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Ebola virus typing detection primer, probe and kit

A technology for Ebola virus detection kits, applied in biochemical equipment and methods, microbe measurement/testing, DNA/RNA fragments, etc., can solve the problems of immune cross-reaction cycle, low sensitivity, false positive, etc., and achieve Small amount of sample, high sensitivity and high accuracy

Pending Publication Date: 2018-06-15
TECH CENT OF GUANGZHOU CUSTOMS
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, antigen-antibody reaction-based detection and virus isolation and culture have disadvantages such as low sensitivity, immune cross-reaction, and long cycle; conventional RT-PCR detection is prone to contamination, resulting in false positive results

Method used

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  • Ebola virus typing detection primer, probe and kit
  • Ebola virus typing detection primer, probe and kit
  • Ebola virus typing detection primer, probe and kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] An embodiment of the Ebola virus typing detection kit of the present invention comprises the following components:

[0025] (1) Detection primers and probes for five subtypes of Ebola virus

[0026] The kit of this embodiment includes the detection of Zaire-type Ebola virus, Sudan-type Ebola virus, Côte d'Ivoire-type Ebola virus, Bundibugyo-type Ebola virus and Reston-type Ebola virus Primers and Probes. The detection primers include upstream primers and downstream primers, the 5' end of the detection probe contains a fluorescent group, and the 3' end contains a quenching group. The detection primer sequences for the five subtypes of Ebola virus are shown in Table 1, and the detection probe sequences are shown in Table 2. The five probes have different fluorescent groups, and can be carried out simultaneously in the same reaction tube for 5 times. Types of Ebola virus. Specific primer and probe information is as follows:

[0027] Table 1 Detection primer sequences f...

Embodiment 2

[0040] In this embodiment, the Ebola virus typing detection kit in Example 1 is used to detect the simulated samples of known Ebola virus types.

[0041] 1. Detection method

[0042] (1) Collect 20 simulated samples of confirmed Ebola virus infection types, and use a commercially available viral RNA extraction kit to extract the viral RNA in the samples, and the operation steps are strictly in accordance with the instructions;

[0043] (2) Prepare 25ul reaction system according to Table 3:

[0044] Table 3 Reaction system

[0045] Element

Volume (ul / test)

DEPC H 2 o

13.7

10×Buffer

2.5

MgCl 2 (25mM)

2

dNTPs

0.5

primer mix

0.2

probe mix

0.1

RT-PCR enzyme

1

RNA template

5

[0046] During the preparation process, the components other than the RNA template were mixed evenly, distributed into each reaction well, and then the corresponding RNA template was added to each reaction we...

Embodiment 3

[0057] The detection primers and probes of the present invention are obtained by the inventor through a large number of experiments and research analysis. The detection primers and probes between various types will not cross-react. In the entire detection system, between primers, between primers and No secondary structure is formed between the probes or between the probes, which ensures the detection efficiency and the accuracy of the detection results, and has good specificity and sensitivity.

[0058] In this example, the detection effect of the detection primers and probes of the present invention is studied, and the detection primers and probes are designed using conventional primer design software, and 2 pairs of them are selected to compare the detection effects with the detection primers and probes of the present invention .

[0059] 1. Experimental design

[0060] Design experimental group 1 and control group 1~10, wherein experimental group 1 adopts detection primer ...

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Abstract

The invention discloses an ebola virus typing detection primer and probe. The ebola virus typing detection primer and probe comprise a detection primer and probe of a Zaire ebola virus, a detection primer and probe of a Sudan ebola virus, a detection primer and probe of a Cote d'Ivoire ebola virus, a detection primer and probe of a Bundibugyo ebola virus and a detection primer and probe of a Reston ebola virus. Meanwhile, the invention further provides a kit comprising the typing detection primer and probe. The ebola virus typing detection primer and probe, provided by the invention, have theadvantages of good specificity, high sensitivity and the like, and can be used for rapidly carrying out typing detection on five types of ebola viruses in the same detection system; the ebola virus detection primer and probe of each subtype do not have a cross reaction, and secondary structures are not formed between the primers, between the primer and the probe and between the probes, so that theaccuracy of detection efficiency and a detection result is ensured.

Description

technical field [0001] The invention belongs to the technical field of nucleic acid detection, and in particular relates to a primer, a probe and a kit for Ebola virus typing detection. Background technique [0002] Ebola virus (EBOV) belongs to the family Filoviridae and belongs to the genus Filovirus. It is a non-segmented single-stranded negative-sense RNA virus. It first occurred in the Ebola River Basin in the Democratic Republic of Congo (formerly Zaire) in 1976. It was named Ebola hemorrhagic fever because it caused systemic bleeding symptoms in patients. The World Health Organization (WHO) has listed it as one of the most serious infectious diseases for humans. Ebola virus is a virus that seriously threatens human life. It is mainly transmitted through the blood and excrement of patients. The main clinical manifestations are acute onset fever, myalgia, bleeding rash, and liver and kidney function damage. The mortality rate is as high as 50%. to 90%. The Ebola viru...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/686C12N15/11
CPCC12Q1/686C12Q1/701C12Q2563/107C12Q2545/113
Inventor 师永霞黄吉城戴俊袁帅孙芳芳黄树祥郑夔李小波黎东红
Owner TECH CENT OF GUANGZHOU CUSTOMS
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