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Characteristic miRNAs in Ebola virus infected blood and application of characteristic miRNAs

A blood, viral load technology, used in the field of biomedicine

Inactive Publication Date: 2017-05-10
MICROBE EPIDEMIC DISEASE INST OF PLA MILITARY MEDICAL ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] At present, many studies on virus pathogenicity are carried out in animal models and in vitro experiments, but there are few related studies on human virus infection

Method used

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  • Characteristic miRNAs in Ebola virus infected blood and application of characteristic miRNAs
  • Characteristic miRNAs in Ebola virus infected blood and application of characteristic miRNAs
  • Characteristic miRNAs in Ebola virus infected blood and application of characteristic miRNAs

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Example 1. Molecular markers related to viral load in the blood of EBOV-infected patients

[0038] Extract total RNA from the whole blood of EBOV-infected patients and non-EBOV-infected patients, use magnetic beads with Oligo(dT) to absorb and purify the mRNA in the total RNA, fragment the mRNA under heating conditions, and use this as a template for inversion Recorded into double-stranded cDNA. The double-stranded cDNA is repaired and filled to phosphorylate the 5' end and add "A" to the 3' end. The double-stranded cDNA adapter with 3'dTMP end was connected to the sequencing adapter, amplified by PCR, enriched and purified with AMPure XP magnetic beads, and the library was identified by PCR reaction. The constructed library was subjected to paired-end sequencing using the Illumina sequencing platform. Use the Perl script to filter adapter sequences, low-quality sequences at both ends (sequences with Q<=20 bases accounting for more than 50% of the entire reads), and l...

Embodiment 2

[0045] Embodiment 2, small RNAs (sRNAs) associated with viral load in the blood of EBOV-infected patients

[0046] Extract total RNA from the whole blood of EBOV-infected patients and non-EBOV-infected patients, use 15% denatured polyacrylamide gel electrophoresis (PAGE) to separate small fragments (15-30nt) RNA from total RNA, and isolate small fragments after purification The fragmented RNA was ligated with adapters at the 3' and 5' ends, and then the RNA with the adapters was reverse-transcribed using SuperScriptII reverse transcriptase (product of Invitrogen) to synthesize cDNA. Then, PCR amplification was carried out, and the amplification program was: pre-denaturation at 98°C for 30s, denaturation at 98°C for 10s, annealing at 60°C for 30s, extension at 72°C for 15s, 14 cycles, and extension at 72°C for 8min, thereby constructing the library. Finally, the extended products were subjected to high-throughput sequencing using the Illumina sequencing platform. Use the Perl ...

Embodiment 3

[0052] Example 3. Molecular markers related to clinical prognosis in the blood of EBOV-infected patients

[0053] Extract total RNA from the whole blood of EBOV-infected patients and non-EBOV-infected patients, use magnetic beads with Oligo(dT) to absorb and purify the mRNA in the total RNA, fragment the mRNA under heating conditions, and use this as a template for inversion Recorded into double-stranded cDNA. The double-stranded cDNA is repaired and filled to phosphorylate the 5' end and add "A" to the 3' end. The double-stranded cDNA adapter with 3'dTMP end was connected to the sequencing adapter, amplified by PCR, enriched and purified with AMPure XP magnetic beads, and the library was identified by PCR reaction. The constructed library was subjected to paired-end sequencing using the Illumina sequencing platform. Use the Perl script to filter adapter sequences, low-quality sequences at both ends (sequences with Q<=20 bases accounting for more than 50% of the entire reads...

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Abstract

The invention discloses characteristic miRNAs in Ebola virus infected blood and an application of the characteristic miRNAs. The miRNAs in blood related to Ebola virus infection provided by the invention include hsa miR 151a 5p, hsa miR 744 5p, hsa miR 423 5p, hsa miR 331 3p, hsa miR 1306 5p, hsa miR 1285 3p, hsa miR 185 5p, hsa miR 550a 5p, hsa miR 3940 3p, hsa miR 574 5p and hsa miR 941; and the eleven miRNAs, as biological molecular markers, are applicable to clinical diagnosis of EBOV infection.

Description

technical field [0001] The invention relates to the characteristic miRNA in Ebola virus-infected blood and its application in the field of biomedicine. Background technique [0002] Ebola virus disease is an acute hemorrhagic infectious disease caused by Ebola virus (EBOV) belonging to the filoviridae family. The fatality rate is very high, up to 50% to 90%. [0003] The Ebola virus genome is a single-stranded negative-strand RNA, about 19kb in length, which can encode seven structures including nucleoprotein (NP), envelope protein (VP35, VP40, VP30, VP24), glycoprotein (GP) and RNA polymerase Protein, among which GP gene has unique coding and transcriptional functions for EBOV replication. The virus replicates, assembles, and releases by budding in the cytoplasm of infected cells. Ebola virus mainly includes Zaire type (EBOV-Z), Sudan type (EBOV-S), Côte d'Ivoire type (EBOV-C) and Reston type (EBOV-R), etc. Different subtypes have different characteristics. Among them, ...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68C12N15/11
CPCC12Q1/701C12Q2600/158C12Q2600/178
Inventor 王慧曹务春李涛刘雄江佳富户义邓永强刘坤宁年智
Owner MICROBE EPIDEMIC DISEASE INST OF PLA MILITARY MEDICAL ACAD OF SCI
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