African Swine Fever Virus Detection Kit

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

African Swine Fever Virus Detection Kit is a fluorescent PCR assay for the detection of African swine fever virus (ASFV) antigens in swine whole blood.

【Product Name】General name: Viral DNA Extraction Kit

English name: Extraction Kit for Virus DNA

[Expected Use] This kit is mainly used for the extraction of viral DNA in the anticoagulant, blood purifier, swab, tissue, feed and environment for suspected pathogens such as viruses.The purified nucleic acid can be suitable for various routine operations and PCR tests.

【Kit Components】

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

1.Adsorption columns 20 50 2.Collection tubes 20 50 3.Lysis solution 5mL 11mL 4.Wash solution I13mL 32mL 5.Wash solution II13mL 32mL 6.Eluent 1.2mL 3mL

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

3.You need to prepare your own materials: anhydrous ethanol, 1.5mL sterile centrifuge tube, 1.5mL sterile centrifuge tube (long arm type), normal saline, mortar, sterile 1mL, 2OOuL, 10uL gun head, marker, etc.

【Operation Steps】

1.Sample preparation

1.Blood sample: Place the anticoagulant sample in a centrifuge tube, centrifuge at 8000 rpm for 2 minutes, and take the upper plasma; place the non-anticoagulant sample in a centrifuge tube, wait for coagulation, centrifuge at 8000 rpm for 2 minutes, take the upper serum, place it in a sterilized centrifuge tube, and number to be tested.

2.Tissue sample: 0.1g of the lesions such as spleen, liver, lymph nodes, tonsils, etc.Collect 0.1g of the tissue homogenizer or mortar to homogenize or grind it thoroughly, add 1mL of normal saline and mix well, centrifuge at 8000 rpm for 2 minutes, take the supernatant into the sterilized centrifuge bone, and number it to be tested.

3.Oral fluid: Before eating, use a swab/oral fluid collection bag or a homemade cotton thread-tied gauze ball to attract animals to chew.Collect oral fluid greater than 500uL, centrifuge at 8000 rpm for 2 minutes.Take the supernatant in a sterilized centrifuge tube and number to be tested.

4.Feed: Take an appropriate amount of ground feed (about 0.2g), put it in a 2mLEP tube containing 1mL of normal saline or PBS (PH is 7.4) buffer, shake and mix well, centrifuge at 8000 rpm for 2 minutes, take the supernatant in a sterilized centrifuge tube, and the number is waiting for inspection.

5.Dust: Dip a sterile cotton swab to appropriate amount of dust on the environmental surface, put it in a 2mLEP tube containing 1mL of normal saline or PBS (PH is 7.4) buffer, shake and mix well, centrifuge at 8000 rpm for 2 minutes, take the supernatant in the sterilized centrifuge tube, and number to be tested.

2.DNA extraction

1.Take 200uL of lysate and add 1.5mL of nuclease-free 1.5mL of nuclease-free, add 200uL of plasma/serum/tissue solution and other sample solution.Shake and mix for 10 seconds or reverse and mix for 10 times.Place at room temperature for 5 minutes (if the indoor temperature is low, place in a 25C water bath for incubation), then add 200uL of anhydrous ethanol and mix for 10 seconds; if the sample is turbid, centrifuge at 12,000 rpm for 1 minute and take the supernatant to avoid blocking 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 of wash solution I to the adsorption column, centrifuge for 1 minute at 12,000 rpm;

4.Discard the liquid in the collection tube, add 600uL of wash solution II to the adsorption column, and centrifuge at 12,000 rpm for 1 minute;

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

6.Transfer the adsorption column into a new nucleic acid-free 1.5mLEP tube (long arm type), add 50uL of eluent dropwise to the center of the column, let stand for 1 minute, centrifuge at 12,000 rpm for 1 minute, and the liquid in the EP tube is viral DNA.

[Precautions] 1.Before the experiment, Nuo carefully read the instructions of this kit and strictly follow the operating steps.During the operation, you can obtain the best results.

2.Samples should be collected and used freshly, or transported and stored at low temperatures;

3.The sample has potential infection risk, and the extraction of nucleic acid should be carried out in the biosafety cabinet of the nucleic acid extraction laboratory.

4.Consumables are not required to ensure high-temperature sterilization.After extraction, the nucleic acid will enter the next test or be frozen and stored for later use.

5.The reagents should be mixed evenly before use, and the reagents are corrosive.Please wear gloves and masks to protect them when using them.

6.It is normal for the lysate to have crystallization at low temperatures, and it can be used after heating and aiding the solution in a 60°C water bath.

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

For veterinary diagnosis only

African swine fever virus fluorescent PCR nucleic acid detection kit instructions [veterinary drug name]

General 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 ingredients and content】The kit contains the following components: kit component content, positive control 250μL/tube × 1 tube, negative control 250μL/tube × 1 tube, PCR reaction solution 850μL/tube × 1 tube, moist mixture solution 150μL/tube × 1 tube, 1 instruction manual/box

[Operation 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) 5mL of anticoagulant or serum collected aseptic samples from live pigs

(2) Anonymary samples of dead pigs or slaughterhouses, and samples of dead pigs such as card, lung, kidney, tonsils, lymph nodes, muscles, etc.were collected aseptic.Transport to the laboratory at low temperature at 2~8°C for testing.

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

1.1.2 Sample storage

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

1.1.3 Sample Transport

Add an ice pack to the foam box and seal it for transportation.Packaging and transportation should comply with the Ministry of Agriculture and Rural Affairs' "Packaging Specifications for the Transportation of Highly Pathogenic Animal Pathogen Microbial Bacteria or Samples" and the relevant regulations of the transportation department on the management of dangerous goods transportation.

1.2 Sample treatment

1.2.1 Blood sample treatment

The anticoagulant sample was placed in a centrifuge tube, and the upper plasma was taken for 2 minutes at 8000 r/min, and the number was waiting for examination.The non-anticoagulant sample is placed in a centrifuge tube.After coagulation, centrifuge at 8000r/min for 2 minutes to take 200 μL of the upper serum and number it to be tested.

1.2.2 Tissue sample treatment

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

1.2.3 Environmental sample processing

1.2.3.1 Methods for treating feces and feed samples

Take an appropriate amount of feces and feed into a grinding tube containing PBS buffer and grind it well, and make about 10% homogenate.Centrifuge at 8000r/min for 2 minutes.Take 200 μL of supernatant in RNase/DNase-free sterile centrifuge tube and set aside.

1.2.3.2 Sewage sample treatment method

Directly take 200 μL of sewage for nucleic acid extraction.

1.3 Extraction of viral nucleic acids is performed by magnetic bead method or column method for DNA nucleic acid extraction.

1.4 Fluorescence PCR reaction:

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

Reagent system

PCR reaction solution 17× (n+1) μL

Enzyme mixture solution 3× (n+1) μL

1.4.2 Add the above reagent to a new RNase/DNase-fire 1.5mL centrifuge tube, mix thoroughly and centrifuge instantly.Aliquot the reagent into the fluorescent PCR reaction tube at a 20 μL/tube aliquot.

1.4.3 Add 5 μL of negative control and positive control to the above-prepared fluorescent PCR reaction tube, 5 μL of treated nucleic acid to be tested, and the final volume is 25 μL/tube.Cover the fluorescent PCR reaction tube cover, mix well and instantly centrifuge, and transfer to the fluorescent PCR instrument.

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

Step condition cycle number UNG treatment 50℃, 2 minutes 1 Predenaturation 95℃, 3 minutes 1 Preamplification 95℃: 8 seconds 55℃: 8 seconds 5 PCR amplification 95℃: 8 seconds 55℃: 8 seconds 40

FAM was selected for fluorescence channel, and the fluorescence signal was collected at 55°C per cycle in the PCR amplification stage.Set the amplification system to 25μL, and at the same time, select the mode of passive reference and quencher as none.(Note: If the fluorescent PCR instrument (such as AB17500) is set according to the reaction procedure in the instruction manual and cannot operate due to short temperature holding time, the pre-amplification and PCR amplification conditions can be changed to 95C: 5 seconds 55°C: 30 seconds)

2 results judgment

2.1 Determination of test effectiveness

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

2.2 Sample determination

2.2.1 Positive: The sample test result Ct value is ≤35 and there is a significant exponential growth period, and it is determined that the nucleic acid of African swine fever virus is detected.

2.2.2 Suspicious: The Ct value of the sample detection result is within the range of 35 to 38.At this time, the sample should be tested repeatedly.If the Ct value of the repeated experiment results is still within the range of 35 to 38, there is a significant exponential growth period.It is determined to be positive, otherwise it is negative.

2.2.3 Negative: The sample test result Ct value is > 38 or there is no Ct value, and it is determined that no African swine fever virus nucleic acid was detected.

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

(2) The laboratory should strictly manage the zoning in accordance with relevant regulations, and conduct genetic testing in accordance with the order of the liquid dispensing area-template extraction area-amplification area-analysis area.The flow direction of personnel, equipment, reagents and air in each section should be strictly required.

(3) The relevant nucleic acid extraction ensures that it is clean and free of DNase/RNase.The extraction process should be as low as possible and fast as possible.After completion, it will enter the next step of experiment or freezing insurance.

(4) For the top lighting instrument, bring new disposable PE gloves to seal the fluorescent PCR tube, and for the bottom lighting instrument,

The device should avoid contact with the bottom of the fluorescent PCR tube with bare hands or used gloves.Disposable latex gloves without fluorescent substances should be used during the detection process.

(5) The frozen reagent should be completely melted at room temperature before use, and the liquid should be completely submerged at the bottom of the tube in an instant.Avoid repeated freeze-thawing to avoid affecting the performance of the reagent.

(6) The samples, positive controls, etc.are sealed in time after use to avoid false positives caused by contamination between components and aerosols.

(7) The amplified products are prohibited from being opened.The waste generated by the experiment should be collected in time and kept away from the PCR laboratory for harmless treatment.

【Specification】50 pieces/box

[Storage and validity period] The kit is stored at -20°C in the light and is valid for 12 months.

[Approval Number] Veterinary Medicine New Word 163668871

For veterinary diagnosis only