Fluorescent quantitative PCR (polymerase chain reaction) method, primer and kit for detecting EBOV (Ebola virus)

An Ebola virus, fluorescence quantitative technology, applied in microorganism-based methods, biochemical equipment and methods, microorganisms, etc., can solve the problem of no patent publication of EBOV detection technology, achieve stable source, increase safety, and save time Effect

Inactive Publication Date: 2013-04-17
SHANGHAI VETERINARY RES INST CHINESE ACAD OF AGRI SCI
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] After searching the literature of the existing technology, it was fou

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  • Fluorescent quantitative PCR (polymerase chain reaction) method, primer and kit for detecting EBOV (Ebola virus)
  • Fluorescent quantitative PCR (polymerase chain reaction) method, primer and kit for detecting EBOV (Ebola virus)
  • Fluorescent quantitative PCR (polymerase chain reaction) method, primer and kit for detecting EBOV (Ebola virus)

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0058] Example 1 Primer Design

[0059] 1. Experimental steps

[0060] EBOV SYBR Green I 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 viruses 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 (reference strain information is shown in Table 1), the primer and probe design software Primer Express 2.0 (Applied Biosystems, USA) was used to design a pair of Z, S, B, C, R 5 subtype specific primers of EBOV. The primer sequences are shown in Table 2, and the primer gene positions in Table 2 refer to the positions on the gene of Zaire Ebola virus strain Mayinga (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. Stra...

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 (marburgvirus, MARV), dengue virus (dengue virus, DHFV), Xinjiang hemorrhagic fever virus (xinjiang hemorrhagic fever virus, XHFV) (see Table 3 for reference strains), according to The sequence indicated by the accession number was completely artificially synthesized by Nanjing Jinsite Technology Co., Ltd. for their full-length NP gene cDNA. 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. Design synthetic primers according to Example 1 (see Table 1), extend outwards to 273nt respectively along the primer amplification region of the EBOV NP gene sequence, design upstream primers and downstream primers connected to the T7 promoter sequence, and the ...

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 and kidney), put it into a grinder, add 1000 μL of DEPC water, and grind. 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 in a 1.5 mL sterilized centrifuge tube, add 1000 μL Trizol, mix well, Let stand 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 DNase and RNas...

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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 SYBR Green I fluorescent quantitative PCR. Background technique [0002] Ebola hemorrhagic fever (EHF), caused by Ebola virus (EBOV), is an acute hemorrhagic infectious disease mainly in humans and non-human primates as susceptible animals. 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 infected with the vir...

Claims

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Application Information

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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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