One-step process real-time fluorescent quantitative RT-PCR (Reverse Transcription-Polymerase Chain Reaction) method and kit for detecting Z/S subtype ebola viruses

A real-time fluorescence quantitative, Ebola virus technology, applied in fluorescence/phosphorescence, biochemical equipment and methods, DNA/RNA fragments, etc., can solve the problem of no patent publication of EBOV detection technology, achieve high specificity and save time , the effect of increasing safety

Inactive Publication Date: 2013-04-17
SHANGHAI VETERINARY RES INST CHINESE ACAD OF AGRI SCI
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  • 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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  • One-step process real-time fluorescent quantitative RT-PCR (Reverse Transcription-Polymerase Chain Reaction) method and kit for detecting Z/S subtype ebola viruses
  • One-step process real-time fluorescent quantitative RT-PCR (Reverse Transcription-Polymerase Chain Reaction) method and kit for detecting Z/S subtype ebola viruses
  • One-step process real-time fluorescent quantitative RT-PCR (Reverse Transcription-Polymerase Chain Reaction) method and kit for detecting Z/S subtype ebola viruses

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0059] Embodiment 1 Primer and probe design

[0060] 1. Experimental steps

[0061] Ebola virus fluorescent quantitative RT-PCR primers refer to: oligonucleotide chains with a length of 25 ± 5 nt, which are completely identical or complementary to the sequence of the NP gene shared by EBOV Z and S subtype viruses. Ebola virus fluorescent quantitative RT-PCR probe refers to: an oligonucleotide chain with a length of 18±5nt, its 5' end is marked with a fluorescent excitation group, and its 3' end is marked with MGB (minor groove binder); It is completely identical or complementary to the sequence of the NP gene shared by EBOVZ and S subtype viruses.

[0062] According to the EBOV, MARV, XFHV, and DHFV NP gene sequences published in the NCBI gene database (see Table 1 for reference strain information), primers and probes were designed using Primer Express 2.0 (Applied Biosystems, Inc.) for Z, S Subtype EBOV specific fluorescent quantitative PCR primers and probes. The primer a...

Embodiment 2

[0070] Example 2 Preparation of RNA Positive Standard Molecules and Negative Standard Molecules

[0071] 1. Experimental steps

[0072] 1. Five subtypes of EBOV, Marburg virus (MARV), dengue virus (DHFV), Xinjiang hemorrhagic fever virus (XHFV) (see Table 3 for reference strains), according to the registration The sequence indicated by No., 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.

[0073] Table 3. The strain information of artificially synthesized full-length NP gene cDNA reference

[0074]

[0075] 2. Design synthetic primers (see Table 1) according to Example 1, extend outwards to 273nt along the primer amplification region on the EBOV NP gene sequence, and design upstream primers and downstream primers connected to the T7 promoter sequence. The primer sequences are listed in Table 4. The primer sequence is located on the Zaire Ebola vi...

Embodiment 3

[0085] The extraction of embodiment 3 sample RNA

[0086] 1. Experimental steps

[0087] Tissue sample processing: take about 100 mg of the tissue sample to be tested (such as liver, kidney), put it in a grinder, add 1000 μL DEPC water and grind it. 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.

[0088] 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 200 μL of chloroform, shake vigorously for 15 seconds, let stand at room temperature for 2-3 minutes, and centrifuge at 12,000 g for 15 minutes at 4°C. Carefully pipette 450 μL of the supernatant into another clean 1.5 mL centrifuge tube free of ...

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Abstract

The invention discloses a one-step process real-time fluorescent quantitative RT-PCR (Reverse Transcription-Polymerase Chain Reaction) method and kit as well as a primer and a probe for detecting Z / S subtype ebola viruses (EBOV). The one-step process real-time fluorescent quantitative RT-PCR method is a general detection method; and the PCR detection process can be used for detecting Z and S subtype EBOVs after being carried out once. A sample is positive as long as any one of the Z and S subtype EBOVs or both the Z and S subtype EBOVs exist in the sample to be detected. The one-step process MGB (Minor Groove Binder) probe fluorescent quantitative RT-PCR technology provided by the invention combines the advantages of efficient amplification of nucleic acids in a PCR technology and sensitivity of a MGB probe and a computer-assisted fluorescence detection technology, overcomes the shortcomings of conventional PCR detection and greatly increases detection sensitivity, specificity and convenience of operation.

Description

technical field [0001] The invention belongs to the field of biological detection, in particular to a real-time fluorescent quantitative RT-PCR method for detecting Z / S subtype Ebola virus in one step, and a kit, primers and probes thereof. 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...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68C12N15/11G01N21/64
Inventor 马志永史子学魏建超邵东华王少辉李蓓蓓刘阳
Owner SHANGHAI VETERINARY RES INST CHINESE ACAD OF AGRI SCI
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