Nucleic acid, kit and method for simultaneously detecting three viruses of Congo fever virus, Copo fever virus type 1 and 2

A technology of Kupo heat and Congo heat, applied in biochemical equipment and methods, microbiological determination/inspection, DNA/RNA fragments, etc., can solve problems such as laborious, high cost, and time-consuming, and achieve rapid screening and low cost Low, good detection effect

Active Publication Date: 2018-06-29
淮安市疾病预防控制中心
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At present, there is no effective and quick way to distinguish which type of Kupo fever virus or Congo fever virus the pathogen causing fever m

Method used

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  • Nucleic acid, kit and method for simultaneously detecting three viruses of Congo fever virus, Copo fever virus type 1 and 2
  • Nucleic acid, kit and method for simultaneously detecting three viruses of Congo fever virus, Copo fever virus type 1 and 2
  • Nucleic acid, kit and method for simultaneously detecting three viruses of Congo fever virus, Copo fever virus type 1 and 2

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0053] The design of embodiment 1 primer, probe

[0054] Fluorescent quantitative PCR detection is based on ordinary PCR detection, further through a specific fluorescent probe, the probe is an oligonucleotide, and the two ends are respectively labeled with a reporter fluorescent group and a quencher fluorescent group. When the probe is intact, the fluorescent signal emitted by the reporter group is absorbed by the quencher group; during PCR amplification, the 5'-3' exonuclease activity of Taq enzyme degrades the probe, so that the reporter fluorescent group and the quencher group The fluorescent group is separated, so that the fluorescence monitoring system can receive the fluorescent signal, that is, every time an RNA strand is amplified, a fluorescent molecule is formed, and the accumulation of the fluorescent signal is completely synchronized with the formation of the PCR product. Therefore, the premise of fluorescent quantitative PCR detection is to carry out PCR amplific...

Embodiment 2

[0081] Real-time fluorescent PCR detection kit of embodiment 2 Congo fever virus, Kupo fever virus type 1 and Kupo fever virus type 2

[0082] The fluorescent quantitative PCR detection kit for the detection of Congo fever virus, Kupo virus type 1 and Kupo fever virus type 2 consists of the following components:

[0083] 2×RT-PCR buffer;

[0084] 25×RT-PCR Enzyme Mix

[0085] The upstream primer sequence of Congo fever virus is shown in SEQ ID No.1, and the downstream primer sequence is shown in SEQ ID No.2, and the probe sequence is shown in SEQ ID No.3, and the 5' mark of the probe SEQ ID No.3 TEXRED, 3' tagged BHQ2;

[0086] The upstream primer sequence of Kupovirus type 1 is shown in SEQ ID No.4, SEQ ID No.4, the downstream primer sequence is shown in SEQ ID No.5, and the probe sequence is shown in SEQ ID No.6. Wherein, the probe 5' mark FAM of SEQ ID No.6, 3' mark TAMRA;

[0087] The upstream primer sequence of Kupovirus type 2 is shown in SEQ ID No.7, the downstream ...

Embodiment 3

[0095] Embodiment 3 simultaneously detects the method for three kinds of virus fluorescent quantitative PCR

[0096] The method for simultaneously detecting three viruses of Congo fever virus, Kupo fever virus type 1 and Kupo fever virus type 2 by fluorescent quantitative PCR comprises the following steps:

[0097] (1) Pathogen RNA extraction

[0098] Roche's viral RNA kit can be used for extraction.

[0099] (2) Fluorescent quantitative PCR amplification

[0100] The mixed design of upstream and downstream primers and probes of Congo fever virus, Kupo fever virus type 1 and type 2, take equal volumes of primers and mix to form primer MIX, the final concentration of each primer is 3.3μmol / L, each take equal volumes The probes were mixed to form a probe MIX, and the final concentration of each probe was 1.0 μmol / L.

[0101] Using AgPath-ID One-step RT-PCR of ABI Company, 25 μL reaction system: primer MIX 2.0 μL; probe MIX 1.0 μL; 2×RT-PCR buffer 12.5 μL; 25×RT-PCR Enzyme Mix...

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Abstract

The invention relates to a set of nucleic acids, a kit and a detection method for simultaneously detecting three viruses of Congo fever virus, Copo fever virus type 1 and Copo fever virus type 2. Thenucleic acids for simultaneously detecting three viruses by multiplex PCR comprise upstream and downstream primers of the three viruses of Congo fever virus, Copo fever virus type 1 and Copo fever virus type 2; nucleic acids for simultaneously detecting the three viruses by fluorogenic quantitative PCR comprise probes corresponding to each pathogen. The invention also establishes a relatively high-efficiency and sensitive detection method for simultaneously detecting the three viruses of Congo fever virus, Copo fever virus type 1 and Copo fever virus type 2. The method can effectively improvethe sensitivity of detection and avoid the occurrence of false negative results, and provides an effective detection method for effectively preventing hemorrhagic fever of an input virus.

Description

technical field [0001] The invention belongs to the technical field of biological detection, and in particular relates to a nucleic acid, a kit and a method for simultaneously detecting Congo fever virus, Kupo fever virus type 1 and Kupo fever virus type 2. Background technique [0002] Shanghai Inspection and Quarantine Bureau's Health Quarantine Central Laboratory conducted overnight tests on persons suspected of being infected with yellow fever from Angola, and worked with Guangdong Bureau's P3 laboratory to rule out yellow fever virus, Rift Valley fever virus, chikungunya virus, dengue virus, Plasmodium and other insect-borne pathogens, influenza A and B viruses, hepatitis viruses and other pathogens. In order to further identify the cause of the fever, the Shanghai Entry-Exit Inspection and Quarantine Bureau and the Chinese Academy of Military Medical Sciences used metagenomic sequencing technology to detect and analyze a sequence that was as high as 96.5% homologous to...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/6851C12N15/11
CPCC12Q1/6851C12Q1/701C12Q2600/16C12Q2531/113C12Q2537/143C12Q2563/107
Inventor 燕清丽杨鹏飞侯海燕李兵兵唐丽赵怀荣姚海波何南江
Owner 淮安市疾病预防控制中心
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