Primer pairs and probes for detecting AIDS treatment drug ddi and tdf resistance mutation sites and their application

A technology for drug resistance mutation sites and therapeutic drugs, applied in the field of biomedicine, can solve the problems of high detection cost, long time consumption, complicated operation, etc., and achieve the effects of high sensitivity, simple operation, and simple and fast operation.

Active Publication Date: 2017-11-10
JIANGSU FAST BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] The object of the present invention is to solve the problems of low sensitivity, poor specificity, cumbersome operation, high detection cost and long time-consuming of the existing methods for detecting AIDS treatment drug DDI and TDF drug-resistant mutation sites. The present invention provides a A fluorescent quantitative PCR detection kit with high sensitivity, good specificity, low detection cost, fast and simple AIDS treatment drug DDI and TDF resistance mutation sites, and also provides a method for using the detection kit

Method used

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  • Primer pairs and probes for detecting AIDS treatment drug ddi and tdf resistance mutation sites and their application
  • Primer pairs and probes for detecting AIDS treatment drug ddi and tdf resistance mutation sites and their application
  • Primer pairs and probes for detecting AIDS treatment drug ddi and tdf resistance mutation sites and their application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0057] Kits for detecting AIDS drug DDI and TDF resistance mutation sites, including:

[0058] (1) 875uL RT-PCR mixture;

[0059] RT-PCR buffer (20mM Tris-Hcl, 100mM NaCl, pH8.3) final concentration 1×;

[0060] The final concentration of dNTPs is 3mM;

[0061] MgCl 2 The final concentration is 1.6mM;

[0062] 3 pairs of primers and 3 probes for the detection of AIDS treatment drug DDI and TDF resistance mutation sites: the final concentration of the upstream and downstream primers of each ARMS primer pair is 200nM; the Taqman and kit of each ARMS primer pair The final concentration of Taqman probes for the quality control primer pair is 300nM;

[0063] Specifically, the RT-PCR mixture comprises: primer sequences SEQ ID No.1, SEQ ID No.2, SEQ ID No.4, SEQ ID No.5, and the respective final concentrations of SEQ ID No.6 are 0.2, 0.4, 0.2, 0.2, 0.2mmol / uL, Taqman probe sequence is SEQ ID No.3, SEQ ID No.7, the final concentration is 0.2, 0.1mmol / uL respectively;

[0064] S...

Embodiment 2

[0097] Example 2 The detection sensitivity of the AIDS treatment drug DDI and TDF drug-resistant mutation site RT-PCR detection kit of Example 1 to K65R

[0098] Prepare 17.5 μL of RT-PCR reaction solution and 2.5 μL of mixed enzyme solution for each sample, and prepare 1% of the positive control substance 1, 0.01% of the positive control substance, 0.0001% of the positive control substance, and 0.0001% of the positive control substance in Example 1. 0.000001% was diluted and used as a template for PCR reaction, and the loading volume of each diluted sample was 5uL to determine the detection sensitivity of the detection kit in the present invention to K65R (the final concentration of each component in the 20μL system was 1×RT -PCR buffer, 3 mM dNTPs, 1.6 mMMgCl2, 2U / μL M-MLV reverse transcriptase, 0.05 U / μL Taq DNA polymerase, 200nM primers, 300nM Taqman probe), the PCR reaction conditions are: 42℃ reverse transcription 10 min, one cycle; pre-denaturation at 95°C for 3 minutes...

Embodiment 5

[0107] The AIDS treatment drug DDI and TDF drug-resistant mutation site RT-PCR detection kit of Example 1 is used for detection.

[0108] RT-PCR reaction solution and mixed enzyme solution were prepared according to 17.5 μL of RT-PCR reaction solution and 2.5 μL of mixed enzyme solution for each sample, and PCR amplification was performed on the positive control substance mixed solution. The PCR reaction conditions were: reverse transcription at 42°C for 10 min, one cycle; pre-denaturation at 95°C for 3 minutes, one cycle; denaturation at 95°C for 10 seconds, annealing and extension at 55°C for 40 seconds, 45 amplification cycles.

[0109] The result is as Figure 4 shown.

[0110] Depend on Figure 4 It can be seen that the Ct values ​​of the positive controls provided in the detection kits are all below 35.

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Abstract

The invention discloses a primer pair and a probe used for detecting AIDS treatment medicine DDI and TDF drug-resistance mutation sites, which comprise an ARMS primer and a Taqman probe of mutation sites K65R, K70E and Q151M at 65th site, 70th and 151st site of pol gene for detecting HIV-1 virus RNA. The invention also provides the application of the primer pair and the probe. The kit has the advantages of high detection sensitivity, good specificity, and low detection cost, and provides a medicine usage guidance for treating clinic AIDS patient, individuation treatment for AIDS patient can be realized, medicine effectiveness can be timely reflected, AIDS patient living quality can be prolonged, and the primer pair and the probe have wide application prospect and long social benefit.

Description

technical field [0001] The invention belongs to the field of biomedicine, and relates to a primer pair and a probe for detecting drug-resistant mutation sites of DDI and TDF for AIDS treatment drugs, and also relates to the use of the primer pair and probe in detecting drug-resistant mutations of DDI and TDF for AIDS treatment drugs application in the site. technical background [0002] Most of the AIDS epidemic in the world is caused by HIV-1. The HIV-1 genome contains three structural genes gag, pol, and env and six regulatory genes tat, rev, nef, vpr, vif, and vpu, with long repeat sequences (LTR) at the 5' end and 3' end. The Pol gene encodes protease, integrase and reverse transcriptase, the three major viral proteases are used for the maturation of protein precursors, the reverse transcription of viral genomic RNA and the integration of viral cDNA on the host genomic DNA. [0003] At present, more than 30 kinds of anti-HIV-1 drugs have been circulated in the market f...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6858C12Q1/703C12Q2600/106C12Q2600/156C12Q2535/137C12Q2561/101C12Q2545/113
Inventor 龚剑李娟唐乃平
Owner JIANGSU FAST BIOTECH
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