A method and kit for the identification and detection of porcine pseudorabies vaccine virus and wild virus
A porcine pseudorabies virus and kit technology are applied in the field of identification and detection of porcine pseudorabies vaccine virus and wild virus, which can solve the problems of complex primers, increased primer mismatch rate, false positive reactions, etc., so as to improve specificity and reliability, speed up The speed of amplification and the effect of on-site realization
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Embodiment 1
[0166] Embodiment 1 is used for the preparation of sensitivity detection PRV UL52 gene and gE gene plasmid
[0167] According to the conventional method of molecular cloning, the pMD18-T cloning plasmid was used as the vector to construct the recombinant plasmids containing the PRVUL52 gene and the gE gene, and named them pMD18-T-UL52 and pMD18-T-gE, respectively. The sequences of the PRV UL52 gene and gE gene were respectively referred to GenBank No.: 47996-48309nt and 122529-122714nt coding sequences of JF797218, the average value of 3 concentration measurements was carried out by ultraviolet spectrophotometer, the base numbers of the two recombinant plasmids were 2915bp and 2892bp respectively, and the plasmid copy number was calculated by the following formula, namely Copy number (copies / μL)=6.02×10 23 ×plasmid concentration (ng / μL)×10 -9 / (plasmid base number × 660) is calculated, and the final two determined 3.3 × 10 9 copies / μL, as the starting sample, adjusted to 3.3...
Embodiment 2
[0168] Embodiment 2 Preparation of SmartMix RPA reaction solution
[0169] The composition of conventional RPA amplification reaction solution system: 100ng / μL recombinase uvsX; 50ng / μL auxiliary protein uvsY; 100ng / μL LGp32 protein (SSB, single-stranded DNA binding protein); 25ng / μL Bsu DNA polymerase; 0.5mM dNTPs; 1mMDTT (dithiothreitol); 3mMATP (adenosine triphosphate); 100mM Tricine (trimethylglycine), pH8.0; 5% PEG20K (polyethylene glycol 20000); 100ng / μL Creatine kinase (creatine kinase); 20mM Phosphokinase (phosphokinase); 80 mM Potassium acetate (potassium acetate). Add 10units of NFO enzyme (endonuclease IV), 50pM UL52primer1, 50pM UL52primer2, 10pM UL52probe, 50pMgEprimer1, 50pM gEprimer2, 10pM gEprobe on the basis of the basic components of conventional RPA, mix well and pack in 45.5μL per tube to obtain RPA or NFO-RPA SmartMix Rapid Reaction Solution should be stored at low temperature for later use. In addition, 100 mM Magnesium Acetate (magnesium acetate) was a...
Embodiment 3
[0170] The magnetic bead method extraction of PRV nucleic acid in the pig serum sample of embodiment 3
[0171] For liquid samples such as plasma, serum, ascites, and cell culture, directly take 200 μl into a 1.5ml nuclease-free centrifuge tube, and then add 500 μl of lysate (10mmol / L Tris.Cl (pH 8.0), 10mmol / L EDTA, 150mmol / L NaCl , 0.5% SDS, 100-200 μg / ml proteinase K), shake and mix well.
[0172] The sample mixture was lysed in a 65-70°C incubation bath for 5 minutes.
[0173] Add 10 μL of well-mixed magnetic bead solution into the centrifuge tube (magnetic beads mark a layer of silica gel membrane, the reaction principle is that nucleic acid and silica gel membrane bind nucleic acid by positive and negative electric adsorption force under the condition of high salt ions, Then the nucleic acid is eluted and separated in an environment of low salt ions to achieve the purpose of nucleic acid separation.), and mixed upside down at room temperature for 10 minutes.
[0174] P...
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