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Preparation and application of hemorrhagic fever associated pathogen identifying gene chip

A gene chip and hemorrhagic fever technology, applied in the direction of microorganism-based methods, biochemical equipment and methods, microorganism measurement/inspection, etc., can solve the problems of insufficient detection flux, limited detection range, complicated operation, etc., and achieve improved detection Efficiency, shorter detection cycle, high specificity effect

Active Publication Date: 2015-11-25
ZHEJIANG CENT FOR DISEASE CONTROL & PREVENTION +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The above methods have their own advantages and disadvantages. Some methods are complicated to operate, time-consuming and laborious; some methods have low specificity and / or sensitivity; some methods use too many samples; Problems with limited detection range

Method used

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  • Preparation and application of hemorrhagic fever associated pathogen identifying gene chip
  • Preparation and application of hemorrhagic fever associated pathogen identifying gene chip
  • Preparation and application of hemorrhagic fever associated pathogen identifying gene chip

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Example 1: Development of gene chip for identification of hemorrhagic fever-related pathogens

[0041] 1. Design and screening of specific primers

[0042] Log on to the NCBI website, search for the whole genome sequences of related viruses in GenBank for comparison and analysis, and download the 18srRNA genes of 4 species of Plasmodium for comparison and analysis in the genus Plasmodium. The AlignX program in the VectorNTIAdvance10 (invitrogen) software package was used to perform a global alignment of the gene sequences of each pathogen according to the default parameter settings. According to the comparison results, specific primers were designed in the conserved region of the gene sequence, and 29 specific primers (as shown in Table 1) were finally determined after screening, and the downstream primers were biotin-labeled as primers for chip use.

[0043] 2. Construction of Reference Products

[0044] Except for some hemorrhagic fever-related pathogens, nucleic ac...

Embodiment 2

[0057] Example 2: Specificity evaluation of gene chips for identification of hemorrhagic fever-related pathogens

[0058] Specificity is the most important assessment index of a diagnostic method. The present invention uses optimized systems and conditions to detect nucleic acids of 16 kinds of hemorrhagic fever-related pathogens. The chip detection results are shown in the attached image 3 . The results show that the invention can correctly distinguish 16 kinds of hemorrhagic fever-related pathogens, and has good specificity.

Embodiment 3

[0059] Example 3: Sensitivity evaluation of gene chip for identification of hemorrhagic fever-related pathogens

[0060] The detection sensitivity of the chip was evaluated using a sensitivity reference product, and it was found that the chip could detect at least 10 of each pathogen. 3 copies / system of RNA or DNA. Taking Ebola virus, Plasmodium and fever with thrombocytopenia syndrome virus as examples, choose 10 4 copies / μl, 10 3 copies / μl, 10 2 copies / μl, 10 1 Copies / μl of pathogenic nucleic acid and negative control for chip detection, the results are shown in the attached Figure 4 . The results showed that the chip could detect at least 10 3 The pathogenic nucleic acid of the copies / system, the detection sensitivity of the chip is 10 2 ~10 3 copies / system.

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Abstract

The invention relates to a hemorrhagic fever associated pathogen identifying gene chip; the preparation method comprises preparation of a specific primer, preparation of a pathogen specific oligonucleotide probe, preparation of an oligonucleotide chip, establishment of an RT-PCR (reverse transcription-polymerase chain reaction) system and establishment of a hybrid system and a signal detection method. The gene chip prepared by the invention can be used for simultaneously identifying 16 hemorrhagic fever associated pathogen microorganisms, including Zaire Ebola virus, Sudan Ebola virus, marburg virus, lassa virus, junin virus, Machupo virus, rift valley fever virus, Crimea-Congo hemorrhagic fever virus, plasmodium, hantaan virus, SFTS (severe fever with thrombocytopenia syndrome) virus, dengue virus, yellow fever virus, Chikungunya virus, influenza A virus and influenza B virus. The gene chip has the characteristics of being rapid and accurate, high in throughput and high in sensitivity; and a new technological means is offered for the diagnosis of hemorrhagic fever pathogen, health supervision and the control and prevention of infectious diseases.

Description

technical field [0001] The invention relates to the preparation and application of a nucleic acid detection gene chip for sixteen kinds of hemorrhagic fever-related pathogens, and belongs to the technical field of gene chip detection. Background technique [0002] Viral Hemorrhagic Fever (VHF) is a group of natural foci diseases caused by different types of viruses, with fever and bleeding as the main clinical symptoms. Hemorrhagic fever viruses are mainly distributed in Filoviridae, Arenaviridae, Bunyaviridae and Flaviviridae. There are many types of viruses that can cause hemorrhagic fever, among which the ones that have a greater global impact include: filoviridae Ebola virus, Marburg virus, arenavirus Kolassa virus, Junin virus, Maqiubo virus, Bursa virus Rift Valley Fever Virus, Crimean-Congo Hemorrhagic Fever Virus, Hantavirus, Fever with Thrombocytopenia Syndrome Virus, Dengue Virus, Flaviviridae, Yellow Fever Virus, Togaviridae Chikungunya virus etc. The early cli...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68C40B40/06C12R1/93
CPCY02A50/30
Inventor 张严峻颜浩王升启刘琪琦陈苏红刘志红孙晓彦
Owner ZHEJIANG CENT FOR DISEASE CONTROL & PREVENTION
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