Method for verification of blood typing in cynomolgus monkeys

Abstract

The present invention provides a method for verifying the blood type of cynomolgus monkeys, and belongs to the technical field of blood type identification of cynomolgus monkeys. The method comprises the following steps: step 1: collecting blood from venous cynomolgus monkeys and storing the blood; step 2: preparing monkey blood, putting monkey blood into a centrifuge for centrifugal separation into serum or plasma; step 3: preparing human blood, and using a centrifuge to extract a mixed liquid of red blood cells; step 4: screening, dripping 2 drops of monkey serum to an anti-A column and an anti-B column for each monkey, dripping 1 drop of 10% human red blood cells of type A blood to an anti-A column, dripping 1 drop of 10% human red blood cells of type B blood to an anti-B column, and then determining the blood type of the cynomolgus monkeys. The identification and screening method of the present invention is simple and fast, and identification is performed by borrowing human red blood cells, and has high identification accuracy, can be quickly and massively identified, and has low identification cost, and can identify a large number of cynomolgus monkey blood types at one time, and is very popularizable.

Description

Technical Field

[0001] The invention relates to the technical field of cynomolgus monkey blood type identification, and in particular to a cynomolgus monkey blood type identification and verification method. Background Art

[0002] Crab-eating macaque (scientific name: Macaca fascicularis): also known as long-tailed macaque. Adult body length is about 40-47 cm, tail length is 50-60 cm. Male weight is 5-7 kg, female weight is about 3-4 kg. Fur color varies from yellow, gray, brown, ranging from gray-brown to reddish brown. The hair on the abdomen and the inside of the limbs is light white; the crown hair is draped behind, the face is brown-gray, with whiskers, the skin around the eyes is naked, and there is a white triangle on the upper side of the eyelids; the ears are erect and the eyes are black. The nose is flat and the nostrils are very narrow. Because they like to go to the beach to forage for crabs and shellfish after low tide, they are called crab-eating macaques.

[0003] Existing methods for identifying cynomolgus macaques are very complicated and require the use of a large amount of experimental equipment. Therefore, it is necessary to design a simple, fast and accurate method for identifying and verifying the blood type of cynomolgus macaques. Summary of the invention

[0004] The purpose of the present invention is to provide a method for identifying and verifying the blood type of cynomolgus monkeys, so as to solve the technical problems that the existing blood type identification methods of cynomolgus monkeys are complicated and have low accuracy.

[0005] In order to achieve the above object, the technical solution adopted by the present invention is as follows:

[0006] A method for identifying and verifying the blood type of cynomolgus monkeys, the method comprising the following steps:

[0007] Step 1: Collect blood from the venous blood of the cynomolgus monkey and store it;

[0008] Step 2: monkey blood preparation, placing the monkey blood in a centrifuge for centrifugation to separate into serum or plasma;

[0009] Step 3: Prepare human blood and use a centrifuge to extract the red blood cell mixed liquid;

[0010] Step 4: Screening, drop 2 drops of monkey serum for each monkey onto the anti-A column and the anti-B column, drop 1 drop of 10% human red blood cells of type A blood onto the anti-A column, drop 1 drop of 10% human red blood cells of type B blood onto the anti-B column, and then determine the blood type of the cynomolgus monkey.

[0011] Furthermore, the specific process of step 1 is to collect 1.5 ml of whole blood from the cynomolgus monkey vein and put it into a 1.7 ml or 2 ml microtube, and let it stand at room temperature for 30 minutes.

[0012] Furthermore, the specific process of step 2 is: in the virus isolation testing room, operate according to the centrifuge operating reference instructions. The operator needs to wear appropriate PPE and ensure that the collected blood samples are separated into serum or plasma. Place the centrifuge tube in the machine, turn on the power, adjust the centrifuge speed to 10000rpm / minute, centrifuge for 5 minutes, and use a pipette to transfer the serum to a new microtube for standby use. Please label the microtube in advance before using it.

[0013] Further, the specific process of step 3 is to operate in the virus isolation test room according to the centrifuge operation reference instructions. The operator needs to wear appropriate PPE and ensure that the collected blood sample is separated into serum or plasma. 1 ml of human whole blood in the EDTA tube is transferred to a 1.7 ml or 2 ml microtube, and then centrifuged. The microcentrifuge is 10000 rpm / minute for 5 minutes. The separated red blood cells are transferred to a microtube containing 1 ml of normal saline with a pipette. The amount of red blood cells is 300 microliters or 0.3 ml. The red blood cells and saline are mixed and the bottom of the test tube is tapped with a finger. Stir evenly up and down, do not shake the test tube vigorously to avoid damaging the red blood cells, then centrifuge the mixed liquid, microcentrifuge 10000rpm / min, centrifuge for 5 minutes, use a pipette to aspirate the supernatant, discard the supernatant, then add 1ml of saline, repeat the operation 3 times, during the last centrifugation, you can see the clear supernatant, transfer 100 microliters or 200 microliters of red blood cells from the treated test tube to a conical tube containing 0.9ml or 1.8ml of saline, the red blood cell content is 10%, when moving 200 microliters of red blood cells, first use a pipette to remove the supernatant.

[0014] Further, in step 4, use a wooden stick to mix the monkey blood serum with the human red blood cells, fix one drop of blood with one stick, do not use the same stick to fix other blood, and slowly shake around the plastic plate. When there is no reaction on both sides, it is AB type, when there is a reaction at the anti-A column, it is B type, when there is a reaction at the anti-B column, it is A type, and when both the anti-A column and the anti-B column react, it is O type. The reading result is completed within 15 to 20 seconds. If the time is too long, the reading result may be wrong because the RBC will settle.

[0015] The present invention has the following beneficial effects due to the adoption of the above technical solution:

[0016] (1) The identification and screening method of the present invention is simple and rapid. It uses human red blood cells for identification and has high identification accuracy. It can be quickly and massively identified. At the same time, the identification cost is low. A large number of cynomolgus macaque blood types can be identified at one time, which is very popularizable.

[0017] (2) The present invention can complete the identification of blood type in the fastest and shortest time. The ABO blood type of crab-eating macaques is detected by conventional immunological detection methods, and the blood type identification of experimental monkeys is essential in research such as organ transplantation. The purpose of this study is to establish a rapid and reliable method for identifying the ABO blood type of crab-eating macaques. Methods Our laboratory has established a new method for identifying ABO blood type. 1 ml of human whole blood (3 ml) in an EDTA tube is transferred to a microtube (1.7 or 2 ml), and then centrifuged (microcentrifuge 10000 rpm 5 min). Molecular detection method, using competitive allele PCR (KASP) technology, by identifying the bases at 2 SNP sites of functional genes closely related to blood type, use a wooden stick to mix monkey blood serum with human red blood cells (one stick is used to fix one drop of blood, do not use the same stick to fix other blood) for identification. DETAILED DESCRIPTION

[0018] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, preferred embodiments are given below to further describe the present invention in detail. However, it should be noted that many details listed in the specification are only for the purpose of enabling the reader to have a thorough understanding of one or more aspects of the present invention, and these aspects of the present invention can be implemented even without these specific details.

[0019] The specific steps of the cynomolgus monkey blood type identification and verification method are as follows:

[0020] 1.1 The theoretical process is organized and the specific steps are listed. The subsequent identification process is identified according to the specific steps. The principle steps of the identification are the steps of the above invention content.

[0021] Instruments / Materials

[0022] 2.1 Centrifuge (Micro II High Speed ​​Centrifuge Smart )

[0023] 2.2 Pipette

[0024] 2.3 Human whole blood (type A, type B)

[0025] 2.4 Monkey whole blood

[0026] 2.5 EDTA tube (3ml), micro tube (1.7ml or 2ml)

[0027] 2.6 Test tube

[0028] 2.7 Saline

[0029] 2.8 Plastic basin

[0030] 2.9 Wooden sticks or toothpicks

[0031] III. Operation Procedure

[0032] 3.1 Monkey venous blood collection

[0033] 1.5 ml of monkey whole blood was collected intravenously and placed in a microtube (1.7 ml or 2 ml) and allowed to stand at room temperature for 30 minutes.

[0034] 3.2 Monkey blood preparation

[0035] 3.2.1 Preparation site: Virus isolation test room, centrifuge operation refers to "Desktop Centrifuge Operation Procedure (Micro High-speed Centrifuge Intelligent )>》Instructions.

[0036] 3.2.2 Operators need to wear appropriate PPE and ensure that the blood samples are separated into serum or plasma.

[0037] 3.2.3 Separation of serum: Place the centrifuge tube into the machine, turn on the power, adjust the centrifuge speed to 10,000 rpm (rotations / minute), and centrifuge for 5 minutes.

[0038] 3.2.4 Use a pipette to transfer the serum to a new microtube for later use.

[0039] Note: Please label the microtubes before use!

[0040] 3.3 Human blood preparation

[0041] 3.3.1 Preparation site: Virus isolation test room, centrifuge operation refers to the "Desktop Centrifuge Operation Procedure (Micro High-speed Centrifuge Intelligent )>》Instructions.

[0042] 3.3.2 Operators need to wear appropriate PPE and ensure that the blood samples are separated into serum or plasma.

[0043] 3.3.3 Transfer 1 ml of human whole blood (3 ml) in the EDTA tube to a microtube (1.7 or 2 ml), and then centrifuge (microcentrifuge 10000 rpm 5 min).

[0044] 3.3.4 Use a pipette to transfer the separated red blood cells into a microtube containing 1 ml of normal saline. The amount of red blood cells is 300 microliters or 0.3 ml.

[0045] 3.3.5 Mixing red blood cells and saline

[0046] 3.3.5.1 Tap the bottom of the test tube gently with your finger.

[0047] 3.3.5.2 Stir slowly up and down to make it evenly

[0048] Note: Do not shake the test tube vigorously to avoid damaging the red blood cells. Handle it with care!

[0049] 3.3.6 Mix according to 3.3.5 and centrifuge (10000 rpm for 5 min).

[0050] 3.3.7 Use a pipette to aspirate the supernatant, discard it, and then add 1 ml of normal saline.

[0051] 3.3.8 Repeat (3.3.5-3.3.7) for 3 times. During the last centrifugation, a clear supernatant can be seen.

[0052] 3.3.9 Transfer 100 μl or 200 μl of red blood cells from the treated test tube to a conical tube containing 0.9 ml or 1.8 ml of normal saline. The red blood cell content is 10%.

[0053] Note: When transferring 200 μl of red blood cells, first remove the supernatant with a pipette to more safely store the red blood cells.

[0054] 3.4 Screening

[0055] 3.4.1 For each monkey, add 2 drops of monkey serum to the column.

[0056]

[0057] 3.4.2 Add 1 drop of 10% human red blood cells (type A blood) to the anti-A column, and add 1 drop of 10% human red blood cells (type A blood) to the anti-A column.

[0058] B blood type) to the anti-B column.

[0059] 3.4.3 Use a wooden stick to mix the monkey blood serum with human red blood cells (use one stick to fix one drop of blood, do not use the same stick to fix other drops of blood)

[0060] 3.4.4 Slowly shake the plastic disk surrounding it.

[0061] 3.4.5 You can see lumps (or condensation) in the reaction area

[0062] -No response from either side: AB type

[0063] -Anti-A reaction: Type B

[0064] - Anti-B reaction: Type A

[0065] - Both sides react: O type

[0066] 3.4.6 Read the result within 15-20 seconds. If it takes too long, the result may be wrong because the RBC will settle.

[0067] 3.4.7 Record all results in the test log.

[0068] The technology of the present invention is a result of my many days of scientific research and technical discovery. It has solved the relatively novel blood type identification method of crab-eating macaques so far, and can complete the blood type identification in the fastest and shortest time. The conventional immunological detection method for ABO blood type of crab-eating macaques, and the blood type identification of experimental monkeys is indispensable in research such as organ transplantation. The purpose of this study is to establish a fast and reliable ABO blood type identification method for crab-eating macaques. Methods Our laboratory has established a new ABO blood type identification method. 1ml of human whole blood (3ml) in the EDTA tube is transferred to a microtube (1.7 or 2ml), and then centrifuged (microcentrifuge 10000rpm 5min) Molecular detection method, using competitive allele PCR (KASP) technology, by identifying the bases on the two SNP sites of the functional gene closely related to the blood type, using a wooden stick, the monkey blood serum is mixed with human red blood cells (one stick is used to fix one drop of blood, do not use the same stick to fix other blood) for identification.

[0069] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for verifying blood typing in cynomolgus monkeys, characterized in that: The method comprises the following steps: Step 1: Collect blood from the venous blood of the cynomolgus monkey and store it; Step 2: monkey blood preparation, placing the monkey blood in a centrifuge for centrifugation to separate into serum or plasma; Step 3: Prepare human blood and use a centrifuge to extract the red blood cell mixed liquid; Step 4: Screening, drop 2 drops of monkey serum from each monkey onto the anti-A column and the anti-B column, drop 1 drop of 10% human red blood cells of type A blood onto the anti-A column, drop 1 drop of 10% human red blood cells of type B blood onto the anti-B column, and then determine the blood type of the cynomolgus monkey; The specific process of step 1 is to collect 1.5 ml of whole blood from the cynomolgus monkey vein and put it into a 1.7 ml or 2 ml microtube, and let it stand at room temperature for 30 minutes; The specific process of step 2 is as follows: In the virus isolation testing room, operate according to the centrifuge operating instructions. The operator needs to wear appropriate PPE and ensure that the blood sample is separated into serum or plasma. Put the centrifuge tube into the machine, turn on the power, adjust the centrifuge speed to 10000rpm / minute, centrifuge for 5 minutes, and use a pipette to transfer the serum to a new microtube for standby use. Please label the microtube in advance before using it; The specific process of step 3 is to operate the centrifuge in the virus isolation test room according to the reference instructions for operation. The operator needs to wear appropriate PPE and ensure that the collected blood samples are separated into serum or plasma. Transfer 1 ml of human whole blood in the EDTA tube to a 1.7 ml or 2 ml microtube, and then centrifuge it. The microcentrifuge is 10000 rpm / minute for 5 minutes. Use a pipette to transfer the separated red blood cells to a microtube containing 1 ml of normal saline. The amount of red blood cells is 300 microliters or 0.3 ml. Mix the red blood cells and saline, tap the bottom of the test tube with your fingers, and then stir it up and down. Stir well, do not shake the test tube vigorously to avoid damage to the red blood cells, then centrifuge the mixed liquid, microcentrifuge 10000rpm / min, centrifuge for 5 minutes, use a pipette to aspirate the supernatant, discard the supernatant, then add 1ml of normal saline, repeat the operation 3 times, during the last centrifugation, you can see the clear supernatant, move 100 microliters or 200 microliters of red blood cells from the treated test tube to a conical tube containing 0.9ml or 1.8ml of normal saline, the red blood cell content is 10%, when moving 200 microliters of red blood cells, first use a pipette to remove the supernatant; In step 4, use a wooden stick to mix the monkey blood serum with human red blood cells. Fix one drop of blood with one stick. Do not use the same stick to fix other blood. Slowly shake the plastic plate. When there is no reaction on both sides, it is AB type. When there is a reaction at the anti-A column, it is B type. When there is a reaction at the anti-B column, it is A type. When both the anti-A column and the anti-B column react, it is O type. Complete the reading within 15 to 20 seconds. If the time is too long, the reading result may be wrong because the RBC will settle.

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