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Method for detecting type-1 human immunodeficiency virus and special reagent set

A complete set of reagent technology, applied in biochemical equipment and methods, microbial determination/inspection, DNA/RNA fragments, etc., can solve the problems of not covering all HIV-1 subtypes, missed detection, etc.

Active Publication Date: 2017-11-24
北京旌准医疗科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there are some real-time fluorescence quantitative PCR kits for HIV-1 detection based on TaqMan probe technology on the market, but due to the large number of HIV genotypes, these kits cannot cover all subtypes of HIV-1, and there are often missed detections

Method used

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  • Method for detecting type-1 human immunodeficiency virus and special reagent set
  • Method for detecting type-1 human immunodeficiency virus and special reagent set
  • Method for detecting type-1 human immunodeficiency virus and special reagent set

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0077] Embodiment 1, the preparation of the kit reagent that detects HIV-1

[0078] 1. Preparation of RT-PCR reaction solution

[0079] The inventor of the present invention has been through a large number of experiments, by comparing and analyzing the nucleotide sequences of each subtype of HIV-1, and screening out a conservative sequence of 209bp in length (the nucleotide sequence after its transcription is as shown in the sequence listing shown in sequence 1). According to the conserved sequence, primer HIV-F, primer HIV-R, probe HIV-1 and internal standard detection probe were designed and synthesized. In the nucleotide sequences of the respective primers and probes, H=A / T / C, R=A / G, K=T / G, Y=C / T, M=A / C.

[0080]RT-PCR reaction solution consists of Tris-HCl buffer, KCl, dATP, dGTP, dCTP, dUTP, MgCl 2 , reverse transcriptase, Taq DNA polymerase, UNG enzyme, primer pair HIV-1 (composed of primer HIV-F and primer HIV-R), probe HIV-1 and internal standard detection probe.

...

Embodiment 2

[0112] Embodiment 2, establishment of the method for detecting HIV-1

[0113] 1. Extraction of total RNA from plasma to be tested

[0114] (1) Take the plasma to be tested, add 10 μL of internal standard, and mix well to obtain a mixed solution.

[0115] (2) Take the mixed solution, extract RNA according to the operation steps of the QIAamp Viral RNA Mini Kit kit, and obtain the total RNA of the plasma to be tested at a concentration of 10 ng / μL.

[0116] 2. Prepare reaction system

[0117] Reaction system A is 25 μL, consisting of 10 μL total RNA of plasma to be tested and 15 μL RT-PCR reaction solution.

[0118] Reaction system B (negative control) was 25 μL, consisting of 10 μL of normal plasma total RNA and 15 μL of RT-PCR reaction solution. The steps for preparing total RNA from normal plasma are as follows: take HIV-1 RNA-free normal human plasma, add 10 μL of internal standard, and mix well to obtain a mixed solution; take the mixed solution, and extract RNA accordin...

Embodiment 3

[0134] Embodiment 3, sensitivity experiment

[0135] 1. Prepare reaction system

[0136] Reaction system a is 25 μL, consisting of 10 μL RNA solution (RNA solution 1, RNA solution 2, RNA solution 3, RNA solution 4 or RNA solution 5) and 15 μL RT-PCR reaction solution.

[0137] Reaction system B is the same as reaction system B in step 2 in Example 2.

[0138] Reaction system C is the same as reaction system C of step 2 in embodiment 2.

[0139] The reaction system D is the same as the reaction system D of step 2 in Example 2.

[0140] 2. Real-time quantitative PCR detection

[0141] Same as Step 3 in Example 2.

[0142] 3. Result judgment

[0143] Same as Step 4 in Example 2.

[0144] HIV-1 detection curve see figure 1 (1 is RNA solution 1, 2 is RNA solution 2, 3 is RNA solution 3, 4 is RNA solution 4, 5 is RNA solution 5). The results show that the sensitivity of the set of reagents provided in Example 1 for detecting HIV-1 is 10 copies / reaction system.

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Abstract

The invention discloses a method for detecting a type-1 human immunodeficiency virus and a special reagent set. The kit comprises a primer pair HIV-1 and a probe HIV-1; the primer pair HIV-1 consists of a primer HIV-F and a primer HIV-R; the primer contains a specific RNA fragment for a target sequence in an HIV-1 genome; a nucleotide sequence of a specific DNA fragment obtained by virtue of reverse transcription of the specific RNA fragment is as shown in sequence 1 of a sequence list; and the probe HIV-1 is a single-chain DNA molecule consisting of 20 to 30 nucleotides and is same with partial sections in the specific DNA fragment. Experiments prove that when the reagent set is used for detecting HIV-1, the specificity is good, and the sensitivity is high (the sensitivity can reach 10 copies / reaction system). Meanwhile, an interior label in the kit is used as a competitive interior label of the target nucleic acid and can be used as a reference to prevent a false-negative result. The method has important application value.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method for detecting type 1 human immunodeficiency virus and a set of special reagents thereof. Background technique [0002] Human Immunodeficiency Virus (HIV), also known as HIV, is a lentivirus that can infect cells of the human immune system and belongs to a type of retrovirus. The virus can destroy or damage the function of the immune system, leading to progressive decline of the immune system, and finally unable to play a role in resisting infection and disease, leading to the emergence of various diseases and cancers in the human body, that is, Acquired Immune Deficiency Syndrome (AQS) Deficiency Syndrome, AIDS), also known as AIDS. [0003] HIV can be divided into two types phylogenetically, human immunodeficiency virus type 1 (HIV-1) and human immunodeficiency virus type 2 (HIV-2), both of which can cause AIDS. HIV-1, the first human retrovirus and associated with the va...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68C12N15/11
CPCC12Q1/686C12Q1/703C12Q2600/166C12Q2561/113C12Q2545/114
Inventor 乔阳余倩葛猛王宏伟
Owner 北京旌准医疗科技有限公司
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