LAMP primer group for detecting porcine circovirus type 3, kit and application thereof
The technology of a porcine circovirus and a kit, applied in the biological field, can solve problems such as long reaction time, low sensitivity, and failure to meet basic needs, and achieve the effects of rapid response, high sensitivity, and easy wide-scale application
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Embodiment 1
[0046] Design and synthesis of embodiment 1LAMP primer
[0047] According to the principle of primer design, aiming at the conserved region sequence of porcine circovirus type 3 ORF2 gene, the LAMP primer online design software PrimerExplorer V5 was used to design the internal and external primers. According to our experience, the GC content of the primers was guaranteed to be between 40% and 60%. %between. After empirically screening the primer set with the optimal theoretical value, continue to use the software PrimerExplorer V5 to design loop primers. The sequences are shown in Table 1. The primers were synthesized by Shanghai Sangon Bioengineering Technology Service Co., Ltd. The synthesized primers were diluted with sterile triple distilled water to a concentration of 10 μM and stored at -20°C.
[0048] Table 1 LAMP detection primer set for porcine circovirus type 3
[0049]
[0050]
Embodiment 2
[0051] Application of embodiment 2 LAMP primers in detecting the virus to be tested
[0052] 1. Preparation of Nucleic Acid Standards
[0053] After measuring the nucleic acid concentration of the porcine circovirus type 3 ORF2 gene synthesized by Shanghai Sangon Bioengineering Technology Service Co., Ltd., dilute it with ultrapure water to 10 5 The concentration of copies / μL was used as a nucleic acid standard.
[0054] 2. Establishment of LAMP detection method
[0055] With a concentration of 10 5 The copy / μL porcine circovirus type 3 ORF2 gene was used as a template, and a set of primers (as shown in Table 1) with the optimal theoretical value obtained by screening was used for cross-primer amplification, and the obtained product was subjected to agarose gel ( Mass volume ratio 2%) electrophoresis detection.
[0056] 3. Optimization of LAMP reaction conditions
[0057] 1. Optimization of reaction temperature
[0058] In order to obtain the optimal reaction temperature...
Embodiment 3
[0074]Example 3 Suspected PCV3 infected pig disease material tissue detection
[0075] Collect 6 lungs of suspected PCV3-infected pigs from the clinic, take 2-3 mg of clinical disease material, grind them thoroughly and add 1 ml of normal saline. Freezing and thawing were repeated 3 times, and the supernatant was removed by centrifugation. Aspirate 200 μL of the supernatant, and extract the nucleic acid of the virus according to the operating instructions of the viral RNA / DNA extraction kit. Take 1 μL of the extracted nucleic acid for LAMP detection (the reaction system is shown in Table 2, except that the template is changed; the reaction conditions are the optimized conditions determined in step 1) and PCR detection. The result is as Figure 7 and 8 As shown, both the LAMP detection method and the PCR agarose gel electrophoresis detection method can detect 5 positive disease materials, and the coincidence rate of the two is 100%.
[0076] In the present invention, the ke...
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