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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

Pending Publication Date: 2019-08-02
JIANGSU FAST BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] The purpose of the present invention is to overcome the problems of low sensitivity, poor specificity, cumbersome operation, high detection cost and long time-consuming of the existing method for detecting the ABC resistance mutation site of AIDS treatment drug in the prior art. The present invention provides a high-sensitivity, The primer pair and probe for ABC drug-resistant mutation site of AIDS treatment drug that has good specificity, low detection cost, fast and simple, and also provides a detection kit including the above primer pair and probe and its use method

Method used

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  • 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
  • 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
  • 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

Examples

Experimental program
Comparison scheme
Effect test

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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Abstract

The invention discloses a primer pair and probe for detecting ABC (Abacavir) drug-resistant mutation sites of AIDS (Acquired immune deficiency syndrome) curing drugs. The primer pair and probe are characterized by comprising an ARMS (Amplification refractory mutation system) primer and a Taqman probe for detecting the mutation sites of K65R, K70E, L74V, L74I, Y115F and Q151M at the 65th, 70th, 74th, 115th and 151th sites of a pol gene of HIV-1 (Human immunodeficiency virus) viral RNA. The invention further provides application of the primer pair and probe in the detection of the ABC major drug-resistant mutation sites of K65R, K70E, L74V, L74I, Y115F and Q151M. According to the primer pair and probe disclosed by the invention, a kit is high in detection sensitivity, good in specificity andlow in detection cost; medication guidance is provided for the treatment of clinical AIDS patients; individualized treatment of the AIDS patients is realized; the effectiveness of the drugs can be improved; the survival time of the AIDS patients is prolonged; wide application prospect and social benefit are realized.

Description

technical field [0001] The invention belongs to the field of biomedicine, in particular to a pair of primers and probes for detecting ABC resistance mutation sites of AIDS treatment drugs and their application. Background technique [0002] AIDS (AIDS) virus, human immunodeficiency virus (HIV-1), is a virus that causes the deficiency of the human immune system. It is a lentivirus that infects cells of the human immune system, and is a type of retrovirus. So far, there is no cure for it. By the end of 2013, the number of HIV-1 infected people in the world has exceeded 35 million, and AIDS has brought a huge disaster to all mankind. [0003] In foreign countries, HIV-1 was first discovered in 1981. So far, no AIDS vaccine has been developed internationally. In the case of widespread and rapid spread of HIV-1 without effective vaccine prevention, chemotherapy drugs have become the most powerful tool against HIV-1 infection. In 1985, scientists discovered the first HIV-1 tre...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/6858C12N15/11
CPCC12Q1/703C12Q1/6858C12Q2600/106C12Q2600/156C12Q2600/166C12Q2531/119C12Q2521/107C12Q2561/101C12Q2545/113
Inventor 武建张军
Owner JIANGSU FAST BIOTECH
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