Detection kit for identifying porcine reproductive and respiratory syndrome virus and application thereof
A technology for respiratory syndrome and pig reproduction, which is applied in the determination/inspection of microorganisms, DNA/RNA fragments, recombinant DNA technology, etc., can solve the problems of inability to identify and detect PRRSV mutant strains at the same time, achieve good application prospects, and improve prevention and control. Control level, reduce detection cost and detection time effect
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Embodiment 1
[0063] Kit sensitivity test
[0064] 1. RNA extraction of PRRSV TJM-F92 strain (commercially available):
[0065] The cultured PRRSV TJM-F92 strain was diluted to 10 5 TCID 50 、10 4 TCID 5 、10 3 TCID 50 、10 2 TCID 50 、10 1 TCID 50 、10 0 TCID 50 . Viral RNA was extracted by the Trizol method,
[0066] Methods as below.
[0067] (1) Add 600 μL Trizol Reagent to every 200 μL sample solution, vortex for 15 seconds, and let stand at room temperature for 5 minutes.
[0068] (2) Add 200 μL of chloroform to the centrifuge tube, mix it upside down, and let it stand at room temperature for 5 minutes.
[0069] (3) Centrifuge at 12,000rpm at 4°C for 10 minutes, take the upper liquid and put it in a new centrifuge tube (be careful not to suck up the protein on the layered surface by mistake, try to stick to the wall as much as possible, and change the pipette tip every time). Add 800 μL of isopropanol (containing 0.01% DEPC), cap and turn it upside down 1 or 2 times,...
Embodiment 2
[0083] Kit specificity test:
[0084] 1. Table 2 of the control strains, including:
[0085]
[0086]
[0087] 2. Virus and bacterial DNA extraction: Use the cell / tissue DNA extraction kit from Tiangen Biological Company, and refer to the instruction manual for the method.
[0088] (1) Take 1ml of the bacterial solution and centrifuge at 12,000rpm for 5 minutes, remove the supernatant, add 200μL GA solution to the pellet, resuspend and mix well.
[0089] (2) Add 20 μL proteinase K solution and mix well. The samples were mixed by inversion 2-3 times in a water bath at 65°C for 1 hour. Remove from the water bath and centrifuge briefly to remove water droplets from the inside of the cap.
[0090] (3) Add 200 μL of buffer GB, mix thoroughly by inversion, and place at 70°C for 10 minutes. The solution should become clear, and briefly centrifuge to remove water droplets on the inner wall of the tube cap.
[0091] (4) Add 200 μL of absolute ethanol, shake and mix well for 1...
Embodiment 3
[0116] RT-PCR Amplification Test to Differentiate PRRSV American Classic Strain, Variant Strain and TJM-F92 Strain
[0117] 1. Table 3 of the control strains, including:
[0118]
[0119] 2. Extraction of porcine reproductive and respiratory syndrome virus RNA: according to the Trizol method, refer to Example 1.
[0120] 3. cDNA synthesis
[0121] Use 20.0 μL cDNA synthesis system, that is, PRRSV RNA template 11 μL, 5×RT Buffer 5.0 μL, Random Primers (50pmol / μL) 1.0 μL, dNTP Mixture 2.0 μL, RNase Inhibitor (40 U / μL) 0.5 μL, MLV Reverse Transcriptase XL (5U / μL) 0.5 μL.
[0122] Reaction program: 10 min at 25°C, 60 min at 42°C, 5 min at 99°C, 5 min at 4°C. After cDNA synthesis, perform PCR amplification or store at -20°C for later use.
[0123] 4. PCR reaction amplification: use the porcine reproductive and respiratory syndrome virus identification and detection kit for PCR amplification.
[0124] The total volume of the amplification reaction is 25 μL, and its various co...
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