LAMP (loop mediated isothermal amplification method) detection kit for ASFV (African swine fever virus) and application of LAMP detection kit
A technology of African swine fever virus and detection kit, which is applied in the direction of recombinant DNA technology, microbial measurement/inspection, DNA/RNA fragments, etc., can solve the problems of strong instrument dependence, low efficiency, and inability to realize on-site detection, etc., to achieve High sensitivity, simple operation, and the effect of avoiding the formation of primer dimers
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Embodiment 1
[0048] Embodiment 1: Sample, primer design and preparation
[0049] 1.1 Plasmid, sample source and experimental site
[0050] According to the African swine fever virus P72 protein gene (shown in SEQ ID No.7) provided on Genebank, the plasmid pUC57-P72 was synthesized by Sangon Bioengineering (Shanghai) Co., Ltd., dissolved and diluted to 1.0×10 7 copies / μL.
[0051] Porcine Japanese encephalitis virus, classical swine fever virus, porcine parvovirus, porcine pseudorabies virus, porcine circovirus, and porcine transmissible gastroenteritis virus were provided by Shaanxi Nuowei Lihua Biotechnology Co., Ltd.
[0052] SPF pig lymph nodes and SPF pig serum were collected from Shaanxi Nuowei Lihua Biotechnology Co., Ltd.
[0053] 1.2 Identification of positive plasmids
[0054] The SalI-XbaI site of the positive plasmid pUC57-P72 was digested to verify the correctness of the plasmid. The SalI-XbaI site of the positive plasmid pUC57-P72 was digested and identified, and the resul...
Embodiment 2
[0059] Embodiment 2: the establishment of African swine fever virus LAMP detection kit
[0060] A detection kit for African swine fever virus LAMP, said kit comprising the above-mentioned primer set.
[0061] Preferably, the kit includes the primer set described in Example 1 above, Bst DNA polymerase, LAMP reaction solution, betaine, positive control and negative control.
[0062] Preferably, the ratio of the outer primer, the loop primer, and the inner primer is: the molar ratio of the outer primer, the loop primer, and the inner primer is 4FIP: 4BIP: 4LoopF; 4LoopB: 4F3; 4B3 is 10:10:4:4:1:1 . When preparing primers, the optimal ratio of 4FIP, 4BIP, 4LoopF, 4LoopB, 4F3, 4B3 to deionized water is 10:10:4:4:1:1:250.
[0063] Preferably, the LAMP reaction solution contains 10mM dNTP, 10×ThermoPol reaction buffer, 150mM MgSO 4 aqueous solution.
[0064] Preferably, the positive control is a plasmid DNA containing the gene fragment of interest, and the negative control is ste...
Embodiment 3
[0071] Embodiment 3: the establishment of African swine fever LAMP detection method
[0072] 3.1 Establishment of LAMP reaction system
[0073] According to the kit of Example 2, use the above primers to determine the content and ratio of each component in the 25 μl reaction system detected by African swine fever virus LAMP, and place it in a constant temperature container for amplification. The test result can be judged by visual observation of white turbidity and gel electrophoresis. The 25 μL reaction system is shown in Table 2.
[0074] Table 2 25μL reaction system
[0075]
[0076] 3.2 LAMP reaction
[0077] 3.2.1 Determination of LAMP reaction time
[0078] According to the LAMP reaction system determined above, assuming that the positive plasmid pUC57-P72 was specifically amplified at 65°C for 20 min, 30 min, 40 min, 50 min, and 60 min, record the results and determine the optimal reaction time.
[0079] Set the concentration to 10 4 The copied positive plasmid...
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