African Swine Fever Virus Detection Kit

African Swine Fever Virus Detection Kit (column membrane method) instruction manual

African Swine Fever Virus Detection Kit was designed with specific primers and probes based on African swine fever genome, and the DNA of African swine fever was amplified in vitro by fluorescent PCR.

[Product name] Common name: Viral DNA extraction kit

English name: Extraction Kit for Virus DNA

[Intended use] This kit is mainly used for the extraction of viral DNA from anticoagulant blood, blood, swabs, tissues, feed and the environment caused by suspected viruses and other pathogenic infections.The purified nucleic acid can be used in various routine operations and PCR tests..

【Kit components】

I.Kit composition and storage conditions name 20T 50T

1.20 adsorption columns and 50 pcs.2.20 collection tubes and 50 pcs.3.Lysis solution 5mL11mL4.Washing solution I13mL32mL5, Washing solution II13mL32mL6.Eluent 1.2mL3mL

2.Storage conditions and validity period: Store at room temperature, validity period is 12 months.

3.You need to prepare your own materials: absolute ethanol, 1.5mL sterile centrifuge tube, 1.5mL sterile centrifuge tube (long arm type), physiological saline, mortar, sterile 1mL, 200uL, 10uL pipette tip, marker, etc.

【Operating steps】

1.Sample preparation

1.Blood samples: Place the anticoagulated blood sample in a centrifuge tube, centrifuge at 8000 rpm for 2 minutes, and take the upper plasma layer; place the non-anticoagulated blood sample in a centrifuge tube, and after coagulation, centrifuge at 8000 rpm for 2 minutes and take the upper layer.Serum, placed in sterilized centrifuge tubes, numbered for inspection.

2.Tissue samples: Collect 0.1g of diseased tissue samples such as spleen, liver, lymph nodes, tonsils, etc.Fully homogenize or grind in a tissue homogenizer or mortar, add 1mL of physiological saline, mix well, centrifuge at 8000 rpm for 2 minutes, and take The supernatant was placed in a sterilized centrifuge and numbered for inspection.

3.Oral fluid: Before eating, use a swab/oral fluid collection bag or homemade cotton thread tied with gauze to attract animals to chew.Collect oral fluid greater than 500uL, centrifuge at 8000 rpm for 2 minutes, and take the supernatant in a sterilized centrifuge tube.Number Pending inspection.

4.Feed: Take an appropriate amount of ground feed (about 0.2g), put it into a 2mL EP tube containing 1mL physiological saline or PBS (pH 7.4) buffer, shake and mix, centrifuge at 8000 rpm for 2 minutes, and take out the tube.Clear it in a sterilized centrifuge tube and number it for inspection.

5.Dust: Dip an appropriate amount of dust on the environmental surface with a sterile cotton swab, put it into a 2mL EP tube containing 1mL physiological saline or PBS (pH 7.4) buffer, shake and mix, centrifuge at 8000 rpm for 2 minutes, and take out The supernatant is placed in a sterilized centrifuge tube and numbered for inspection.

2.DNA extraction

1.Take 200uL of lysis solution and add 1.5mL of nuclease-free centrifuge solution.Add 200uL of plasma/serum/tissue fluid and other sample solutions respectively.Vortex and mix for 10 seconds or invert and mix 10 times.Leave it at room temperature for 5 minutes (if the room temperature is When the internal temperature is low, it needs to be placed in a 25C water bath for incubation), then add 200uL absolute ethanol, invert and mix for 10 seconds; if the sample is turbid, centrifuge it at 12000 rpm for 1 minute, and take the supernatant to avoid clogging the column.;

2.Transfer the liquid into an adsorption column with a collection tube, and centrifuge at 12,000 rpm for 1 minute:

3.Discard the liquid in the collection tube, add 600uL washing solution I to the adsorption column, and centrifuge at 12000rpm for 1 minute;

4.Discard the liquid in the collection tube, add 600uL Washing Solution II to the adsorption column, and centrifuge at 12000rpm for 1 minute;

5.Discard the liquid in the collection tube and centrifuge at 12,000 rpm for 2 minutes to completely remove the residual washing liquid;

6.Transfer the adsorption column to a new nucleic acid-free 1.5mL EP tube (long-arm type), drop 50uL eluent into the center of the column, let it stand for 1 minute, and centrifuge at 12000rpm for 1 minute.The liquid in the EP tube is the viral DNA..

[Notes] 1.Read the instructions of this kit carefully before the experiment and strictly follow the operating steps.The best results can be obtained by accurately controlling the time, reagent volume, etc.during the operation.

2.Samples should be collected freshly for use or transported and stored at low temperature;

3.Samples have potential infection risks, and nucleic acid extraction should be performed in a biosafety cabinet in a nucleic acid extraction laboratory.

4.All consumables for nucleic acid extraction must be sterilized at high temperatures.After extraction, the nucleic acid can enter the next step of testing as soon as possible or be frozen and stored for later use.

5.The reagent should be mixed evenly before use.The reagent is corrosive to a certain extent.Please wear gloves and a mask for protection when using it.

6.It is normal for the lysate to crystallize at low temperatures and can be used after heating in a 60°C water bath to help dissolve it.

7.The waste generated by the experiment should be collected in time and treated away from the PCR laboratory for harmless treatment.

For Veterinary Diagnostic Use Only

African Swine Fever Virus Fluorescent PCR Nucleic Acid Detection Kit Instructions [Veterinary Drug Name]

Generic name: African swine fever virus fluorescent PCR nucleic acid detection kit

Chinese Pinyin: Feizliouzhuwen Bingdu Yingguang PCR Hesuan Jianceshijihe

English name: African swine Fever Virus (ASFV) PCR Nucleic Acid Diagnostic Kit

Product name: None

[Main components and content] The kit contains the following components: kit component content, positive control 250 μL/tube × 1 tube, negative control 250 μL/tube × 1 tube, PCR reaction solution 850 μL/tube × 1 tube, mixed Solution 150μL/tube × 1 tube, 1 instruction manual/box

[Function and use] This kit can be used for the detection of African swine fever virus nucleic acid in blood, spleen, liver, lymph nodes, and samples.Tonsils, kidneys, muscles, environmental samples, etc.

【Usage and Judgment】

1 Usage

1.1 Collection, storage and transportation of samples to be inspected

1.1.1 Sample collection

(1) Aseptically collect 5mL of anticoagulated blood or serum from live pig samples

(2) Necropsy samples of dead pigs or slaughterhouse necropsy samples, and sterile collection of tissue samples such as dead pigs, lungs, kidneys, tonsils, lymph nodes, muscles, etc.Transport to the laboratory at low temperature of 2~8°C for testing.

(3) Collect feces, feed, and sewage samples from places related to sick pigs in the surrounding environment contaminated by sick pigs.Transport to the laboratory at low temperature of 2~8°C for testing.

1.1.2 Sample preservation

The collected samples should be stored at 2~8°C for no more than 24 hours and at -70°C to avoid repeated freezing and thawing.

1.1.3 Sample transportation

The foam box is packed with ice packs and then sealed for transportation.Packaging and transportation should comply with the Ministry of Agriculture and Rural Affairs' "Specifications for Transport and Packaging of Highly Pathogenic Animal Pathogenic Microorganisms (Viruses) Species or Samples" and the relevant regulations of the transportation department on the transportation management of dangerous goods.

1.2 Sample processing

1.2.1 Blood sample processing

Place the anticoagulated blood sample in a centrifuge tube, centrifuge it at 8000r/min for 2 minutes, take out the upper plasma, and number it for testing.Place the non-anticoagulated blood sample in a centrifuge tube.After coagulation, centrifuge at 8000r/min for 2 minutes to take out 200 μL of upper serum, and number it for testing.

1.2.2 Tissue sample processing

Take an appropriate amount of spleen, liver, lymph nodes, tonsils, muscles and other tissues, grind them in a grinder or grinding tube, add an appropriate amount of physiological saline and mix well to make about 10% tissue homogenate, centrifuge at 8000r/min for 2 minutes, and take Dispense 200 μL of supernatant into an RNase/DNase-free sterile centrifuge tube and number it for later use.

1.2.3 Environmental sample processing

1.2.3.1 Methods for processing feces and feed samples

Take an appropriate amount of feces and feed and put it into a grinding tube containing PBS buffer, grind and mix to make a homogenate of about 10%.Centrifuge at 8000r/min for 2 minutes, take 200μL of the supernatant and centrifuge in RNase/DNase-free sterile In the tube, the number is reserved.

1.2.3.2 Sewage sample processing methods

Directly take 200 μL of sewage for nucleic acid extraction.

1.3 Extraction of viral nucleic acid Use magnetic beads or column method to extract DNA nucleic acid.

1.4 Fluorescent PCR reaction:

1.4.1 Take out the fluorescent PCR reaction solution and enzyme mixture from the kit.After melting at room temperature, centrifuge at 2000r/min for 5 seconds.Assuming that the sum of the tested sample, positive control and negative control is n, the reaction system is formulated as follows:

Reagent system

PCR reaction solution 17× (n+1) μL

Enzyme mixture 3× (n+1) μL

1.4.2 Add the above reagents to a new RNase/DNase-fire 1.5mL centrifuge tube, mix thoroughly and centrifuge immediately.Dispense the reagent into fluorescent PCR reaction tubes according to the aliquot volume of 20 μL/tube.

1.4.3 Add 5 μL of each negative control, 5 μL of positive control, and 5 μL of processed nucleic acid to be tested into the fluorescent PCR reaction tube prepared above, with a final volume of 25 μL/tube.Cover the fluorescent PCR reaction tube and mix immediately.Centrifuge and transfer to fluorescence PCR instrument.

144 Place the PCR reaction tube in the sample tank of the fluorescent PCR instrument, and run the following program on the fluorescent PCR instrument:

Step conditions cycle number UNG treatment 50℃, 2 minutes 1 Pre-denaturation 95℃, 3 minutes 1 Pre-amplification 95℃: 8 seconds 55℃: 8 seconds 5 PCR amplification 95℃: 8 seconds 55℃: 8 seconds 40

Select FAM as the fluorescence channel, and collect fluorescence signals at 55°C in each cycle of the PCR amplification stage.Set the amplification system to 25 μL, and select the mode of passive reference and quencher as none.(Note: If the fluorescence PCR instrument (such as AB17500) cannot operate due to a short temperature holding time after being set according to the reaction program in the instruction manual, the pre-amplification and PCR amplification conditions can be changed to 95C: 5 seconds and 55°C: 30 seconds)

2 Result Judgment

2.1 Determination of test validity

The positive control has a typical amplification curve and a Ct value ≤ 30.The negative control has no Cl value or amplification curve.The line shape is a straight line or a slight slope, and there is no exponential growth phase.The test results are deemed valid.Otherwise, the test will be deemed invalid.

2.2 Sample determination

2.2.1 Positive: The Ct value of the sample test result is ≤35 and there is an obvious exponential growth period, and the African swine fever virus nucleic acid is determined to be detected.

2.2.2 Suspicious: The Ct value of the sample test result is in the range of 35~38.At this time, the sample should be tested repeatedly.If the Ct value of the repeated test result is still in the range of 35~38 and there is an obvious exponential growth period, it will be judged as positive, otherwise it will be negative.

2.2.3 Negative: The Ct value of the sample test result is >38 or there is no Ct value, and it is determined that the African swine fever virus nucleic acid has not been detected.

[Notes] (1) Please read the instructions of this kit carefully before experimenting, strictly follow the operating steps, and accurately control the time, reagent volume, etc.during the operation to obtain the best results.

(2) The laboratory should be managed in strict accordance with relevant regulations and carry out genetic testing in the order of solution preparation area - template extraction area - amplification area - analysis area.There should be strict requirements for personnel, equipment, reagents and air flow in each section.

(3) The waste related to nucleic acid extraction must be clean and free of DNase/RNase.The extraction process should be as low-temperature and fast as possible.After completion, proceed to the next step of experiment or cryopreservation.

(4) For top lighting instruments, new disposable PE gloves should be worn to cover the fluorescent PCR tubes.For bottom lighting instruments,

Avoid touching the bottom of the fluorescent PCR tube with bare hands or used gloves.Use disposable latex gloves without fluorescent substances during the detection process.

(5) The cryopreserved reagents should be completely thawed at room temperature before use, and centrifuged momentarily to allow the liquid to completely sink to the bottom of the tube.Avoid repeated freezing and thawing to avoid affecting reagent performance.

(6) Samples, positive controls, etc.should be sealed promptly after use to avoid false positives caused by contamination between components and aerosols.

(7) It is forbidden to open the lid of the amplification product, and the waste generated by the experiment should be collected in time and treated away from the PCR laboratory for harmless treatment.

[Specification] 50 heads/box

[Storage and Validity Period] The kit is stored in the dark below -20°C and is valid for 12 months.

[Approval Number] Veterinary Drug No.163668871

For Veterinary Diagnostic Use Only