Primer pair and probe for detecting ABC (Abacavir) drug-resistant mutation sites of AIDS (Acquired immune deficiency syndrome) curing drugs and application of primer pair and probe
A drug-resistant mutation site and therapeutic drug technology, applied in the field of biomedicine, can solve problems such as long time-consuming, high detection cost, and poor specificity, and achieve the effect of simple operation, high sensitivity, and low cost
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Embodiment 1
[0053] A kit for detecting ABC resistance mutation sites of AIDS treatment drugs, including:
[0054] 1. RT-PCR mixture 700μL
[0055] RT-PCR mix includes
[0056] RT-PCR buffer (20mM Tris-HCl, 50mM KCl pH8.3) final concentration 1×
[0057] The final concentration of dNTPs is 2mM;
[0058] MgCl 2 Final concentration 2mM;
[0059] The final concentrations of the upstream and downstream primers of the ARMS primer pair and the upstream and downstream primers of the kit quality control primer pair are both 200nM; the final concentration of Taqman probes corresponding to each ARMS upstream and downstream primers and the kit quality control primers 175nM;
[0060] Specifically, the RT-PCR mixture contains: primer sequences SEQ ID No.1, SEQ ID No.2, SEQ ID No.3, SEQ ID No.4, SEQ ID No.5, SEQ ID No.7 , SEQ ID No.8, SEQ ID No.9 and their respective concentrations are 0.2,0.4,0.2,0.2,0.4,0.4,0.2,0.2mmol / μL, Taqman probe sequence is SEQ ID No.6 and SEQ ID No .10, the final concen...
experiment example 2
[0106] Use the primer probes SEQ ID No.6 and SEQ ID No.10 of the quality control product of the kit as the primer probe pair, and use the negative control prepared above as the template after dilution to carry out RT-ARMS-Taqmanq PCR. In the PCR reaction, MgCl 2 The concentrations were 1.5mM, 2.0mM, 2.5mM, 3.0mM and 3.5mM, and the concentrations of the other components were the same (the final concentrations of each component in the 20μL system were 1×RT-PCR buffer, 0.8mM dNTPs, 1.65U / μL mixed enzyme, 200nM primer, 175nM Taqman probe, 0.1~10ng / μL positive control substance). The PCR reaction conditions were: reverse transcription at 42°C for 10 min, one cycle; pre-denaturation at 95°C for 3 min, one cycle; deformation at 95°C for 10 s, annealing and extension at 55°C for 30 s, and 40 amplification cycles. The result is as figure 1 shown by figure 1 It can be seen that in MgCl 2 There are amplification curves in the concentration range of 1.5mM~3.5mM, when MgCl 2 The best ...
experiment example 3
[0108] Use the primer probes SEQ ID No.6 and SEQ ID No.10 of the quality control product of the kit as the primer probe pair, and use the negative control prepared above as a template after dilution to carry out RT-ARMS-Taqmanq PCR. The concentration of dNTPs in the PCR reaction 0.6mM, 0.8mM, 1.0mM, and 1.2mM, respectively, and the concentrations of the other components were the same (the final concentrations of each component in the 20μL system were 1×RT-PCR buffer, 2mM MgCl 2 , 1.65U / μL mixed enzyme, 200nM primer, 175nM Taqman probe, 0.1~10ng / μL positive control substance). The PCR reaction conditions were: reverse transcription at 42°C for 10 min, one cycle; pre-denaturation at 95°C for 3 min, one cycle; deformation at 95°C for 10 s, annealing and extension at 55°C for 30 s, and 40 amplification cycles. The result is as figure 2 shown by figure 2 It can be seen that there are amplification curves in the range of dNTPs concentration from 1.5mM to 3.5mM, and the result is...
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