1.  Common sample types

Secretions, blood, spleen, tonsils, kidneys and lymph nodes samples

2.1 Methods of Pretreatment of samples

2.1.1 Samples pretreatment

Tissue Samples: about 1g tissue samples are taken from three different parts of each sample, and then cut and mixed by Surgical scissors, take 0.5g and grinded in a grinder, continue to grind after adding 1.5ml saline water. transferred to a 1.5mL sterilized centrifugal tube after homogenizing. 8000rpm for 2 min, transfer 200 uL supernatant to a 1.5mL sterilized centrifugal tube. The samples are extracted directly on machine ( The Committee of Jiangsu Taizhou Agricultural)

2.1.2 Whole blood sample: Transfer the supernatant (serum) of whole blood after centrifugation to a 1.5mL sterilized centrifugal tube.

Related lab equipments: animal tissue grinder, Handheld Electric Tissue Grinder.

Centrifuge the samples to different levels and take the supernatant after break the samples by highthrouput tissue breaker or handheld tissue grinder.

2.2 The storage and transportation of the samples

The samples could be stored at -20℃ for short term, at -70℃ for long term, but no more than 12 months, the samples should be transported with ice bags and dry ices, avoid repeatedly freeze and thaw.

2.3 DNA Extration

Transfer the pretreated samples to one 96 deep-well plate prepacked with magnetic beeds reagents, and put the plate on the automated nucleic acid purification system for DNA extraction.

Automated nucleic acid purification sytem-96 is used for virus DNA/RNA extraction, related reagent for extraction can be provide in unpacked or prepacked boxes, 96 high-throughput pipette system can be used for repack of the unpacked reagents.
3. Preparation of reagents(Reagent preparation part)

Set the number of PCR tube as N(N=sample amount+1 negtive control+1 positive control; prepare one more for each 7 samples), premix the forward primer, reverse primer, qPCR probe, enzymes, sub-pack the mixed reagents to PCR tubes, for expample

Reagents	Amounts
Mix of ASFV qPCR primer and probes	1.6 uL
ASFV qPCR Premix	10 uL
Extracted DNA	2 uL
RNase free H2O	6.4 uL
Volume	20 uL

Sub-pack the mix using 96 high-throughput pipette system or pipettors.

4. Sampling(sample treatment part)
Take certain amount of extracted DNA, positibe control, negative control, and sub-pack it to certain tubes, mix and short centrifuge.

5. PCR reaction(PCR part)

5.1 Place the prepared tubes in the real-time PCR reaction wells;

5.2 Set the channels, sample information, reaction system to 20μL；

Select the fluorescence channel: Detect channel to（Reporter Dye）FAM, （Quencher Dye）to NONE, select None for ABI instruments, but not ROX reference fluorescence.

5.3 Related  equipments

BTK-6 Real-time PCR System, BTK-10 ultrafast real-time PCR system, ABI7500、LightCycler, Bio-Rad, eppendorf Real-time PCR systems.

6. Results

Read the results according to the guide from ASFV nucleic acid testing kit.

Relavent equipment and reagents

For more information, please contact: service@bioteke.com.