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Recommended overall plan for African swine fever virus nucleic acid detection practice (1)
◆ Origin of African Swine Fever ASF:
African swine fever (African suwinefever, ASF) is a severe infectious disease caused by African swine fever virus infecting domestic and wild boars. The epidemic is on the rise rapidly. Since it was first introduced to my country in August 2018, it has rapidly swept the pig industry, causing unpredictable economic losses. African fever virus is a double-stranded DNA virus with an enveloped structure that causes high fever hemorrhage and death after infecting domestic pigs and wild boars. The PCR method is currently designated for the detection of African swine fever virus, which has the characteristics of high sensitivity, high specificity and rapidity.
Here, through the actual case, I will share with you the efficient and reliable African swine fever virus detection solution combined by the cooperative company.
◆ Pre-processing of African swine fever-infected tissue samples:
The susceptible objects of African swine fever are domestic pigs and wild boars, and the samples usually collected are plasma and tissue. For tissue samples, it needs to be fully ground and crushed before nucleic acid extraction to ensure sufficient nucleic acid release. After nucleic acid extraction, it can be used for subsequent detection. If the nucleic acid is not sufficiently ground, false negative results of nucleic acid detection may be caused. Using the DHFSTPRP series automatic sample rapid grinding machine, the power is upgraded and the grinding method is improved. The samples are cracked in a fully enclosed state to avoid cross-contamination between samples and external contamination, and ensure the safety of samples. On the premise of the premise, the sample can be ground more fully and thoroughly, and the grinding effect is stable; there are various specifications of adapters, grinding beads and grinding jars, which can quickly process 24-64 tissue samples at the same time, and the liquid crystal display can store 10 data. There is also a DHFSTPRP-II series liquid nitrogen freeze grinding machine, which is truly fully automatic freeze grinding, with a low temperature of -196°C, which can easily handle heat-sensitive samples and difficult-to-grind samples.
◆ Nucleic acid extraction from African swine fever infected tissue samples:
TGuide S32 Automatic Nucleic Acid Extractor adopts the latest magnetic bar adsorption transfer nucleic acid purification method, using 96 deep-well reaction plates, with different types of magnetic bead methods Nucleic acid extraction reagent, operate 1-32 samples at the same time, and automatically separate, extract and purify nucleic acid from various samples such as blood, cells, tissues, viruses, etc.
◆ High sensitivity and high specificity real-time PCR detection:
End-point PCR and real-time quantitative PCR can both detect the amount of PCR product and infer the content of the initial template. Due to the limitation of the sensitivity of the gel imaging method itself, the addition of real-time fluorescent quantitative PCR fluorescent dyes enables the instrument to monitor the entire PCR reaction in real time with higher accuracy.
Ningbo Zhongdao African Swine Fever Virus Fluorescence PCR Detection Kit (Box B) is a fluorescent quantitative detection kit based on fluorescent probes, which is mainly used to detect suspected infected pigs with anticoagulation and clinical diseases African Swine Fever Virus (ASFV, African Swine Fever Virus) nucleic acid in feed is suitable for detection, diagnosis and epidemiological investigation of African swine fever virus.
Kits are stored at -20° for a limited period of 12 months. Negative and positive controls are provided in the box for quality control of the entire experiment. In the evaluation results of the first batch of African swine fever on-site rapid detection reagents, Zhengzhou Zhongdao African swine fever fluorescent quantitative PCR rapid detection kit performed excellently in both diagnostic sensitivity and diagnostic specificity.
◆ Fluorescence quantitative detection instrument: Roche universal fluorescence quantitative PCR instrument:
Roche Light Cycler 96 fluorescence quantitative PCR instrument is a full wavelength, high light intensity PCR instrument, cost-effective, long service life, All samples are detected at the same time with high repeatability, and the industry's unique optical fiber guide is used to avoid the loss of optical signal; using the well-received silver temperature control module on the LightCycler 480, the thermal conductivity is fast and uniform, and the temperature control difference between wells is ±0.2°, heating rate4.4°/s, cooling rate2.2°/s,96hole detection≤40min (35 Cyclers), more than many machines type can save half the time. Light Cycler 96 can sensitively detect single-copy genes, copy number difference ≤ 1.5 times, confidence ≥99.8%, and no need< Passive dye correction such as span style="text-align: justify;">ROX is convenient, fast and stable.
◆ Small system loading operation tool: eppendorf pipette:
Tips:
Real-time fluorescence quantitative PCR is usually a small system experiment. For small samples, especially the 1-10ul sample loading operation, there will be danger of pipetting and blowing. Accurate pipetting is an important element for accurate results. Therefore, It is recommended to use Eppendorf Reference2 series pipettes with original imported tips for pipetting operations and contact Quantum team engineers to give special lectures on pipetting operations to ensure the accuracy of the results.
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