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Primer for detecting main drug-resistant mutation sites of aids therapeutic nucleoside reverse transcriptase inhibitor and application thereof

A technology of reverse transcriptase inhibition and drug-resistant mutation sites, which is applied in the field of medical detection, can solve the problems of long detection time and high cost, and achieve the effects of low cost, easy mastery and fast operation

Inactive Publication Date: 2016-06-01
GUANGXI MEDICAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

This technology uses specialized probes that bind with certain parts of DNA molecules called bases or sites on their own. These probes have unique sequences that allow them to distinguish between different types of cancer cells by measuring how many copies they produce each other during cell division. By doing this, research scientists aimed at developing new treatments against these diseases without having to wait too much time before getting tested again due to rare variants discovered earlier.

Problems solved by technology

This patented technical problem addressed by this patents relates to finding out if an individual who suffers from Acquired Immune Deficiency Syndrome Virus (AIDSV), known commonly referred to as Human immunodeficiencies syndromes type I/IIa, needs effective treatments against viruses like Zidoviridae and Lassa fever. Current techniques involve testing each patient individually but cannot accurately identify their own susceptibility risk due to multiple resistant variants caused by various factors associated with disease progression.

Method used

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  • Primer for detecting main drug-resistant mutation sites of aids therapeutic nucleoside reverse transcriptase inhibitor and application thereof
  • Primer for detecting main drug-resistant mutation sites of aids therapeutic nucleoside reverse transcriptase inhibitor and application thereof
  • Primer for detecting main drug-resistant mutation sites of aids therapeutic nucleoside reverse transcriptase inhibitor and application thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Embodiment 1: Construction of HIV-1 wild-type plasmid and mutant plasmid

[0037] (1) Ligate the reverse transcriptase gene sequence (1.1 kb in length, relative to HXB2 position: 2243-3304) identified by sequencing to the pUCm-19 vector, then transform, extract the plasmid DNA, and verify the plasmid connection by sequencing Correct, obtain the HIV-1 wild-type cloning plasmid and dilute to 0.01ng / uL.

[0038] (2) Through the method of artificial site-directed mutagenesis, the bases corresponding to the 41st, 65th, 70th, 184th, 210th, and 215th amino acids of the reverse transcription gene in the HIV-1 wild-type cloning plasmid were mutated to obtain M41L, D67N, and K65R , K70R, K70E, M184V, M184I, L210W, T215Y, T215F mutant plasmids, diluted to 0.01ng / uL.

Embodiment 2

[0039] Embodiment 2: ASPCR method detects the specificity and sensitivity of M41L site

[0040] (1) Using the HIV-1 wild-type cloning plasmid and the M41L site mutant plasmid as templates, real-time PCR detection was performed using the M41L site wild-type amplification primers SEQ ID NO.1 and SEQ ID NO.21. The result is as figure 1 As shown in -(1), the amplification curve of the wild-type template showed an S-shaped growth, and the CT value of the number of cycles experienced by the fluorescence signal reaching the set threshold was 24.8. The mutant template had no amplification curve, and the fluorescence signal did not reach the set threshold. The threshold value is set, and there is no CT value. The difference between the two is obvious, and the amplification specificity is high.

[0041] (2) Using the HIV-1 wild-type cloning plasmid and the M41L site mutant plasmid as templates, the M41L site mutant amplification primers SEQ ID NO.2 and SEQ ID NO.21 were used for real-t...

Embodiment 3

[0043] Embodiment 3: ASPCR method detects the specificity and sensitivity of D67N site

[0044] (1) Using the HIV-1 wild-type cloning plasmid and the D67N site mutant plasmid as templates, real-time PCR detection was performed using the D67N site wild-type amplification primers SEQ ID NO.3 and SEQ ID NO.21. The result is as figure 2 - As shown in (1), the primer pair specifically amplifies the HIV-1 wild-type sequence.

[0045] (2) Using the HIV-1 wild-type cloning plasmid and the D67N site mutant plasmid as templates, real-time PCR detection was performed using the D67N site mutant amplification primers SEQ ID NO.4 and SEQ ID NO.21. The result is as figure 2 -As shown in (2), the primer pair specifically amplifies the D67N site mutant sequence.

[0046] (3) Using the 1%-10% mutation samples of the D67N site as a template, real-time PCR detection was performed using the D67N site mutant amplification primers SEQ ID NO.4 and SEQ ID NO.21. The result is as figure 2 -As s...

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Abstract

The invention relates to a primer for detecting main drug-resistant mutation sites of an aids therapeutic nucleoside reverse transcriptase inhibitor. The primer comprises ASPCR primers for detecting the mutation sites M41L, D67N, K65R, K70R, K70E, M184V, M184I, L210W, T215Y and T215F of HIV-1 virus pol genes. Each mutation site comprises a pair of wild PCR amplification primers and a pair of mutation PCR amplification primers. Specific upstream primers of each mutation site primer pair are shown in SEQ ID NO.1 to SEQ ID NO.20, and a universal downstream primer is shown in SEQ ID NO.21. The primer is used for detecting the drug-resistant mutation sites of the aids therapeutic nucleoside reverse transcriptase inhibitor and has the advantages of being high in specificity, high in flexibility, easy, convenient and rapid to use, low in cost and the like.

Description

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Claims

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Application Information

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Owner GUANGXI MEDICAL UNIVERSITY
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