company news about [Testing] The primary factor affecting ASF test results: correct sample collection and storage

Testing is the most crucial component of African swine fever prevention and control. However, the scientific and standardized nature of sampling not only impacts test results but can also impact the safety of pig farms.

In 2020, China issued the national standard "African swine fever diagnostic technology," GB/T 18648-2020. This standard was promulgated and implemented by the State Administration for Market Regulation and the Standardization Administration of China on December 14, 2020.

This national standard covers biosafety measures, clinical diagnosis, laboratory diagnostic sample collection and processing, methods such as fluorescence quantitative PCR, and comprehensive assessment.

A complete ASF pathogen fluorescence quantitative PCR detection process generally includes: sample collection and storage; sample pretreatment; nucleic acid extraction and solution preparation; real-time fluorescence quantitative PCR amplification reaction; and test result output and analysis.

From a laboratory perspective, proper sample collection and transportation are paramount to ensuring accurate laboratory test results.

What samples should be collected? What is the appropriate quantity? How should they be collected in a standardized manner? What should be done after collection? If samples are not tested promptly, how should they be stored? What should be done with the tested samples?

By interpreting the national standard for ASF diagnostic technology and the "Technical Specifications for African Swine Fever Quarantine" (SN/T 1559-2010) issued in 2010, we present the key points to consider regarding clinical sample collection, storage, and transportation:

#01 Anticoagulated Blood (Whole Blood):

Collection: Draw 5 ml of whole blood from the jugular vein, inferior vena cava, or marginal auricular vein using a sterile tube (vacutainer) containing an anticoagulant (EDTA-purple cap). If the animal is deceased, blood can be collected from the heart, but this must be done immediately. Transport blood refrigerated and, upon arrival at the testing laboratory, store it at -70°C.

Note: Do not use sterile blood collection tubes with heparin (green cap), as it will inhibit the PCR reaction and easily cause false negatives.

#02 Serum:

Collection: Collect a 5ml blood sample using an unanticoagulant vacutainer from the jugular vein, inferior vena cava, or marginal auricular vein, or during necropsy. It is recommended to first incubate the sample at 4°C for 14-18 hours to separate the clot. Discard the clot and centrifuge for 10-15 minutes. Collect the clear supernatant (serum). Serum samples can be tested immediately after separation or shipped refrigerated. Upon arrival at the laboratory, they can be stored frozen at -70°C.

Note: If the serum is red, it indicates hemolysis and cannot be used for ELISA testing.

#03 Organ and Tissue Samples

Collection: Target organs include the spleen, lymph nodes, tonsils, and kidneys. The spleen and lymph nodes typically have the highest viral loads. Transport samples refrigerated and submit them to the laboratory as soon as possible. If timely delivery is not possible, samples can be stored in a freezer or in liquid nitrogen. Viruses can be inactivated by soaking in 10% buffered formalin for at least 30 minutes before PCR testing.

#04 Soft Ticks

Collection: Collect soft ticks, keeping them alive as much as possible to ensure the preservation of viruses within them and to prevent DNA degradation. Collected soft ticks can be placed in a screw-capped vial/tube with a small amount of soil, lined with gauze, and stored at room temperature for transport. Upon arrival at the testing laboratory, freeze at -70°C or place in liquid nitrogen. If only morphological observation is being conducted, samples can be stored in 100% alcohol.

Note: The same procedure applies to other insects, such as mosquitoes and flies. Keep them alive as much as possible for testing, or store directly in liquid nitrogen.

#05 Environmental Samples

Collection: Collect multiple environmental swabs, such as feces, swill, and feed, and store at 4°C.

#06 Saliva and Swab Samples

Collection: Secure a cotton rope or gauze to the railing. After the pig chews naturally, collect the liquid extracted from the rope or gauze. Store the sample in a clean test tube or ziplock bag. Seal and store in a refrigerated container for transportation.

For nasal swabs, use a sterile cotton swab. Insert it 3-5 cm into the pig's nasal cavity at a 45° angle. Gently rotate the swab 3-5 times to stimulate the pig's sneezing reflex. Wait until the swab is moistened. Store the cotton swab sample in a separate clean test tube or sealed bag. Seal and store in a refrigerated container for transportation.

For anal swabs, use a sterile cotton swab soaked in saline solution. Insert the sample 2-3 cm into the pig's anus. Gently rotate the swab and rub it. Then, store the sample in a clean test tube or sealed bag. Store in a refrigerated container for transportation.

High-quality samples are the key basis for ensuring the accuracy of laboratory test results. Through standardized sample collection, preservation and pretreatment quality control, the integrity of biological molecules can be effectively maintained, providing sufficient experimental support for subsequent accurate nucleic acid detection and reliable diagnosis.

Sample Storage and Transportation

To ensure the accuracy of test results, we recommend that samples be sent to a designated laboratory for testing as soon as possible after collection. Use a next-day delivery method and utilize cold chain transportation to prevent sample degradation and loss of efficacy.