African Swine Fever Virus Rapid Test Kit

African Swine Fever Virus Rapid Test Kit Instructions

African Swine Fever Virus Rapid Test Kit is used for the qualitative detection of African swine fever virus DNA extracted from spleen, whole blood and whole blood collected from pigs.

(Quick nucleic acid amplification kit)

【Product Name】

Common name: African Swine Fever Virus Rapid Test Kit (PCR Fluorescence Probing) English name: Diagnostic Kit for African Swine Fever Virus (PCR Fluorescence Probing)

[Expected use]

This kit is mainly used to detect African Swine Fever Virus nucleic acids in suspected infected pig anticoagulation and clinical data.It is suitable for the detection, auxiliary diagnosis and epidemiological investigation of African swine fever virus.

【Kit Components】

1.Kit composition and storage conditions

2.You need to bring your own materials: normal saline, sterile gun tip, fluorescent PCR special reaction tube, marker, etc.

【Operation Steps】1.Sample Preparation

1.Blood sample: collect anticoagulant (anti-coagulant is EDTA) or serum sample and number it for later use.

2.Tissue sample: collect 0.1g of lesions such as spleen, liver, lymph nodes, tonsils or soft ticks (sized by soybeans), cut and crush the ophthalmic scalds and cut them into a tissue homogenizer or mortar and mix them thoroughly.Add 1 mL of normal saline and mix them well.Centrifuge them at 8000rpm/min for 2 minutes at 4°C.Take the supernatant in a sterilized centrifuge tube and number them for later use.

2.DNA extraction

Take 20μL of reaction solution A and add to a new 1.5mL centrifuge tube, add anticoagulation, or 2μL of serum or tissue supernatant and mix well.Let stand at room temperature (about 20°C) for 3 minutes, add 20μL of reaction solution B, and mix well.If it is anticoagulated whole blood, it takes 8000rpm/min and centrifuge for 2 minutes before serving as the DNA solution to be tested.

2.PCR amplification

1.Dispose of liquid: Take out the PCR reaction solution and enzyme mixture, mix thoroughly after the room temperature is completely melted, and then mix it thoroughly.You can take out all the enzyme mixture and add it directly to the PCR reaction solution, mix it thoroughly, and then use it according to the instantaneous separation.Or take a certain amount of test dose according to the ratio of 17 μL of each PCR reaction solution and 3 μL of enzyme mixture.Mix the two reaction solutions.After instantaneous separation, fill it into a special fluorescent PCR reaction tube, and freeze the remaining reagent immediately; (Precautions should be avoided from light during operation)

2.Add sample to amplification: Add 5 μL of sample DNA or negative control or positive control to the above PCR reaction tube, mix well and disconnect instantly, and put it on a fluorescent PCR instrument to run the following procedures:

Step Conditions Cycle Number UNG Processing 50°C: 2 minutes 1 Predenaturation 95°C: 3 minutes 1 Preamplification 95°C: 8 seconds 55°C: 8 seconds 5 PCR Amplification 95°C: 8 seconds 55°C: 8 seconds 40

FAM was selected for the fluorescence channel, and the fluorescence signal was collected at 55°C per cycle phase.Set the amplification system to 25μL, and select the mode of passive reference and quencher as none.

(Note: If the fluorescent PCR instrument (such as ABI7500) is set according to the reaction procedure in the manual and cannot run due to short temperature holding time, the pre-amplification and PCR amplification conditions can be changed to 95°C: 5 seconds, 55°C: 30 seconds.)

[Result Determination] 1.Baseline and threshold setting; baseline adjustment takes 6-15 cycles of fluorescence signals, and the threshold setting is based on the principle that the threshold line just exceeds the highest point of the negative control detection fluorescence curve.

2.Quality control: After the reaction is over, the test results of the negative control should have no specific amplification curve, with a Ct value of >38 or no; the Ct value of the positive control should be ≤30.0, and a significant amplification curve; otherwise the experiment will be deemed invalid.

3.Sample determination: The fluorescence signal of the sample to be tested has an exponential increase, and the result shows that the Ct value is <35.0, and it is reported as positive; no Ct value or Ct value >38 or no significant amplification curve is detected, the sample is determined to be negative.When 35≤Ct value is ≤8, re-test will be performed once.If the repetition result is positive, it will be determined as positive, otherwise it will be determined as negative.

[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 be managed strictly in accordance with relevant regulations.The flow of personnel, equipment, reagents and air in each range should have product requirements.

3.The relevant consumables must be clean and sterile.After the nucleic acid extraction is completed, the next step of test or cryopreservation will be carried out as soon as possible.

4.The premixed reagents should be used in a short period of time.Outdoor use should be stored in a foam box with an ice pack, and should be frozen and stored in a timely manner after daily tests.

5.For fluorescent PCR tubes, avoid contact with bare hands or used gloves.Disposable latex gloves without fluorescent substances should be used during the detection process.

6.Before use, the frozen reagent should be completely melted at room temperature, mixed thoroughly, and instantly centrifuged to completely sink the liquid to the bottom of the tube.

7.After the sample and negative control are sealed, add a positive control in a ventilated environment and seal it in time to avoid false positive contamination such as components and aerosols.

8.It is strictly forbidden to open the cover of the PCR tube after the amplification reaction.It should be collected in time with other waste generated in the test and be harmlessly treated away from the PCR laboratory.

【Specification】50 pieces/box.

【Storage and validity period】Storage below -20°C away from light, valid for 12 months.

For veterinary diagnosis only