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Human immunodeficiency virus type 1 one-step fluorescence quantitative RT-PCR detection kit

A technology of human immunodeficiency and detection kits, which is applied in the direction of fluorescence/phosphorescence, microbial measurement/inspection, biochemical equipment and methods, etc. It can solve the problems of loss of re-amplification ability, etc., and achieve easy operation and high reliability , The effect of preventing multiple operation pollution

Inactive Publication Date: 2014-08-06
SHANGHAI XINGYAO MED TECH DEV CO LTD +1
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  • Claims
  • Application Information

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Problems solved by technology

[0009] At the same time, using the anti-pollution characteristics of UNG enzyme, dUTP is used instead of dTTP in the PCR amplification reaction, so that only the amplified fragment contains dUTP; and dUTP makes the polluted amplicon easy to be degraded by UNG enzyme before amplifying the target DNA: This dUTP-containing PCR product was incubated with UNG enzyme, because UDG enzyme can cleave the N-glycosyl bond between the uracil base and the sugar-phosphate backbone, and can eliminate dUTP from single-stranded or double-stranded DNA. Taq Elongation by DNA polymerase, thereby losing the ability to be reamplified

Method used

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  • Human immunodeficiency virus type 1 one-step fluorescence quantitative RT-PCR detection kit

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Embodiment 1

[0026] Example 1 A one-step fluorescent quantitative PCR detection kit for human immunodeficiency virus type 1.

[0027] 1. Extraction of Human Immunodeficiency Virus Type 1 (HIV-1) RNA

[0028] Nucleic acid was extracted from HIV-1 positive serum samples using column purification reagents (silica membrane adsorption method) from Shanghai Xingyao Medical Technology Development Co., Ltd.

[0029] 2. Reverse transcription and fluorescent quantitative PCR amplification (25ul system per person)

[0030] a. Preparation of one-step fluorescent quantitative RT-PCR reaction solution:

[0031]

[0032] b. One-step fluorescent quantitative RT-PCR reaction procedure:

[0033] [1] 37°C 10 min

[0034] [2] 50°C for 15 minutes

[0035] [3] 95℃ for 2 minutes

[0036] [4] 94°C 10s

[0037] [5] 60°C 45s

[0038] [6] Go to [4] ,45 cycles

[0039] In the fifth step, the fluorescence signals of FAM and JOE channels are collected,

[0040] [7] End.

[0041] 3. Detection:

[0042] ...

Embodiment 2

[0045] Example 2 clinical testing.

[0046] Using the above method to detect 130 clinical samples, including 32 cases of human immunodeficiency virus type 1 patients, the positive rate of detection was 24.6%, and the accuracy rate was 100%. Accurate quantitative analysis is far superior to traditional methods such as ELISA. The detection method and kit of the present invention have strong specificity, high sensitivity, simple operation and high repeatability, can perform rapid qualitative and quantitative detection of human immunodeficiency virus type 1 (HIV-1) RNA, and can replace the conventional Traditional ELISA diagnostic method. Nucleic acid was extracted from HIV-1 positive serum samples using column purification reagents (silica membrane adsorption method) from Shanghai Xingyao Medical Technology Development Co., Ltd. to ensure the purity of the template. At the same time, a one-step fluorescence quantitative RT-PCR technique was used. After the nucleic acid extr...

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Abstract

The present invention provides a human immunodeficiency virus type 1 one-step fluorescence quantitative RT-PCR detection kit, which comprises a RT-PCR reaction solution, a RT-PCR enzyme mixture, a primer probe, a negative control, a strong positive control, a weak positive control, and calibration substances No.1-5. According to the present invention, a one-step fluorescence quantitative reaction can be directly performed on the extracted HIV-1RNA, the virus loading of the HIV-1RNA in a sample can be detected, the reference gene is adopted as the internal control, and the UNG enzyme is adopted to prevent pollution; and the kit has characteristics of simple one-step amplification method, short procedure, easy operation, pollution prevention, strong detection result specificity, high sensitivity, clear result and high result reliability, and can be used for detection of the virus loading of the human immunodeficiency virus type 1 (HIV-1) RNA in blood serum.

Description

technical field [0001] The invention belongs to the field of biological technology, and relates to a biological detection technology, in particular to a fluorescent quantitative detection kit for human immunodeficiency virus type 1 (HIV-1) RNA. Background technique [0002] Human Immunodeficiency Virus (HIV) is a lentivirus that infects cells of the human immune system and belongs to a type of retrovirus. The virus destroys the immune function of the human body, leading to the loss of immune system resistance, which leads to the survival of various disease pathogens in the human body, and finally develops into AIDS (acquired immunodeficiency syndrome, Acquired Immunodeficiency Syndrome, AIDS), It is a deadly infectious disease for which there is no effective treatment so far. Since it was first discovered and identified in the United States in 1981, AIDS has claimed the lives of more than 30 million people, making it one of the most devastating global epidemics in history, ...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68G01N21/64
CPCC12Q1/70C12Q1/6848C12Q1/6851
Inventor 迟大利吴大治夏懿
Owner SHANGHAI XINGYAO MED TECH DEV CO LTD
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