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The effectiveness of African swine fever detection equipment largely depends on the standardization of operation. Although there are differences in the operating procedures of different types of instruments, the core revolves around three key steps: sample processing, detection operation, and result interpretation. The details of each step directly affect the accuracy of the detection results, and beginners need to pay special attention when operating them.

Preparation work before use. Firstly, check the equipment status to ensure stable power connection and unobstructed heat dissipation holes. The fluorescence quantitative PCR instrument needs to be preheated for 30 minutes in advance to ensure the stability of the temperature control system; The colloidal gold rapid detection instrument needs to be placed at room temperature (20-25 ℃) in advance to avoid the temperature being too low and affecting the reaction effect. At the same time, it is necessary to prepare specialized sample collection tools, disposable gloves, etc., and take personal protective measures to prevent sample cross contamination. The preparation of reagents must strictly follow the instructions. Freeze the primers, probes, and other frozen reagents in an ice bath slowly, then gently invert and mix to avoid violent shaking that may damage the activity of the reagents.

Taking mainstream fluorescent quantitative PCR detection instruments as an example, the core operating steps can be divided into four steps. Sample collection and processing are the foundation of detection. Suitable sample types should be selected based on the testing purpose. Live pigs should be given priority for collecting anticoagulant samples, while dead pigs should choose tissues with high viral content such as spleens and lymph nodes. The collected samples are treated with specialized lysis buffer and nucleic acid is extracted through centrifugation, adsorption, elution and other steps. During the process, the centrifugation speed (usually 12000r/min) and time (3-5 minutes) need to be strictly controlled to ensure the purity of nucleic acid extraction and avoid interference from impurities such as proteins in subsequent testing.

The second step is to prepare the reaction system, which is the key to ensuring detection accuracy. In a sterile operating table, the extracted nucleic acid template, PCR reaction solution, and enzyme preparation are added to the reaction tube in proportion, and a new pipette tip is used at each step to prevent cross contamination. After the system configuration is completed, gently tap the bottom of the reaction tube to mix the liquid, then centrifuge for a few seconds to eliminate bubbles and avoid bubbles affecting the collection of fluorescence signals.

The third step is to perform machine testing and program setup. Place the reaction tube into the sample slot of the instrument, ensuring that the number corresponds correctly to the sample information. Set up the detection program according to the reagent manual, including pre denaturation (95 ℃, 30 seconds), denaturation (95 ℃, 5 seconds), annealing and extension (60 ℃, 30 seconds) stages, with a typical cycle of 40 times, while selecting the corresponding fluorescent channel (such as FAM channel). After completing the program settings, start the detection and avoid touching the instrument during the process to prevent sample slot displacement from causing detection failure.

The fourth step is result interpretation and recording. After the detection is completed, the instrument will automatically generate an amplification curve and CT value, and the judgment criteria must strictly follow the reagent requirements: CT value ≤ 37 and amplification curve showing a typical "S" shape is positive; CT value>37 or no amplification curve is negative; If there is an atypical curve, it needs to be retested and confirmed. After the testing is completed, the data should be exported in a timely manner, and records of sample information, testing time, instrument status, etc. should be kept for easy traceability in the future.

The operation of colloidal gold rapid detection instrument is more convenient and suitable for rapid screening at the grassroots level. After sample processing, use a pipette to draw an appropriate amount of sample liquid droplets and add them to the sampling hole of the test strip. The droplet amount should be controlled at 3-4 drops to avoid excessive liquid overflow. After being left to stand for 15-20 minutes, the observation result shows that both the quality control line and the detection line show positive color; Only the color of the quality control line is negative; If there is no color development of the quality control line, it indicates that the test is invalid and the test strip needs to be replaced for retesting. After use, the used test strips and sample containers should be disposed of according to medical waste regulations to avoid the spread of viruses.