Specification for identification and detection of African swine fever virus I177L gene deletion strains

This specification applies to the fluorescence PCR detection of African swine fever virus I177L gene deletion strains.

1 main reagent

1.1 Viral DNA extraction kit.

1.2 Nuclease-free water.

1.3 Probe method fluorescent PCR master mix.

1.4 Primers and probes: P72 and I177L gene amplification primers and probes were designed and provided by the National African Swine Fever Reference Laboratory.

1.5 Positive and negative controls: For each fluorescent PCR test, positive and negative controls should be added. The positive control was ASFV genomic DNA standard material. The negative control replaces the sample DNA with water.

2 Main instruments and equipment

2.1 Fluorescence PCR instrument.

2.2 Desktop low-temperature high-speed centrifuge.

2.3 Tissue homogenizer.

2.4 Automatic nucleic acid extraction instrument.

2.5 Micro-adjustable pipettes.

2.6 Nuclease-free centrifuge tubes, PCR tubes and tips.

3 Sample collection and transportation preservation

During biopsy, pig anticoagulated whole blood (EDTA anticoagulation), oral and nasal swabs or serum can be collected; dead pigs can be collected from spleen, lymph nodes, tonsils, liver and other tissue materials. Send it to the laboratory as soon as possible for viral nucleic acid testing.

The collection, transportation and storage of samples should meet the relevant requirements of NY/T 541 "Technical Specifications for Collection, Storage and Transportation of Veterinary Diagnostic Samples". Samples can be stored at 4°C for up to 2 days, and should be stored frozen below -20°C for long-term storage.

4 Sample processing

4.1 Tissue samples

Take 0.1g-0.2g tissue samples and cut them into small pieces, add 1mL-2mL of pre-cooled PBS buffer (pH7.4) and homogenize them at high speed with a tissue homogenizer to make 10% tissue homogenate, and centrifuge at 5000r/min After 10 min, 200 μL of the supernatant was taken for nucleic acid extraction.

4.2 Liquid samples

For liquid samples such as anticoagulated whole blood and serum, directly take 200 μL to extract nucleic acid.

5 Virus nucleic acid extraction

Use viral DNA extraction kit or automated nucleic acid extraction instrument to extract viral nucleic acid. The extracted nucleic acid solution should be used immediately or stored frozen below -20°C (use within 6 months).

6 Fluorescent PCR detection

6.1 Preparation of fluorescent PCR reaction solution

Prepare fluorescent PCR reaction mix in nuclease-free centrifuge tubes, consisting of

Including: P72 gene and I177L gene probe master mix, 2× probe method fluorescent PCR master mix and nuclease-free water. The preparation of the reaction system is shown in the table below.

Table 1 Configuration of each reaction system

6.2 Fluorescent PCR amplification conditions

50°C for 2 minutes; 95°C for 5 minutes; 95°C for 15 seconds, 60°C for 1 minute, 40 cycles, FAM and VIC fluorescence signals were collected at 60°C in each cycle.

6.3 Analysis of results

The Ct value was automatically determined by the fluorescence PCR instrument software.

6.3.1 Conditions for the establishment of the test

The Ct values of P72 and I177L genes in the positive control should both be <35 and specific amplification curves should appear, while the negative control P72 and I177L genes should have no Ct values or Ct values ≥ 40 and no specific amplification curves. When the above conditions are met at the same time, the test result is valid, otherwise the test should be repeated.

6.3.2 Determination of test results

The FAM channel corresponds to the P72 gene, and the VIC channel corresponds to the I177L gene. When the Ct value of the tested sample is ≤38 and the specific amplification curve appears, the target gene is judged to be positive; when there is no Ct value or the Ct value is ≥40, the target gene is judged to be negative; 38

6.3.3 Comprehensive judgment

The final judgment should be combined with the detection results of P72 and I177L for a comprehensive analysis. The specific judgment criteria are shown in Table 2.

Table 2 Comprehensive Judgment Criteria

Note: "+" represents positive; "-" represents negative.