surface of the spherical blood collection device is covered with nylon, and its diameter is 2 mm. The diameter of the connection between the frustum and the spherical blood collection device is 1 mm, and the height of the frustum is 8.4 mm. The diameter of the connection between the frustum and the transition area is 3 mm. The transition area is 4.6 mm long, 3 mm wide, and 3 mm thick, with recesses on both sides at a depth of 0.5 mm. The handheld area is 15 mm long, 3 mm wide, and 1 mm thick.

[0039] Example 5 This embodiment prepares a dried avian blood spot swab. like Figure 1 and Figure 2 As shown, the blood collection area, transition area, and handheld area are connected sequentially. The blood collection area consists of a spherical blood collection device and a frustum. The entire surface of the spherical blood collection device is covered with nylon, and its diameter is 1 mm. The diameter of the connection between the frustum and the spherical blood collection device is 0.5 mm, and the height of the frustum is 10 mm. The diameter of the connection between the frustum and the transition area is 2 mm. The transition area is 5 mm long, 2 mm wide, and 2 mm thick, with recesses on both sides at a depth of 0.55 mm. The handheld area is 20 mm long, 2 mm wide, and 2 mm thick.

[0040] Example 6 This embodiment prepares a dried avian blood spot swab. like Figure 1 and Figure 2 As shown, the blood collection area, transition area, and handheld area are connected sequentially. The blood collection area consists of a spherical blood collection device and a frustum. The entire surface of the spherical blood collection device is covered with nylon, and its diameter is 3 mm. The diameter of the connection between the frustum and the spherical blood collection device is 1.5 mm, and the height of the frustum is 6 mm. The diameter of the connection between the frustum and the transition area is 4 mm. The transition area is 4 mm long, 4 mm wide, and 2 mm thick, with recesses on both sides at a depth of 0.6 mm. The handheld area is 10 mm long, 4 mm wide, and 2 mm thick.

[0041] Example 7 This embodiment prepares a dried avian blood spot swab, which differs from Example 1 only in that the flocking material is cotton, and all other aspects are the same as in Example 1.

[0042] Example 8 This embodiment prepares a dried avian blood spot swab, which differs from Example 1 only in that the flocking material is artificial fiber, and all other aspects are the same as in Example 1.

[0043] Example 9 This embodiment prepares a dried avian blood spot swab, which differs from Example 1 only in that the polyester fiber flocking covers only half of the area; otherwise, it is the same as Example 1.

[0044] Example 10 This embodiment prepares a dried avian blood spot swab, which differs from Example 1 only in that the handheld area is a cylinder with a length of 4.6 mm and a diameter of 3 mm; otherwise, it is the same as Example 1.

[0045] Example 11 This embodiment prepares a dried avian blood spot swab, which differs from Example 1 only in that the diameter of the spherical blood collection device is 0.5 mm, and all other aspects are the same as in Example 1.

[0046] Example 12 This embodiment prepares a dried avian blood spot swab, which differs from Example 1 only in that the diameter of the spherical blood collection device is 5 mm, and all other aspects are the same as in Example 1.

[0047] Comparative Example 1 This comparative example prepares a dried avian blood spot swab, which differs from Example 1 only in that it does not include a transition zone and the handheld area is 19.6 mm long; otherwise, it is the same as Example 1.

[0048] Application Example 1 This application example involves extracting total DNA from avian blood. Holding the hand area of ​​the dried avian blood spot swab prepared in Example 1, 10 μL of chicken blood was drawn using a spherical blood sampler. Six blood samples were collected for testing, ensuring the flocked area was completely covered with blood during sampling. After blood collection, the sampler was placed in a deep-well plate, ensuring the blood collection area was completely within the plate. The plate was left open to dry the blood for 3 hours before being sealed with a silicone cap for storage.

[0049] The DNA extraction kit was used for detection. 500 μL of PL9 Buffer and 30 μL of Proteinase KSolution (20 mg / mL) were added to a 96-well round plate. Figure 5As shown, after vortexing for 2 min to mix thoroughly, incubate in a 65℃ water bath for 60 min, removing the tube every 5 min and vortexing to mix. After the water bath, briefly centrifuge. After removing from the water bath, add 500 μL of PP Buffer, vortex to mix, let stand at room temperature for 1 min, and then briefly centrifuge. Add 150 μL of PP Buffer solution to the above centrifuge tube, vortex for 30 s, let stand at room temperature for 1 min, and centrifuge at 4000 rpm for 10 min to obtain the supernatant. Transfer 600 μL of the supernatant to a centrifuge tube, add 360 μL of pre-chilled isopropanol and 20 μL of Magnetic Beads Solution. Vortex to mix for 30 s, let stand at room temperature for 1 min, centrifuge, and then place the centrifuge tube on a magnetic rack for 3 min until the magnetic beads are completely adsorbed. Discard the supernatant. Remove the centrifuge tube from the magnetic rack, add 600 μL of WBI Buer, vortex to mix for 30 s, let stand at room temperature for 2 min, centrifuge, place the centrifuge tube on the magnetic rack and let stand for 3 min until the magnetic beads are completely adsorbed, discard the supernatant, and repeat twice.

[0050] Remove the centrifuge tube from the magnetic rack, add 600 μL of WBII Buer, vortex to mix for 30 s, let stand at room temperature for 1 min, centrifuge, then place the centrifuge tube on the magnetic rack and let stand for 3 min until the magnetic beads are completely adsorbed. Discard the supernatant, and repeat twice. Keep the centrifuge tube on the magnetic rack to further remove any residual liquid in the tube, and air dry at room temperature for 10 min until no ethanol residue remains.

[0051] Remove the centrifuge tube from the magnetic rack, add 100 μL of 1×TE Buer, vortex to mix for 30 s, incubate at room temperature for 10 min, centrifuge, then transfer the centrifuge tube to the magnetic rack and incubate for 3 min until the magnetic beads are completely adsorbed. Transfer the DNA elution buffer to a new centrifuge tube. Application Example 2 This application example involves extracting total DNA from avian blood. Holding the hand area of ​​the dried avian blood spot swab prepared in Example 2, 10 μL of chicken blood was drawn using a spherical blood sampler. Six blood samples were collected for testing, ensuring the flocked area was completely covered with blood during sampling. After blood collection, the sampler was placed in a deep-well plate, ensuring the blood collection area was completely within the plate. The plate was left open to dry the blood for 2 hours before being sealed with a silicone cap for storage.

[0052] DNA extraction was performed using a DNA extraction kit. 500 μL of PL9 Buffer and 30 μL of Proteinase KSolution (20 mg / mL) were added to a 96-well round plate. After vortexing for 2 min to mix thoroughly, the plate was incubated at 65°C for 60 min, with the plate removed and vortexed every 5 min during the incubation. After the incubation period, a short centrifugation was performed. After removing the plate from the incubation period, 500 μL of PBD Buffer was added, vortexed, and allowed to stand at room temperature for 1 min before a short centrifugation. 150 μL of PP Buffer solution was added to the centrifuge tube, vortexed for 30 s, allowed to stand at room temperature for 1 min, and centrifuged at 4000 rpm for 10 min to obtain the supernatant. 600 μL of the supernatant was transferred to a centrifuge tube, and 360 μL of pre-chilled isopropanol and 20 μL of Magnetic Beads Solution were added. Shake to mix for 30 seconds, let stand at room temperature for 1 minute, centrifuge, then place the centrifuge tube on a magnetic rack and let stand for 3 minutes until the magnetic beads are completely adsorbed. Discard the supernatant. Remove the centrifuge tube from the magnetic rack, add 600 μL of WBI Buer, shake to mix for 30 seconds, let stand at room temperature for 2 minutes, centrifuge, then place the centrifuge tube on a magnetic rack and let stand for 3 minutes until the magnetic beads are completely adsorbed. Discard the supernatant and repeat twice.

[0053] Remove the centrifuge tube from the magnetic rack, add 600 μL of WBII Buer, vortex to mix for 30 s, let stand at room temperature for 1 min, centrifuge, then place the centrifuge tube on the magnetic rack and let stand for 3 min until the magnetic beads are completely adsorbed. Discard the supernatant, and repeat twice. Keep the centrifuge tube on the magnetic rack to further remove any residual liquid in the tube, and air dry at room temperature for 10 min until no ethanol residue remains.

[0054] Remove the centrifuge tube from the magnetic rack, add 100 μL of 1×TE Buer, vortex to mix for 30 s, incubate at room temperature for 10 min, centrifuge, then transfer the centrifuge tube to the magnetic rack and incubate for 3 min until the magnetic beads are completely adsorbed. Transfer the DNA elution buffer to a new centrifuge tube. Application Example 3 This application example involves extracting total DNA from avian blood. Holding the hand area of ​​the dried avian blood spot swab prepared in Example 3, 10 μL of chicken blood was drawn using a spherical blood sampler. Six blood samples were collected for testing, ensuring the flocked area was completely covered with blood during sampling. After blood collection, the sampler was placed in a deep-well plate, ensuring the blood collection area was completely within the plate. The plate was left open to dry the blood for 4 hours before being sealed with a silicone cap for storage.

[0055] DNA extraction was performed using a DNA extraction kit. 500 μL of PL9 Buffer and 30 μL of Proteinase KSolution (20 mg / mL) were added to a 96-well round plate. After vortexing for 2 min to mix thoroughly, the plate was incubated at 65°C for 60 min, with the plate removed and vortexed every 5 min during the incubation. After the incubation period, a short centrifugation was performed. After removing the plate from the incubation period, 500 μL of PBD Buffer was added, vortexed, and allowed to stand at room temperature for 1 min before a short centrifugation. 150 μL of PP Buffer solution was added to the centrifuge tube, vortexed for 30 s, allowed to stand at room temperature for 1 min, and centrifuged at 4000 rpm for 10 min to obtain the supernatant. 600 μL of the supernatant was transferred to a centrifuge tube, and 360 μL of pre-chilled isopropanol and 20 μL of Magnetic Beads Solution were added. Shake to mix for 30 seconds, let stand at room temperature for 1 minute, centrifuge, then place the centrifuge tube on a magnetic rack and let stand for 3 minutes until the magnetic beads are completely adsorbed. Discard the supernatant. Remove the centrifuge tube from the magnetic rack, add 600 μL of WBI Buer, shake to mix for 30 seconds, let stand at room temperature for 2 minutes, centrifuge, then place the centrifuge tube on a magnetic rack and let stand for 3 minutes until the magnetic beads are completely adsorbed. Discard the supernatant and repeat twice.

[0056] Remove the centrifuge tube from the magnetic rack, add 600 μL of WBII Buer, vortex to mix for 30 s, let stand at room temperature for 1 min, centrifuge, then place the centrifuge tube on the magnetic rack and let stand for 3 min until the magnetic beads are completely adsorbed. Discard the supernatant, and repeat twice. Keep the centrifuge tube on the magnetic rack to further remove any residual liquid in the tube, and air dry at room temperature for 10 min until no ethanol residue remains.

[0057] Remove the centrifuge tube from the magnetic rack, add 100 μL of 1×TE Buer, vortex to mix for 30 s, incubate at room temperature for 10 min, centrifuge, then transfer the centrifuge tube to the magnetic rack and incubate for 3 min until the magnetic beads are completely adsorbed. Transfer the DNA elution buffer to a new centrifuge tube. Application Example 4-12 The total DNA extracted from avian blood in this application example differs from Application Example 1 only in that the dried avian blood spot swabs prepared in Examples 4-12 are used; otherwise, they are identical to Application Example 1.

[0058] Comparative Application Example 1 The total DNA extracted from avian blood in this comparative application example differs from application example 1 only in that the avian dried blood spot swabs prepared in comparative example 1 are used; otherwise, they are identical to application example 1.

[0059] Test Example 1 This test case measures the content of extracted DNA. This test case detects the DNA content in the areas of the above application examples and comparative application examples. The specific detection results are shown in Table 1.

[0060] Table 1 The above test results show that: (1) By comparing Application Example 1 with Application Examples 2-3, it can be seen that the method of the present invention can effectively extract DNA from blood and the extraction concentration is high, which is beneficial to subsequent detection and analysis.

[0061] (2) By comparing application example 1 with application examples 4-6, it can be seen that using nylon for the surface of the spherical blood collection device can achieve better results than using polyester fiber.

[0062] (3) By comparing Application Example 1 with Application Examples 7, 8 and 9, it can be seen that when cotton or artificial fiber is used for flocking and the flocking coverage is reduced, the liquid absorption rate will decrease, thereby reducing the concentration of extracted DNA. The low DNA concentration will prevent subsequent detection methods such as qPCR from being performed.

[0063] (4) A comparison between Application Example 1 and Application Example 10 shows that the cylindrical tail can cause the tip of the automated liquid aspiration device to collide with the tail of the swab when it is inserted, resulting in damage to the tip and affecting liquid aspiration. Due to the low success rate of liquid aspiration, the extraction results are poor and cannot meet the requirements of subsequent testing.

[0064] (5) By comparing Application Example 1 with Application Examples 11 and 12, it can be seen that if the diameter of the sphere is too large, the flocking distribution of the head will be uneven, the extraction concentration will be unstable, and the purity will be poor. If the diameter of the sphere is too small, the extraction concentration will be low and the stability of the extraction results will be poor.

[0065] (6) By comparing Application Example 1 with Comparative Application Example 1, it can be seen that when the tail of the swab is long, the deep well plate cannot be sealed. Therefore, during the shaking operation, the liquid inside the deep well plate is easily lost, and there is also a risk of sample contamination caused by liquid splashing.

[0066] Test Example 2 This test case measures the success rate of liquid aspiration. This test example uses a 96-well deep-well plate (0.8 cm diameter, 4.2 cm height) for automated pipetting device compatibility testing. The specific operation is as follows: dried avian blood spot swabs prepared in Example 1 and flocked swabs with cylindrical tail ends were used respectively. Figure 1As shown, the samples were connected to an automated pipetting device to aspirate the supernatant after centrifugation during the extraction process, totaling 200 μL. A total of 96 samples were collected and subjected to 20 aspiration tests using the automated pipetting device. The aspiration success rate was calculated (successful aspiration from all 96 wells was considered sufficient). Specific test results are shown in Table 2.

[0067] Table 2 The results show that the poultry dried blood spot swabs prepared by this invention are better suited for 96-well plates, and the success rate of liquid aspiration is significantly improved compared with commercially available products.

[0068] Test Example 3 This test case examines DNA in blood samples under different conditions. This test case examines the concentration and purity of nucleic acid in blood extracted from avian dried blood spot swabs preserved under different environments. The specific groups are: room temperature group, high temperature group, low temperature group, and high humidity group. The environmental conditions for these groups are as follows: room temperature group: stored at room temperature; high temperature group: stored at 60°C; low temperature group: stored at -20°C; high humidity group: stored at room temperature with humidity adjusted to 50%.

[0069] After preserving the above groups for 1 day and 7 days, blood was collected using dried poultry blood spot swabs prepared in Example 1 (the blood should completely cover the flocked area). The blood-soaked swabs were placed in 96-well plates and sealed with silicone caps. Nucleic acid extraction was performed after storing the 96-well plates at -20°C for 1 day and 7 days, and the extracted nucleic acid concentration was measured to calculate the extraction success rate. The nucleic acid extraction method described in Example 1 was used for extraction, and the specific results are shown in Table 3.

[0070] Table 3 The above experimental results show that the avian dried blood spot swabs of the present invention can be used to extract blood DNA under different preservation conditions without affecting the purity, and can be used for subsequent testing.

[0071] Test Example 4 This test case involved sampling and extracting nucleic acids from 100 duck samples. The blood collector restrained the duck to be blooded, ensuring the duck's legs, wings, and head were still. The brachial vein (medial wing vein) was located. The blood collection site was cleaned and disinfected with 75% alcohol swabs. After the site had air-dried, the vein was punctured with a lancet, and the dried avian blood spot swab prepared in Example 1 was immediately used to collect blood, ensuring the flocked area of ​​the swab tip was completely covered with blood. After collection, the swab was quickly placed into a 96-well deep plate, allowed to air-dry, and then sealed with a silicone cap for storage, awaiting nucleic acid extraction. Nucleic acid extraction was performed using the method described in Example 1, and the specific results are shown in Table 4.

[0072] Table 4 Prepare a 1% agarose gel. Mix the extracted nucleic acids and loading buffer, and then transfer the mixture to the same gel well using a pipette. Electrophoresis for 21 minutes, and then photograph the gel using an imager. Figure 3 As shown, M represents a 10 K marker. The results demonstrate that the avian dried blood spot swabs of this invention can effectively extract nucleic acids from avian blood in large quantities, and the extracted nucleic acids are highly intact and concentrated, suitable for subsequent testing.

[0073] Test Example 5 This test case involved sampling and extracting nucleic acids from 96 duck samples. The blood collector restrained the duck to be blooded, ensuring the duck's legs, wings, and head were still. The brachial vein (medial wing vein) was located. The blood collection site was cleaned and disinfected with a 75% alcohol swab. After the site had air-dried, the vein was punctured with a lancet, and the dried avian blood spot swab prepared in Example 4 was immediately used to collect blood, ensuring the flocked area of ​​the swab tip was completely covered with blood. After collection, the swab was quickly placed into a 96-well deep plate, allowed to air-dry, and then sealed with a silicone cap for storage, awaiting nucleic acid extraction. Nucleic acid extraction was performed using the method described in Example 1, and the specific results are shown in Table 5.

[0074] Table 5 Prepare a 1% agarose gel. Mix the extracted nucleic acids and loading buffer, and then transfer the mixture to the same gel well using a pipette. Electrophoresis for 21 minutes, and then photograph the gel using an imager. Figure 4 As shown, M represents a 10 K marker. The results demonstrate that the avian dried blood spot swabs of this invention can effectively extract nucleic acids from avian blood in large quantities, and the extracted nucleic acids are highly intact and concentrated, suitable for subsequent testing.

[0075] In summary, this invention divides the dried poultry blood spot swab into a blood collection area, a transition area, and a handheld area. The entire surface of the spherical blood collection device in the blood collection area is covered with flocking, and blood is drawn using flocking. The transition area isolates the handheld area from the blood collection area, reducing the probability of environmental pollution. Using this dried poultry blood spot swab for blood collection results in a small sampling wound, a small blood volume, and does not affect poultry growth. Furthermore, it enables rapid and large-scale extraction of poultry blood nucleic acid.

[0076] The applicant declares that the above description is only a specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention fall within the protection and disclosure scope of the present invention.

Claims

1. A dried blood spot swab for poultry, characterized in that, The avian dry blood spot swab includes a blood collection area (1), a transition area (2) and a handheld area (3). The blood collection area includes a spherical blood collector (4) and a frustum (5). The surface of the spherical blood collector is completely covered with flocking.

2. The dried blood spot swab for poultry according to claim 1, characterized in that, The flocking material includes polyester fiber or nylon; Preferably, the diameter of the spherical blood collection device (4) is 1-3 mm.

3. The dried avian blood spot swab according to claim 1 or 2, characterized in that, The diameter of the connection between the frustum (5) and the spherical blood collection device (4) is 0.5-1.5 mm; Preferably, the height of the frustum (5) is 6-10 mm; Preferably, the diameter of the connection between the frustum (5) and the transition zone (2) is 2-4 mm.

4. The dried blood spot swab for poultry according to any one of claims 1-3, characterized in that, The transition region (2) has a length of 4-5 mm, a width of 2-4 mm, and a thickness of 2-3 mm; Preferably, the transition region (2) is recessed on both sides; Preferably, the depth of the depression is 0.5-0.6 mm.

5. The dried blood spot swab of poultry according to any one of claims 1-4, characterized in that, The handheld area (3) is 10-20 mm long, 2-4 mm wide, and 1-2 mm thick.

6. The use of dried avian blood spot swabs according to any one of claims 1-5 in the extraction of avian blood nucleic acid.

7. A method for extracting nucleic acid from avian blood, characterized in that, The method includes collecting blood using the dried avian blood spot swabs as described in any one of claims 1-5 and then performing the test.

8. The method for extracting avian blood nucleic acid according to claim 7, characterized in that, The method includes using a spherical blood collection device made from dried avian blood spot swabs to collect blood, placing the spherical blood collection device completely inside a deep-well plate after blood collection, drying the blood, and then extracting nucleic acid.

9. The method for extracting avian blood nucleic acid according to claim 8, characterized in that, The amount of blood drawn is 5-10 μL.

10. The method for extracting avian blood nucleic acid according to claim 8 or 9, characterized in that, The drying time is 2-4 hours.

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