DPO primer sequences for salmonella detection by using DPO-PCR method, and detection kit thereof
A DPO primer, Salmonella technology, applied in the direction of using vectors to introduce foreign genetic material, microorganism-based methods, biochemical equipment and methods, etc., can solve the problems of low sensitivity, high false positive rate, poor specificity, etc., and achieve good repeatability and stability effects
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Embodiment 1
[0047] Preparation of Salmonella DNA template
[0048] Extract and purify bacterial genomic DNA according to the instructions of TIANamp Bacteria DNA Kit, and add the corresponding reagents in order:
[0049] (1) Take 1.5 mL of bacterial liquid, centrifuge at 10,000 r / min for 1 min, discard the supernatant, add 200 μL of buffer GA, and suspend the bacterial cells thoroughly;
[0050] (2) Add 20 μL proteinase K (20mg / mL), and add 220 μL buffer solution GB, mix thoroughly, and act in a 70°C water bath for 10 minutes;
[0051] (3) Add 220 μL of absolute ethanol, mix thoroughly for 15 seconds, transfer the resulting solution (including flocculent precipitate) to the adsorption column CB3 after brief centrifugation, centrifuge at 12000 r / min for 30 seconds, and discard the liquid in the collection tube;
[0052] (4) Add 500 μL buffer GD (containing absolute ethanol) to the adsorption column CB3, centrifuge at 12000r / min for 30s, and discard the liquid in the collection tube;
[0...
Embodiment 2
[0057] Example 2 Design and synthesis of PCR primers
[0058] The Salmonella FimY gene was selected as the target gene, and after BLAST analysis, the following primers were designed and synthesized:
[0059] (1) Prepare PCR amplification primers for the positive control substance of Salmonella FimY gene (the size of the PCR product is 227bp):
[0060] SA-CGF: 5′-TATTTTCCGGAAAACGTACCGCAGCATTCCGC-3′
[0061] SA-CGR: 5′-AGCCATACGGATAAACTGTGTTATAGCGGAGGT-3′
[0062] (2) DPO primers for Salmonella DPO-PCR method identification (PCR product size is 237bp):
[0063] SA-DPOF: 5'-TATTTTCCGGAAAACGTACCGCAIIIIIGCATTCCGC-3',
[0064] SA-DPOR: 5'-AGCCATACGGATAAACTGTGTTATIIIIIAGCGGAGGT-3';
Embodiment 3
[0065] Example 3 Preparation of positive control substance
[0066] Using SA-CGF and SA-CGR as primers, the FimY gene of Salmonella was amplified by PCR.
[0067] Utilize target gene amplification primer SA-CGF / SA-CGR, adopt PCR method to amplify Salmonella FimY gene, the reaction system of PCR is:
[0068] 10×PCR Buffer 2.5μL dNTP (2.5mM for each) 2.0 μL Upstream primer SA-CGF (10μM) 1.0 μL Downstream primer SA-CGR (10μM) 1.0 μL Taq DNA Polymerase (5U / μL) 0.5μL DNA template 0.5μL wxya 2 o 17.5μL
[0069] The reaction conditions of PCR were: 95°C for 5min; 35 cycles of 94°C for 15s, 57.6°C for 20s, and 72°C for 30s; 72°C for 10min.
[0070] The PCR amplification result was detected by agarose gel electrophoresis, and the size of the PCR product was 227bp.
[0071] Connect the PCR product to the pMD-19-T vector, transform competent Escherichia coli JM109, and obtain positive recombinant bacteria, and prepare positi...
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