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HIV-1 viral load real-time fluorescence quantitative PCR detection specific primer pair and kit

A real-time fluorescence quantification, HIV-1 technology, applied in microbial determination/inspection, biochemical equipment and methods, DNA/RNA fragments, etc., can solve the problem that primers are difficult to be widely used and unsuitable for detection of HIV-1 infected persons. , to achieve the effect of shortening detection time, accurate viral load level, and reducing pollution

Inactive Publication Date: 2020-09-25
BEIJING LIANGXIN BIOLOGICAL TECH DEV CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] Although the sequences of each subtype of HIV-1 virus have been published, the sequence changes are still very large for individuals infecting humans. Therefore, it is difficult to design primers based on the conserved sequences of HIV-1 viruses obtained from published data. Widely applicable, especially not suitable for detection of HIV-1 infection unique to China

Method used

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  • HIV-1 viral load real-time fluorescence quantitative PCR detection specific primer pair and kit
  • HIV-1 viral load real-time fluorescence quantitative PCR detection specific primer pair and kit
  • HIV-1 viral load real-time fluorescence quantitative PCR detection specific primer pair and kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0055] The RNA extracted from 16 HIV-1 positive serum samples was detected by using the human immunodeficiency virus type Ⅰ one-step fluorescent quantitative PCR detection kit.

[0056] Real-time fluorescence quantitative PCR was performed using Applied Biosystems QuantStudio5 Real-Time PCR Systems, and the fluorescence signal of the FAM channel was collected at 72°C, and the melting curve step was performed after the cycle process was completed.

[0057] After the RT-PCR program of the instrument is completed, save the results and analyze the data according to the requirements of the instrument and software. Taking the fluorescence value higher than the sample noise line and the negative control as the detection threshold, the analysis software automatically combined the two standard curves to calculate the HIV-1 RNA content C (copies / mL) of each sample extract, and took the average as the final result. Then analyze the melting curve to ensure that there is no primer-dimer in...

Embodiment 2

[0065] Embodiment 2, to the detection result of various subtype HIV-1

[0066] The samples were subjected to first-generation sequencing and the resulting sequences were submitted to the Stanford University HIV Drug Resistance Database (https: / / hivdb.stanford.edu / hivdb) for subtype analysis. Afterwards, the viral load of each sample was detected using the kit provided by the present invention (the method and procedure are the same as in Example 1), and the results are as follows.

[0067] (1) Sample number R16, sequencing result (SEQ ID No: 5).

[0068] HIV-1 subtype is B; amplification result; Ct=26.681

[0069] (2) Sample number T1516, sequencing result (SEQ ID No: 6).

[0070] HIV-1 subtype is CRF01_AE; amplification result: Ct=23.259

[0071] (3) Sample number: Y4, sequencing result (SEQ ID No: 7).

[0072] HIV-1 subtype is CRF07_BC; amplification result: Ct=27.806

[0073] (4) Sample number: Y10, sequencing result (SEQ ID No: 8).

[0074] HIV-1 subtype is B+C; ampli...

Embodiment 3

[0076] Embodiment 3, sensitivity and stability verification test:

[0077] 1. Sensitivity verification test: The template DNA was serially diluted with a 10-fold gradient, starting from 5.92×10 3 ~5.92×10 9 In the copies / mL interval, 5 μL of each order of magnitude dilution was taken as the amplification template. The test conditions are the same as in Example 1. The result shows: obvious amplification curve can be seen at Ct=30 place (see Image 6 ), demonstrating that the lowest dilution that can be detected is 3 copies / mL (approximately equivalent to the minimum detection limit of 30 DNA copies per reaction), with good sensitivity, each reaction can detect a minimum of 30 copies of DNA.

[0078] 2. Stability verification test: Three sets of primers and standard products provided by the present invention are divided into three groups at one time, and stored at -20°C. On the 0th, 3rd, and 6th day, carry out RT-PCR amplification test respectively (test method and program a...

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Abstract

The invention relates to the field of biomolecular detection, in particular to a HIV-1 viral load real-time fluorescence quantitative PCR detection specific primer pair and kit. The specific primer pair is used for precisely, simply and conveniently quantitative detection of human immunodeficiency virus type I (HIV-1) RNA. A one-step fluorescence quantitative RT-PCR technology is used to detect HIV-1 viral load. The operation ensures the accuracy and sensitivity of amplification results, the time is shortened, the pollution is reduced, the simplicity and convenience of a fluorescence quantitative PCR detection method are improved, and the HIV-1 viral load real-time fluorescence quantitative PCR detection specific primer pair and kit can be used for the quantitative detection of HIV-1 and as a clinical auxiliary diagnosis method on HIV-1 infection and a monitoring method of clinical treatment effect.

Description

technical field [0001] The invention relates to the field of biomolecular detection, in particular to a pair of specific primers and a kit for real-time fluorescent quantitative PCR detection of HIV-1 viral load. Background technique [0002] Human Immunodeficiency Virus (HIV) is the main pathogen that induces human acquired immunodeficiency syndrome (Acquired Immunodeficiency Syndrome, AIDS), and belongs to the genus Lentivirus of the family Retroviridae in taxonomy. According to the genotype, HIV can be divided into two types: HIV-1 and HIV-2. HIV-1 has four different groups (M, N, O, and P), and each group is caused by independent cross-species transmission. HIV-1 has higher virulence and is easier to spread, but it also causes the vast majority of HIV infections in the world. [0003] Subtype C accounts for 46.6% of all HIV-1 infections worldwide. Subtype B caused 12.1% of infections, followed by subtype A, CRF02_AG, CRF01_AE, subtype G, subtype D. F, H, J, K subtype...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/6851C12N15/11
CPCC12Q1/703C12Q1/6851C12Q2531/113C12Q2563/107C12Q2561/113
Inventor 于斌姜淼
Owner BEIJING LIANGXIN BIOLOGICAL TECH DEV CO LTD
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