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Fluorescent quantitative PCR (Polymerase Chain Reaction) kit for detecting Zaire Ebola virus

An Ebola virus, fluorescence quantitative technology, applied in the field of biochemistry, can solve the problems of long time, poor specificity, low sensitivity, etc., and achieve good specificity and high specificity

Inactive Publication Date: 2017-05-31
SOUTHERN MEDICAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] At present, the routine detection methods of Zaire Ebola virus in my country include serological reaction, nucleic acid hybridization, antigen-antibody ELISA and other technologies, but these tests or methods are cumbersome to operate and take a long time (about 2 days or more).
Existing Zaire-type Ebola virus detection technologies are still few, and most of them are unreliable, with low sensitivity and poor specificity
The nucleic acid detection method promoted in the past proved to have serious false negatives in the 2014 West African epidemic, indicating that the detection primers and probes for Zaire Ebola virus urgently need to be updated

Method used

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  • Fluorescent quantitative PCR (Polymerase Chain Reaction) kit for detecting Zaire Ebola virus
  • Fluorescent quantitative PCR (Polymerase Chain Reaction) kit for detecting Zaire Ebola virus
  • Fluorescent quantitative PCR (Polymerase Chain Reaction) kit for detecting Zaire Ebola virus

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Experimental program
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Effect test

Embodiment 1

[0033] Embodiment 1: (preparation kit)

[0034] 1. The composition of the fluorescent quantitative PCR kit for detection of Zaire Ebola virus:

[0035] Upstream primer: 5'-GAACCACATGATTGGACCAAGA-3', 10μM;

[0036] Downstream primer: 5'-TAACTCCAATACCTGCCGGTATC-3', 10μM;

[0037] Fluorescent probe: 5'FAM-TTGTTGATAAAACCCTTCCGGACCAGG-BHQ 3', 10μM;

[0038] Positive standard: pUC57 (Amp + ) recombinant plasmid, the concentration is 10 6 copies / μl.

[0039] Polymerase mixture: containing 5U / μl Taq DNA polymerase and 200U / μl reverse transcriptase, purchased from Beijing Kangrun Chengye Biotechnology Co., Ltd.;

[0040] PCR reaction buffer (5×): purchased from Beijing Kangrun Chengye Biotechnology Co., Ltd.;

[0041] The above nucleic acids were all synthesized by Sangon Bioengineering (Shanghai) Co., Ltd.

[0042] 2. The preparation method of the quantitative positive template is:

[0043] (1) According to the coding gene sequence of the Zaire Ebola virus envelope glycoprotei...

Embodiment 2

[0077] Embodiment 2: (in vitro detection experiment)

[0078] 1. Preparation of the template:

[0079] a. Reagents: RNA extraction kit, purchased from Axygen.

[0080] b. Cultivation and preparation of standard strains:

[0081] Positive standard: 10 6 copies / μl recombinant plasmid;

[0082] Samples to be tested: DH5ɑ engineering bacteria containing recombinant plasmids are used as positive samples, animal cell culture medium of type I dengue virus and Zika virus and DH5ɑ strains without recombinant plasmids are used as mock samples, and the bacteria do not need to extract RNA and use the whole genome as a template ;

[0083] Negative control: deionized water.

[0084] c. RNA extraction: take 200 μl of animal cell culture medium containing type I dengue virus and Zika virus, and extract viral RNA with an RNA extraction kit, with a final volume of 30 μl (for detailed steps, please refer to the kit instruction manual). The extracted RNA template was stored at -80°C for lat...

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Abstract

The invention relates to a fluorescent quantitative PCR (Polymerase Chain Reaction) kit for detecting a Zaire Ebola virus. The kit comprises primers, a TaqMan fluorescent probe and a positive standard substance. The fluorescent quantitative PCR kit is characterized in that: (1) the primers are a pair of primers for amplifying a highly conserved segment of a coding gene of a Zaire Ebola virus envelope glycoprotein, wherein an upstream primer is as shown in SEQ NO.1 and a downstream primer is as shown in SEQ NO.2; (2) a TaqMan fluorescent probe sequence is as shown in SEQ NO.3; and (3) the standard positive substance is a recombinant plasmid containing a segment DNA of the sequence as shown in SEQ NO.4. The kit has the advantages of good specificity and high sensitivity.

Description

technical field [0001] The invention relates to the field of biochemistry, in particular to a detection reagent for a determination or detection method comprising nucleic acid, enzyme or microorganism. Background technique [0002] Ebola virus is a filoviridae virus that can cause severe zoonotic infectious diseases. It can cause Ebola hemorrhagic fever. It has the characteristics of high pathogenicity and high fatality rate. One of the most serious viruses for humans, biosafety level 4 (BSL-4). [0003] At present, the routine detection methods of Zaire Ebola virus in my country include serological reaction, nucleic acid hybridization, antigen-antibody ELISA and other technologies, but these tests or methods are cumbersome to operate and take a long time (about 2 days or more). Existing Zaire-type Ebola virus detection technologies are still few, and most of them are unreliable, with low sensitivity and poor specificity. The nucleic acid detection method promoted in the p...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68
CPCC12Q1/686C12Q1/701C12Q2561/101C12Q2545/113
Inventor 万成松李以圣张宝赵卫张其威
Owner SOUTHERN MEDICAL UNIVERSITY
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