Primers, probes and kits for detection and typing of 5 subtypes of Ebola virus by one-step reverse transcription PCR
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A technology of Ebola virus, reverse transcription, applied in the field of biology
Inactive Publication Date: 2015-12-02
YANGZHOU UNIV
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Problems solved by technology
But so far, there is still no RT-PCR method that can simultaneously detect and type all 5 Ebola virus subtypes
Method used
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preparation example Construction
[0064] 1) Preparation of FRET-PCR standard
[0065] Synthesized by GenScript (GenScript Biotechnology, Nanjing, China) carrying 5 subtypes of Ebola virus (Zaire type, Sudan type, Reston type, Tai Forest type and Bundibugyo type) A plasmid for the DNA sequence of interest. Based on the molecular weight and absolute weight of the composition, the absolute number of gene copies contained in the composition is calculated. Subsequently, the composite was diluted, and each 10 μl of the composite dilution reagent was prepared to contain 10,000 copies, 1,000 copies, 100 copies, 10 copies, and 1 copy of the target gene as a standard. Provide concentration in the kit of the present invention is 10 4 / μl of the plasmid standard.
[0066] 2) Preparation of reverse transcription PCR standard
[0067] After infecting chicken embryos with avian influenza virus (H1N1), the allantoic fluid was collected, and RNA nucleic acid was extracted with a commercial RNA extraction kit (HighPureRNAIs...
Embodiment
[0082] Embodiment: transcription method verification reverse transcription PCR system of the present invention
[0083] GenScript (GenScript Biotechnology, Nanjing, China) synthesized the plasmid PUC57 containing the Ebola virus Zaire target fragment DNA and the T7 promoter, and used appropriate restriction enzymes (SacI, Baobio, Dalian) ) to digest the plasmid; increase the concentration of the target DNA by PCR amplification; use a commercial transcription kit ( Kit, bylife The United States) transcribed the PCR amplification product and obtained the RNA product; after the transcription was completed, the transcription product was directly used for reverse transcription PCR to amplify.
[0084] 1) Dilution of plasmid
[0085] Centrifuge the microcentrifuge tube containing the synthetic plasmid at 4000 rpm for 2 minutes, then add 40 μl of double distilled water or TE buffer (PH8.0) to make a solution with a plasmid concentration of 100 ng / μl;
[0086] 2) Digestion of p...
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Abstract
The invention provides primers, probes and a reagent kit for one-step method reverse transcription PCR detection and classification of five Ebola virus subtypes. Nucleotide sequences of the primers and the probes are shown from the figure one to the figure eight. The reagent kit comprises a sample DNA template or quantitative standard reagent, a PCR buffer solution, the FRET-upstream primer, the five FRET-downstream primers, the reverse transcription PCR 6-FAM probe, the LCRed 640 probe, a commercialization Taq enzyme, dNTP, a commercialization reverse transcriptase and a commercialization RNA enzyme inhibitor. According to the primers, the probes and the reagent kit for one-step method reverse transcription PCR detection and classification of the five Ebola virus subtypes, the upstream primer, the pair of probes, the five downstream primers for the five Ebola virus subtypes are ingeniously designed according to conservative intervals in the five Ebola virus subtypes, and therefore the five Ebola virus subtypes can be detected at the same time.
Description
technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a primer, a probe and a kit for detecting and typing 5 subtypes of Ebola virus based on a conserved interval of Ebola virus. Background technique [0002] Ebola virus disease (Ebola virus disease, EVD) is caused by Ebola virus (Ebola virus, EBOV), an acute hemorrhagic infection with high infectivity and high mortality, shared by humans and non-human primates sick. It is named after the first outbreak along the Ebola River (Ebola) in the northern Democratic Republic of the Congo (formerly Zaire) in Africa in 1976. The main features of EVD are rapid fever, headache and muscle pain, followed by sore throat, nausea, vomiting, diarrhea and abdominal pain, and nearly half of the patients will have bleeding symptoms. The disease has a sudden onset, a short course, and a mortality rate as high as 30%-90%. It is a severe viral infectious disease. EVD is currently endemic, mainl...
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