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Marker genes related with Ebola virus infection and applications thereof

A gene, viral load technology, applied in the field of biomedicine

Inactive Publication Date: 2017-03-29
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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  • Marker genes related with Ebola virus infection and applications thereof
  • Marker genes related with Ebola virus infection and applications thereof
  • Marker genes related with Ebola virus infection and applications thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] Example 1. Molecular markers related to viral load in the blood of EBOV-infected patients

[0050] 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

[0056] Embodiment 2, small RNAs (sRNAs) associated with viral load in the blood of EBOV-infected patients

[0057] 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

[0064] Example 3. Molecular markers related to clinical prognosis in the blood of EBOV-infected patients

[0065] 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 marker genes related with Ebola virus infection and applications thereof. The disclosed marker genes related with Ebola virus infection in blood are 10 following genes: DPAGT1 gene, GPR125 gene, GYP4B1 gene, PTPRK gene, NEK10 gene, NCAPD3 gene, ABCA13 gene, DNAH11 gene, KDELC1 gene, and MTA3 gene. The expression amount of the 10 genes in EBOV infected patients is obviously increased, compared with non-EBOV infected patients, and thus the 10 molecules can be used as biological molecular markers applied to the clinical diagnosis of EBOV infected patients.

Description

technical field [0001] The invention relates to marker genes and applications related to Ebola virus infection 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, the Zaire ty...

Claims

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Application Information

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IPC IPC(8): C12Q1/68G01N33/68C12R1/93
CPCC12Q1/6883C12Q2600/118C12Q2600/158G01N33/6893G01N2800/26G01N2800/52
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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