Kit and method for detecting genetic mutation of PR regions and RT regions of HIV-1 genes

A technology of HIV-1 and detection kits, applied in biochemical equipment and methods, microbe determination/testing, DNA/RNA fragments, etc., can solve problems such as drug resistance, protease and reverse transcriptase structural changes

Active Publication Date: 2019-03-08
DAAN GENE CO LTD
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  • Summary
  • Abstract
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  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Mutations in the PR region and RT region of the pol gene may lead to changes in the structure of protease and reverse transcriptase, and thus change the HIV-1 virus' response to clinical protease inhibitors PIs, nucleoside NRTIs and non-nucleosides Changes in the sensitivity of the three types of antiviral drugs, the reverse transcriptase inhibitor NNRTI, may lead to the emergence of drug resistance

Method used

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  • Kit and method for detecting genetic mutation of PR regions and RT regions of HIV-1 genes
  • Kit and method for detecting genetic mutation of PR regions and RT regions of HIV-1 genes
  • Kit and method for detecting genetic mutation of PR regions and RT regions of HIV-1 genes

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preparation example Construction

[0158] Step 2, preparation of RT-PCR system

[0159] Preparation before RT-PCR system preparation: Take out the RT-PCR reaction solution and RT-PCR enzyme system in the kit, melt at room temperature, vortex and oscillate to mix, and then centrifuge for 10 seconds to prepare the RT-PCR system; the RT-PCR system contains RT-PCR reaction solution 27 μL; and RT-PCR enzyme system 3 μL.

[0160] Step 3, RT-PCR loading

[0161] Take 20 μL of the sample prepared in step 1 and each control sample RNA template in the kit and add it to the eight-tube tube of the RT-PCR reaction system prepared in step 2, so that the total volume of each tube of RT-PCR reaction system is 50 μL; Close the cap of the eight-tube tube tightly. The control samples of the kit are shown in Table 6.

[0162] Step 4, RT-PCR amplification

[0163] Put the eight tubes into the PCR machine for amplification. The reaction conditions for RT-PCR amplification are: 50°C for 30 minutes, 95°C for 15 minutes for pre-den...

Embodiment 1

[0192] This embodiment provides a test kit for detecting gene mutations in the RT region and PR region of HIV patients, its composition, packaging and quantity (24 reactions / box), as shown in Table 7,

[0193] Table 7 The composition, packaging and quantity of the kit

[0194]

[0195]

Embodiment 2

[0196] Example 2: Nucleic acid sensitivity and mutation sensitivity detection experiments

[0197] The nucleic acid detection limit sample is a virus-like particle containing the PR region of the HIV-1 virus pol gene and the wild-type fragment of the RT region, diluted to 2×10 5 copies / ml, 2×10 4 copies / ml, 2×10 3 Copies / ml is used as a reference product for nucleic acid detection limit; virus-like particles containing HIV-1 virus pol gene PR region and RT region wild-type fragments and virus-like particles containing HIV-1 virus pol gene PR region and RT region mutant fragments are diluted to 1×10 5 Copies / ml, and then mix HIV-1 wild and mutant target fragments of poisonous particles according to the ratio of 100%, 80%, 60%, 40%, and 20% of the mutation respectively as a reference for mutation sensitivity, and the negative control is negative plasma ;

[0198] Take 20 μL of the extracted negative control and sensitivity reference nucleic acid, add the sample to the eight-...

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Abstract

The invention provides a kit and method for detecting genetic mutation of PR regions and RT regions of HIV-1 genes, and particularly discloses method for detecting genetic mutation of PR regions and RT regions of HIV-1 virus pol genes in plasmas of HIV patient, a primer and a kit comprising mixed liquor of the primer. Based on a generation of sequencing platform, the method has the advantages of being multiple in mutation detection sites, high in accuracy and easy in sample acquisition.

Description

technical field [0001] The invention belongs to the field of biotechnology, in particular, the invention relates to a kit and a method for detecting gene mutations in the RT region and the PR region of HIV-1 virus pol gene. Background technique [0002] Human Immunodeficiency Virus (Human Immunodeficiency Virus; abbr: HIV), that is, AIDS (AIDS, Acquired Immunodeficiency Syndrome) virus, is a virus that causes human immune system deficiency. As of 2014, about 36.9 million people around the world were infected with HIV, an average of 6,000 people were infected every day, and the number of people who died of AIDS was as high as 12 million. According to relevant reports in January 2016, the total number of people infected with HIV in China has exceeded the 1 million mark. [0003] HIV treatment, the current first-line HIV treatment drugs are mainly protease inhibitors PIs, nucleoside NRTIs and non-nucleoside reverse transcriptase inhibitors NNRTI3 antiviral drug combination the...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/6858C12N15/11
CPCC12Q1/6858C12Q1/701C12Q2531/113C12Q2535/122C12Q2521/107C12Q2545/113Y02A50/30
Inventor 蒋析文刘悦李瑞玲董子维朱小亚
Owner DAAN GENE CO LTD
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