Genfine, 2022, June, 24th, Beijing
African swine fever (ASF) is a serious zoonotic disease caused by the African swine fever virus (ASFV). The morbidity and mortality of ASF virulent-infected pigs are as high as 100%. Worldwide, there is currently no vaccine that can effectively prevent African swine fever. African swine fever is preventable, controllable, and incurable. Therefore, monitoring and actively detecting viruses is the key to preventing and controlling ASF.
African swine fever virus belongs to a special kind of DNA virus-nucleocytoplasmic large DNA virus (NCLDV). The diameter of the entire virus reaches 260-300nm, which is equivalent to 730 hepatitis A viruses, 140 ZIKA viruses, and 10 HSV herpes viruses.
Figure 1 “Overall structure of African swine fever virus (left: 5-layer section view; right: capsid layer structure)” (Wang et al., 2019)
African swine fever occurs primarily through direct oral or nasal contact, or as a result of exposure to contaminated agents (tissue, blood, feces and secretions of infected pigs). Viruses enter host cells through a complex process involving dynein- and clathrin-mediated endocytosis and macropinocytosis. After the virus infects the body, the virus infection pathway starts from the mononuclear macrophages in the tonsils and mandibular lymph nodes of pigs. As lymph and blood enter the circulatory system, it invades the endothelial cells of capillaries, arteries, veins and lymph nodes, resulting in Pathological changes such as hemorrhage, serous exudation, thrombus, and infarction appear in the corresponding tissues and organs, resulting in systemic hemorrhagic lesions.
Figure 2 “Diagram of ASFV entering host cells through endocytosis (Galindo and Alonso, 2017)”
ASFC Sample Extraction and Purification
Due to the characteristics of ASFV's strong transmission ability, high morbidity rate, strong lethality rate, strong virus replication ability and difficulty in eradication, etc., it is recommended that all qualified pig farms (with their own laboratories) maintain certain ASFV virus detection. ability. Although regular testing will cost a lot of manpower, material and financial resources, if the virus is detected, it is usually at an early stage to avoid greater losses. There are many methods for virus detection based on different principles. The magnetic bead method is mainly automated extraction., this method has simple operation, high sensitivity and wide application in ASFV nucleic acid extraction with downstream fluorescence quantitative PCR for detection. Because it is easy to operate, has high throughput, and can save manpower. It is favoured by researchers. There are three determinants of successful nucleic acid extraction, namely pretreatment, reagents, and operating procedures.
1. Sample Type and Pre-processing
01 Blood Swab and Whole Blood
In the stage of virus detoxification, the virus content in the blood is relatively high, the venous blood collection is stressful and there is a risk of the virus spreading due to dripping, so the tail tip swab blood collection is easy to operate, less stressful, and less risky of contamination. As a nucleic acid extraction sample, blood swabs are similar to diluted whole blood samples. Due to the difficulty of sampling and the problem of virus lag in blood, blood samples are generally not suitable for early monitoring. The blood sample can be directly extracted. If the sample is clotted, it can be left standing or centrifuged for a short time, and the supernatant can be extracted for nucleic acid extraction.
02 Mouth and nose swab
During the detoxification process of the ASF wild virus, the virus can be detected in the oral and nasal fluids a little earlier than that in the blood. Pig farms often use nose and mouth swabs for virus testing.
Oral and nasal swabs may contain porcine-derived cells as nucleic acid extraction samples, so the downstream virus amplification kit can use endogenous internal reference ASF amplification kits, which can monitor the viral nucleic acid extraction steps. In addition, the sample will also be mixed with feed, soil, feces, etc. near the snout of the pig. If there are more such inhibitors, it may influence nucleic acid extraction and amplification differently. Oral and nasal swabs can be directly extracted. If there are too many impurities, they can be left standing or centrifuged for a short time, and the supernatant can be extracted for nucleic acid extraction.
03 Throat swab
The mutated ASF strain will have a lower detection rate in the oral and nasal fluid. At this time, the tonsil virus content in the pharynx is high. Similar to the collection of coronavirus samples in humans, sampling from the pharynx is conducive to the detection of the virus.
Pharyngeal swabs as nucleic acid extraction samples are similar to oral and nasal swabs.
04 Oral Fluid
Pig oral fluid is also pig saliva, which is generally collected by rope bites or saliva collection bags.
Saliva samples not only contain a variety of enzymes, but are also mixed with soil, feces, vomit, and other ingredients containing more inhibitors, so the extraction of viral nucleic acid from pig saliva samples requires high nucleic acid extraction efficiency and purity. The saliva can be directly extracted. If it contains too many impurities, it can be left standing or centrifuged for a short time, and the supernatant can be taken as a sample for nucleic acid extraction.
05 Environmental Samples
ASFV is more resistant to temperature, pH, and spoilage. In addition to collecting samples from pigs, environmental sampling also needs to be carried out from pig farms and places with a large flow of people. Testing environmental samples can effectively monitor the virus infection in pig farms. Generally, saline gauze or saline swabs are used to test the pig house, doorway, living area, buffer zone, vehicle, personnel and other locations for sampling and testing. Environmental samples are often used for daily virus testing and are also necessary for pathogen testing for pig farms to resume production.
The types of environmental samples are complex and diverse. Dirty environmental gauze samples will contain more inhibitors than swab samples. The collected samples will contain a series of humic acid-containing components such as feces, soil, feed, and dust, so they are dirtier. The requirements for nucleic acid extraction reagents for environmental samples are also relatively high. Environmental samples can be extracted directly. If there are too many impurities, they can be left standing or centrifuged for a short time, and the supernatant can be extracted for nucleic acid extraction.
06 Tissue Samples
Studies have shown that after pigs are infected with ASFV, the organs in the body are infected in the order of spleen, tonsils, lymph nodes, etc. Over time, the effects detected in different tissues are no longer different.
Tissue samples are used as nucleic acid extraction samples. Generally, 1-2 soybean-sized tissues need to be cut off with scissors, and the tissue is ground with about 2 ml of normal saline. After grinding, the supernatant is centrifuged for viral nucleic acid extraction and detection.
07 Other Samples
Other samples such as stool, semen, and castration fluid. Due to the low viral content of fecal samples after gastrointestinal digestion, it is difficult to collect samples such as semen, so such samples are rarely used.
It is also difficult to extract nucleic acid from such samples. The content of inhibitors such as humic acid in feces is very high, and the nucleic acid after extraction is easy to inhibit downstream amplification. It is difficult for ordinary lysate to release nucleic acid from lysed semen cells, and DTT is generally used as the Sample preprocessing steps.
The method of extracting nucleic acid by magnetic bead method is based on superparamagnetic nano-magnetic particles, which can adsorb nucleic acid through hydrogen bonding and electrostatic specificity under the condition of high concentration of the chaotropic agent, while protein or another non-specific adsorption. A small number of impurities are removed by washing, and finally, the nucleic acid is eluted with a low-salt buffer or RNase Free ddH2O. The purified nucleic acid can be suitable for various routine operations, including fluorescence quantitative PCR, and various downstream molecular experiments related to NGS.
This method has the following advantages:
• Safe and non-toxic, no toxic reagents such as phenol/chloroform are used in traditional methods;
• Simple operation, pre-packaged reagents are provided, no need to contact experimental reagents for a long time, and the integration process is automated;
• High-throughput extraction, batch processing 8/16/32/48/64/96 samples at the same time;
• Meet the requirements for nucleic acid extraction from microbiological samples.
Figure 3 “Illustration of nucleic acid extraction by magnetic bar method (picture from the Internet)”
Generally, magnetic beads in nucleic acid extraction by the magnetic bead method are magnetic beads coated with silanol groups or carboxyl groups. Its surface-coated groups can specifically bind to nucleic acids under the action of salt bridges. Different production processes of magnetic beads will result in different effects such as the size of magnetic beads, shape of magnetic beads, magnetic response, and amount of group coating. In addition, the same magnetic beads will have different results under different reagent systems. Given the extremely complex and diverse types of pig farm samples, we recommend testing different magnetic beads under the same sample and reagent system to screen out magnetic beads with better performance. The data in the table below illustrate that different magnetic beads have different effects on sample compatibility. It can be clearly seen from the data in Table 1 that magnetic beads H have better extraction efficiency and sample compatibility than other magnetic beads.
Table 1 “Comparison of the extraction effects of different magnetic beads for ASF quality control products with different background dilutions”
01 Lysis Steps and Binding Steps
Lysis steps and binding steps in the magnetic bead method nucleic acid extraction process can be separated or combined, the main difference is which step the magnetic beads start to participate in nucleic acid extraction. Generally, two steps of cleavage and binding are selected to be performed together, and the effect of magnetic beads and nucleic acid binding steps can be maximized.
In the lysing step, the lysing solution generally contains high concentrations of chaotropic salts and surfactants, which not only provide a high-salt environment for the binding of magnetic beads to nucleic acids but also lyse and release nucleic acids from cells.
The need to add proteinase K in the cleavage step is generally related to the type of sample. Since blood samples contain a lot of protein and cellular components, the auxiliary addition of proteinase K will promote the release of nucleic acids from nucleic acid-protein complexes. For environmental samples with a clean background, since the lysis buffer is sufficient for the release of nucleic acids, the addition of proteinase K has little effect on the results.
Secondly, cracking time and cracking temperature also have a great influence on insufficient cracking. The length of the lysis time at the same lysis temperature will affect the lysis effect. If the time is too short, the lysis will not fully affect the release of nucleic acids, and if the time is too long, it may cause nucleic acid fragmentation; the lysis temperature at the same lysis time will also affect the lysis effect. If the temperature is too low It may cause insufficient lysis to affect the release of nucleic acid, and if the temperature is too high, it may also cause the risk of nucleic acid degradation, but in fact, the lysis temperature may be 50-90°C. According to the differences in the performance of nucleic acid extractors and pre-packing consumables of different manufacturers, the program setting temperature may be 10-20°C different from the actual operating temperature in pre-packing. The lysis temperature and duration need to be selected according to the lysis capacity of the lysis solution.
02 Rinse Step
The rinsing step is also an important step for complex types of pig farm samples. The rinsing ability of the rinsing solution needs to be compatible with samples with clean backgrounds and samples with complex backgrounds. The effect of sample decontamination. Likewise, the number of rinses and the length of rinse time need to be compatible for simple and complex samples.
03 Elution step
Nucleic acid extraction reagent eluent is generally low salt buffer or RNase Free ddH2O. The factors affecting nucleic acid extraction in the elution step are elution time and temperature. Similar to the lysis step, the elution time is too long or too short, and the elution temperature is too high or too low, which may affect the elution of nucleic acids from the magnetic beads. efficiency and nucleic acid degradation. The selection elution time and temperature need to be selected according to the characteristics of the extracted virus and the results of downstream amplification.
In addition to nucleic acid extraction, the output of real results also requires sensitive and efficient fluorescent quantitative detection reagents after extraction. As mentioned above, the environmental gauze samples and saliva samples of pig farms contain a lot of soil, feed, salivary mucin, feces and other components. If the nucleic acid extracted by the extraction reagent is not pure enough for such samples, it contains relatively Inhibitors such as polyhumic acid, or downstream amplification kits with poor resistance to stress, which can easily inhibit amplification. Therefore, the selection of amplification kits with good stress resistance and high amplification sensitivity for downstream amplification is also crucial for the accuracy of the results.
Genfine Biotech is committed to becoming a leading scientific and technological innovation company with overall solutions for the preservation, extraction and detection of biological samples in China. After long-term exploration and innovation, Genfine now has a mature nucleic acid purification system, which is well recognized by customers for its excellent operation convenience, sample compatibility and high detection rate. So far, more than 15 million reagents have been provided. Virus extraction reagents. A large number of data show that the Genfine magnetic bead method animal virus DNA/RNA extraction kit has excellent extraction performance.
1. Good sample compatibility: general extraction of various DNA and RNA virus samples, including saliva, swabs, gauze, whole blood, serum, tissue homogenate and other samples or samples in various virus preservation solutions; 2. Operation Simple and fast: with the automatic extraction instrument, only need to add the sample, and the extraction can be completed in 15 minutes; 3. High detection rate: The virus extraction efficiency is high, especially for low-copy virus samples, it has better detection performance.
▼Good Sample Compatibility
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