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A fluorescent quantitative PCR detection method for detecting Ebola virus and its primers and kit

A technology of Ebola virus and kit, which is applied in the field of Ebola virus SYBRGreenI fluorescence quantitative PCR detection, can solve the problems of no EBOV detection technology patent announcement, and achieve the effects of stable source, time saving and high sensitivity

Inactive Publication Date: 2016-06-08
SHANGHAI VETERINARY RES INST CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] After searching the literature of the existing technology, it was found that there is no patent publication of EBOV detection technology in China

Method used

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  • A fluorescent quantitative PCR detection method for detecting Ebola virus and its primers and kit
  • A fluorescent quantitative PCR detection method for detecting Ebola virus and its primers and kit
  • A fluorescent quantitative PCR detection method for detecting Ebola virus and its primers and kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0058] Example 1 Primer Design

[0059] 1. Experimental steps

[0060] EBOVSYBRGreenI fluorescent quantitative PCR primers refer to: oligonucleotide chains with a length of 25±5nt, which are completely identical or complementary to the NP gene sequences of the five subtypes of EBOV, Z, S, B, C, and R.

[0061] According to the NP gene sequences of EBOV, MARV, XFHV, and DHFV published in the NCBI gene database (see Table 1 for reference strain information), the primer and probe design software PrimerExpress2. S, B, C, R 5 subtype specific primers of EBOV. The primer sequence is shown in Table 2, and the primer gene position in Table 2 refers to the position on the gene of ZaireEbolavirusstrainMayinga (NCBI accession number AF499101.1). The results of comparing the designed primers with the corresponding gene sequences of EBOV, MARV, XFHV, and DHFV are shown in figure 1 .

[0062] Table 1. Strain information for designing primers and probe references

[0063]

[0064] ...

Embodiment 2

[0069] The preparation of embodiment 2RNA positive standard molecule and negative standard molecule

[0070] 1. Experimental steps

[0071] 1. Five subtypes of EBOV, as well as Marburg virus (MARV), dengue virus (DHFV), Xinjiang hemorrhagic fever virus (XHFV) (see Table 3 for reference strains), according to the accession numbers The sequence of their full-length NP gene cDNA was completely artificially synthesized by Nanjing Jinsite Technology Co., Ltd. MARV, XHFV, and DHFV were used as negative standard molecules.

[0072] Table 3. The strain information of artificially synthesized full-length NP gene cDNA reference

[0073]

[0074] 2. According to embodiment 1, design the synthetic primers (see Table 1), extend outwards to 273nt respectively along the EBOVNP gene sequence primer amplification region, design upstream primers and downstream primers connected to the T7 promoter sequence, and the primer sequences are shown in Table 4. The primer sequence is located on th...

Embodiment 3

[0084] The extraction of embodiment 3 sample RNA

[0085] 1. Experimental steps

[0086] Tissue sample processing: Take about 100 mg of the tissue sample to be tested (such as liver, kidney) and put it into a grinder and add 1000 μL DEPC water for grinding. Take 100 μL of the supernatant of the tissue to be tested that has been ground, put it in a 1.5 mL sterilized centrifuge tube, add 1000 μL Trizol, mix well, and let it stand for 10 min.

[0087] Liquid sample processing: Take 100 μL of the liquid sample to be tested (such as blood, physiological saline dilution of nasal swab, and physiological saline dilution of respiratory secretions), put it into a 1.5mL sterilized centrifuge tube, add 1000 μL Trizol, mix well, and statically Set for 10min. Add 200μL of chloroform, shake vigorously for 15s, let stand at room temperature for 2-3min, and centrifuge at 12000g for 15min at 4°C. Carefully pipette 450 μL of the supernatant into another clean 1.5 mL centrifuge tube free of DN...

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Abstract

The invention discloses a fluorescent quantitative PCR (polymerase chain reaction) method, primer and kit for detecting the EBOV (Ebola virus). The general method can be used for detecting that the sample to be detected is positive as long as the sample contains one or more of the five types of subtype EBOVs which are Z, S, B, C and R at the same time. The method overcomes the defects of the conventional PCR method for detecting by adopting the advantages of high-efficiency nucleic acid amplification of the PCR technology and the sensitivity of the fluorescence-dye SYBR Green I and the computer-aided fluorescent technology for detecting and improves the detection sensitivity, specificity and operation convenience greatly. In addition, the positive control adopted by the method is a section of RNA molecules transcribed in vitro of a NP gene, and the method is safer than the method for detecting by taking the inactivated virus solution as the positive control. The RNA molecules transcribed in vitro can be prepared in quantity, and the sources of the positive control are stable and reliable.

Description

technical field [0001] The invention belongs to the field of biological detection, in particular to a method for detecting Ebola virus SYBRGreenI fluorescent quantitative PCR. Background technique [0002] Ebola hemorrhagic fever (Ebola hemorrhagic fever, EHF) is caused by Ebola virus (Ebola virus, EBOV), mainly in humans and non-human primates as an acute hemorrhagic infectious disease. The disease first occurred in the Democratic Republic of the Congo (formerly Zaire) in the Ebola River Basin in 1976. It was named Ebola hemorrhagic fever because the infected person showed bleeding symptoms all over the body. It is speculated that the initial human infection with EBOV was mainly due to human contact with the body of an animal host or an indirect host infected with the virus. This is followed by person-to-person transmission through direct contact with the body of someone who has already been infected with the virus. After an incubation period of 2-21 days, patients infect...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/70C12Q1/68C12N15/11C12R1/93
Inventor 马志永史子学魏建超邵东华王少辉李蓓蓓刘阳王宗尧
Owner SHANGHAI VETERINARY RES INST CHINESE ACAD OF AGRI SCI
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