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Quantitative detection kit for human immunodeficiency virus HIV-1

A technology for human immunodeficiency and HIV-1, which is applied in the field of diagnosis and can solve problems such as complex nucleic acid steps

Active Publication Date: 2015-11-25
FAPON BIOTECH INC +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Traditional HIV virus quantitative detection kits usually need to extract purified nucleic acid molecules (HIV-RNA) from samples, and the steps to extract nucleic acids from samples are complicated

Method used

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  • Quantitative detection kit for human immunodeficiency virus HIV-1
  • Quantitative detection kit for human immunodeficiency virus HIV-1
  • Quantitative detection kit for human immunodeficiency virus HIV-1

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0058] Embodiment 1 reaction system and reaction condition optimization

[0059] The optimization of the detection system for HIV-1 virus nucleic acid free nucleic acid extraction is shown in Table 1 below:

[0060] The reaction liquid configuration of the sample detection of table 1 HIV-1 virus nucleic acid

[0061]

[0062]

[0063] Among them, the optimized concentration of each component in the sample treatment agent is respectively: the concentration of guanidine isothiocyanate is 2mol / L, the concentration of sodium citrate (PH7.0) is 12mmol / L, sodium lauryl creatine The concentration of β-mercaptoethanol is 0.2%, the concentration of β-mercaptoethanol is 0.05mol / L, the concentration of proteinase K is 1mg / mL, and the concentration of HIV-1 internal standard is 5×10 2 copies / mL;

[0064] Among them, the optimized concentrations of each component in the 5×RT-PCR reaction buffer are:

[0065] The final concentration of Tris-HCl in the PCR reaction buffer solution i...

Embodiment 2

[0078] The minimum detectable amount of embodiment 2 kit

[0079] The HIV-1 positive serum calibrated with the lowest detection amount quantitative standard in the "HIV-1 RNA National Standard" of the National Institute of Inspection and Quarantine was used as the initial sample, and was serially diluted downwards to 1.0×10 7 IU / mL, 1.0×10 6 IU / mL, 1.0×10 5 IU / mL, 1.0×10 4 IU / mL, 1.0×10 3 IU / mL, 1.0×10 2 IU / mL, then use the linear minimum concentration template (1.0×10 2 IU / mL) as the first template of the lowest detection amount was diluted to 100IU / mL, 50IU / mL, 25IU / mL, and 10 parallel tubes were detected for each concentration of the template, and the experimental operation was described in Example 1 Consistent, the detection results take the lowest template concentration that can be fully detected as the lowest detection limit of the kit.

[0080] Test results such as Figure 2a , Figure 2b , Figure 2c and Figure 2d , where 2a indicates that the kit can detect...

Embodiment 3

[0082] The precision of embodiment 3 kits

[0083] The HIV-1 positive serum calibrated with the lowest detection amount quantitative standard in the "HIV-1 RNA National Standard" of the National Institute of Inspection and Quarantine was used as the initial sample, and was serially diluted downwards to 1.0×10 7 IU / mL, 1.0×10 5 IU / mL, 1.0×10 3 IU / mL, three concentrations of the template each detect 8 parallel tubes, the experimental operation is consistent with the description in Example 1, and the test results are calculated by calculating the coefficient of variation of the Ct value of each concentration <5% as the precision of the test kit index of.

[0084] Test results such as image 3 shown at 1.0 x 10 7 IU / mL, 1.0×10 5 IU / mL, 1.0×10 3 IU / mL respectively detected the Ct values ​​obtained by 8 parallel tubes to calculate the coefficient of variation, which were 0.7% (CP22.59), 0.76 (CP29.27), 0.88% (CP35.34), and the coefficients of variation were all <5%. It shows ...

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Abstract

The invention discloses a quantitative detection kit for human immunodeficiency virus HIV-1. The quantitative detection kit comprises sample treating agent, an upstream primer HIV-F used for target nucleotide amplification, a downstream primer HIV-R used for target nucleotide amplification, a Taqman probe HIV-P for detecting target nucleotide and RNA one-step reaction buffer, wherein the sample treating agent comprises guanidinium isothiocyanate, sodium citrate, dodecyl creatine sodium, beta-mercaptoethanol and proteinase K; a fluorescent group and fluorescence quencher are respectively combined to two ends of the Taqman probe HIV-P. When the quantitative detection kit is used for detecting, a to-be-detected sample is directly mixed with the sample treating agent and then is used for subsequent detection. Compared with a traditional quantitative detection kit for HIV virus, the quantitative detection kit has the advantages that the step of extracting and purifying nucleic acid molecules from the sample is omitted, and time-saving and simple operation is achieved.

Description

technical field [0001] The invention relates to the technical field of diagnosis, in particular to a quantitative detection kit for human immunodeficiency virus HIV-1. Background technique [0002] HIV infection can be determined by detecting virus antibodies, antigens, and nucleic acids. At present, the commonly used detection method is serological HIV antibody detection, which is the main indicator and standard detection item for diagnosing HIV infection and AIDS. [0003] The principle of HIV virus nucleic acid detection is to use the method of nucleic acid detection to directly detect the nucleic acid RNA of HIV virus. Nucleic acid detection has the advantages of fast, efficient, sensitive and specific. During the infection process, the first thing in the patient's body is viral RNA, which can be detected by nucleic acid detection method, and then IgM antibody appears, using the double antigen sandwich method After detection, IgG antibodies appear, which are detected b...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68C12R1/93
CPCC12Q1/6851C12Q1/703C12Q2527/127C12Q2531/113C12Q2561/101C12Q2545/101
Inventor 李泓彦邓艳华刘莉莉
Owner FAPON BIOTECH INC
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