Bovine viral diarrhea virus nano-PCR detection kit and preparation method thereof
The technology of a bovine viral diarrhea and detection kit is applied in the detection field of bovine viral diarrhea virus, which can solve the problems of time-consuming and laborious, cross-infection, easy misdiagnosis, etc., and achieves the avoidance of cross-infection, high use safety, and high specificity Sex and Sensitivity Effects
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Embodiment 1
[0045] Embodiment 1 Assembly of bovine viral diarrhea virus nano-PCR detection kit of the present invention
[0046] (1) Preparation of PCR reaction solution
[0047] ①A pair of specific primers were designed according to the conserved gene sequence of the BVDV virus 5'-UTR group. In order to identify the primers P1 and P2 of bovine viral diarrhea virus (BVDV), the sequences of the primers are as follows:
[0048] P1: 5'-GGTAGCAACAGTGGTGAGTTC-3',
[0049] P2: 5'-CTCAGGTTAAGATGTGCTGTG-3'.
[0050] ② Preparation of nano-PCR reaction solution: The reaction solution is composed of MightyAmp enzyme, gold nanoparticle sol (20nm, concentration 0.5μg / μL), 2xBufferMix buffer solution, sterile double distilled water and a pair of specific primers in the above ①.
[0051] (2) Construction of positive control recombinant plasmid
[0052] ①Preparation of bovine viral diarrhea virus (BVDV) positive control plasmid: use MDBK cells to proliferate bovine viral diarrhea virus, and after cult...
Embodiment 2
[0055] Embodiment 2 The use method of bovine viral diarrhea virus nano-PCR detection kit of the present invention
[0056] (1) Extract sample RNA from the sample to be tested. The sample to be tested includes cell culture, blood, tissue culture, nasal swab and fecal swab, etc.
[0057] (2) Determination of nano-PCR reaction conditions
[0058] After optimizing the conditions of reaction concentration and annealing temperature, it was determined that in a 25 μL reaction system, 10.5 μL of primer P, 20.5 μL of primer P, 0.5 μL of MightyAmp enzyme, 12.5 μL of 2×buffer, 1 μL of template, 0.7 μL of gold nanoparticle sol, Make up the reaction system to 25 μL with deionized water, and use the BRSV standard positive plasmid as the positive template. The reaction conditions were set as follows: 98°C for 2 min; 98°C for 10 s, annealing temperature range of 52°C to 62°C, annealing time of 2s to 10 s, 68°C for 1 min, 30 cycles, 68°C total extension for 10 min, and double distilled water ...
Embodiment 3
[0062] Embodiment 3 The specificity experiment of bovine viral diarrhea virus nano-PCR detection kit of the present invention
[0063] Using IBRV, BVDV, BRSV and BPI3 positive control recombinant plasmids and negative control (distilled water) as templates respectively, add primers P1 and P2 for identifying bovine viral diarrhea virus (BVDV), and carry out the specificity experiment of the nano-PCR detection kit;
[0064] Experimental results: if figure 1 As shown, the corresponding negative, 136bp, negative and negative bands appeared when IBRV, BVDV, BRSV and BPI3 positive control recombinant plasmids were used as templates, and no band was amplified in the negative control.
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