Recombinant standard plasmid for detecting anthrax bacillus, kit and construction method for the plasmid
A technology of Bacillus anthracis and standard plasmid, applied in the field of bioengineering, can solve the problems of difficulty in general use, difficulty in detection sensitivity of kit detection performance, and difficulty in selecting a kit that meets your own needs, and achieves high specificity and high sensitivity. Effect
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Embodiment 1
[0050] Example 1: Recombination of standard plasmid PGEM-T-easy-RCP
[0051] The recombinant standard plasmid for detecting Bacillus anthracis of this embodiment is as figure 1 As shown, it consists of the PGEM-T easy vector and the PA gene, capA gene, and rpoB gene in series, denoted as PGEM-T-easy-RCP; wherein the PA gene sequence is shown in SEQ.ID.No.1, capA The gene sequence is shown in SEQ.ID.No.2, and the rpoB gene sequence is shown in SEQ.ID.No.3.
[0052] The 5'end of the PA gene is connected to the PGEM-T easy vector via the Sal I restriction enzyme cutting site, and the 3'end of the PA gene is connected to the 5'end of the capA gene via the HindIII restriction enzyme cutting site; the 3 of the capA gene The'end is connected to the 5'end of the rpoB gene through the Nhe I restriction enzyme cut site; the 3'end of the rpoB gene is connected to the PGEM-T easy vector through the Sac I restriction enzyme cut site; among them, HindIII restriction enzyme cuts The site sequenc...
Embodiment 2
[0054] Example 2: Construction of recombinant standard plasmid PGEM-T-easy-RCP
[0055] In this example, a method for constructing a recombinant standard plasmid for detecting Bacillus anthracis was used to construct a recombinant standard plasmid PGEM-T-easy-RCP, and the method includes the following steps:
[0056] (1) Design primer pairs: design primer pairs pa-F and pa-R for PA gene sequence; design primer pairs capa-F and capa-R for capA gene sequence; design primer pairs rpob-F and rpob- for rpoB gene sequence R;
[0057] The PA gene sequence is shown in SEQ.ID.No.1, the capA gene sequence is shown in SEQ.ID.No.2, the rpoB gene sequence is shown in SEQ.ID.No.3; the sequence of pa-F is shown in SEQ. ID.No.9, the sequence of pa-R is shown in SEQ.ID.No.10, the sequence of capa-F is shown in SEQ.ID.No.11, and the sequence of capa-R is shown in SEQ.ID. No. 12, the sequence of rpob-F is shown in SEQ. ID. No. 13, and the sequence of rpob-R is shown in SEQ. ID. No. 14;
[0058] (2) Am...
Embodiment 3
[0093] Example 3: Kit for routine PCR detection of Bacillus anthracis
[0094] The kit of this example includes the recombinant standard plasmid PGEM-T-easy-RCP of the present invention (including but not limited to the recombinant standard plasmid of Example 1).
[0095] The kit of this embodiment also includes a DNA extraction solution containing NaOH at a concentration of 20-30 mM (preferably 25 mM), Tris-HCl at a concentration of 5-15 mM (preferably 10 mM), and a volume percentage of 0.5-1.5% ( Preferably 1%) Triton X-100, 0.5-1.5% by volume (preferably 1%) NP-40, EDTA with a concentration of 0.05-0.15mM (preferably 0.1mM), 1-3% by weight (preferably 2%) Chelex-100. It should be noted that if other existing commercial DNA extraction kits are used to extract DNA, the kit of this embodiment may not include the DNA extraction solution.
[0096] The kit of this embodiment also includes 2×Premix Taq V2.0 (premix containing reagents required for PCR, and dNTPs composed of dATP, dTTP,...
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