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Primer pair and probe used for detecting AIDS treatment medicine 3TC and FTC drug-resistance mutation sites and application thereof

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

Active Publication Date: 2015-09-16
江苏亦文生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] Purpose of the invention: to solve the problems of low sensitivity, poor specificity, cumbersome operation and detection cost in the existing methods for detecting AIDS treatment drug 3TC and FTC drug-resistant mutation sites High, time-consuming and other problems, the present invention provides a high sensitivity, good specificity, low detection cost, fast, simple primer pairs and probes for AIDS treatment drug 3TC and FTC drug-resistant mutation sites, and also provides A detection kit comprising the above-mentioned primer pair and probe

Method used

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  • Primer pair and probe used for detecting AIDS treatment medicine 3TC and FTC drug-resistance mutation sites and application thereof
  • Primer pair and probe used for detecting AIDS treatment medicine 3TC and FTC drug-resistance mutation sites and application thereof
  • Primer pair and probe used for detecting AIDS treatment medicine 3TC and FTC drug-resistance mutation sites and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0071] Preparation and screening of the main components of the detection kit for AIDS treatment drug 3TC and FTC resistance mutation sites:

experiment example 1

[0073] The cDNA sequence of HIV-1 pol RT gene is shown in SEQ ID No.14. According to the mutation types of the four point mutations M184V, M184I, Q151M and K65R, primers and probes for RT-ARMS-Taqman qPCR were designed using Primer premier 5.0 software, so that the mutation site was located at the last position of the upstream or downstream primers, and the Missense mutations were carried out at the 1-2 bases upstream of the mutation site. The specific primer probe sequences are shown in SEQ ID No. 1 to SEQ ID No. 10.

[0074] Two point mutations, M184V and Q151M, were introduced into the HIV-1 pol RT gene by artificial synthesis to obtain the mutant positive control substance 1, whose sequence is shown in SEQ ID No. 15. The synthetic HIV-1 pol RT mutant positive control substance 1 containing two point mutations of M184V and Q151M was cloned into the pGM-T vector to construct a recombinant plasmid, and then the recombinant plasmid was transformed into DH5α Escherichia coli, ...

experiment example 2

[0081] Use the primer probes SEQ ID No. 11, SEQ ID No. 12, and SEQ ID No. 13 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 for RT-ARMS-Taqman qPCR. MgCl in PCR reaction 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 a 20μL system were 1×RT-PCR buffer, 0.8 mM dNTPs, 1.6 U / μL M-MLV reverse transcriptase, 0.04 U / μL TaqDNA polymerase, 200nM primers, 100nM Taqman probe, 0.1-10ng / μL positive control). The PCR reaction conditions were: reverse transcription at 42°C for 30 min, one cycle; pre-denaturation at 95°C for 3 minutes, one cycle; 40 amplification cycles, deformation at 95°C for 10 seconds, annealing and extension at 55-60°C for 30 seconds. The result is as figure 2 shown. Depend on figure 2 It can be seen that in MgCl 2 There are amplification curves ...

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Abstract

The invention discloses a primer pair and a probe used for detecting AIDS treatment medicine 3TC and FTC drug-resistance mutation sites, which comprise an ARMS primer and a Taqman probe of mutation sites M184V, M184I, Q151M and K65R at 184th site, 151st site and 65th site of pol gene for detecting HIV-1 virus RNA. The invention also provides the application of the primer pair and the probe on detection of 3TC and FTC main drug-resistance mutation sites M184V, M184I, Q151M and K65R. The kit has the advantages of high detection sensitivity, good specificity, and low detection cost, and provides medicine usage guidance for treating clinic AIDS patient, individuation treatment for AIDS patient can be realized, medicine effectiveness can be increased, AIDS patient living time can be prolonged, and the primer pair and the probe have wide application prospect and social benefit.

Description

technical field [0001] The invention belongs to the field of biomedicine, in particular to a pair of primers and probes for detecting drug-resistant mutation sites of AIDS treatment drugs 3TC and FTC and their application. Background technique [0002] AIDS is an immunodeficiency syndrome caused by HIV infection. HIV belongs to the Retroviridae family and the Lentivirus genus. The currently discovered HIV virus has two types, HIV-1 and HIV-2, but most of the prevalent AIDS in the world are caused by HIV-1. The HIV-1 genome is about 9.7kb and consists of two single-stranded RNA strands forming a dimer. The genome contains three structural genes gag, pol, and env and six regulatory genes tat, rev, nef, vpr, vif, vpu, and long repeat sequences (LTR) at the 5' end and 3' end. Gag is initially expressed as a 55Kd protein precursor, and then cleaved by viral proteases into 17Kd (p17) matrix protein (MA), 24Kd (p24) capsid protein (CA), 7Kd (p7) nucleocapsid protein (NC ) and ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11
Inventor 龚剑唐乃平谈小龙李娟
Owner 江苏亦文生物科技有限公司
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