DTMUV non-diagnostic detection method based on metagenomics

A duck Tembusu virus and metagenomics technology, applied in the field of biological virus detection, can solve the problems of lack of genome sequence information, unavailable epidemic situation, epidemic spread economy, etc., and achieve the effect of wide sample coverage

Inactive Publication Date: 2016-01-06
INSPECTION & QUARANTINE TECH CENT SHANDONG ENTRY EXIT INSPECTION & QUARANTINE BUREAU
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the initial discovery of the unknown pathogen was limited by the limitations of conventional detection techniques, th

Method used

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  • DTMUV non-diagnostic detection method based on metagenomics
  • DTMUV non-diagnostic detection method based on metagenomics
  • DTMUV non-diagnostic detection method based on metagenomics

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Example 1 Application example of the method of the present invention in the confirmation and biotyping of Tembusu virus in virus-infected cell culture samples

[0043] 1 Materials and methods

[0044] 1.1 Disease data

[0045] The virus-infected cell culture samples were sent for inspection by a company in Qingdao. The pathogen was not identified by virus isolation, nor by conventional PCR diagnostic methods. Duck embryo fibroblasts and BHK cells are preserved by the applicant's laboratory;

[0046] 1.2 Molecular biology reagents

[0047] DNaseⅠ and RnaseA were purchased from Tiangen Biotechnology Co., Ltd.; XPReagent is a product of Thermo Company; DneasyKit and RNeasyMiniKit are products of QIAGen Company; OvationRNA-SeqSystemV2 genome amplification box is purchased from Nugen Company; IonShear TM PlusReagentKit, IonXpress TM BarcodeX, PCRSuperMix, LibraryAmplificationPrimerMix, IonLibrary All qPCRMix kits are products of Life Technologies.

[0048] 1.3 Mai...

Embodiment 2

[0065] Embodiment 2, the non-diagnostic detection method of the present invention detects different types of samples

[0066] The established non-diagnostic detection method will detect different regions and different types of poultry tissue samples, and compare with the conventional virus isolation method to evaluate the sensitivity and specificity of the method.

[0067] 1. Materials

[0068] 1.SPIA amplification kit, IonTorrent sequencer (embodiment 1)

[0069] 2. The disease materials submitted for inspection were 53 tissue samples of diseased and dead ducks from different chicken flocks in Hebei, Shandong, Beijing, etc. with clinical manifestations suspected of being infected with DTMUV. Avian reovirus (ARV), infectious bronchitis virus (IBV), infectious laryngotracheitis virus (ILTV), infectious anemia virus (CAV), chicken infectious bursal virus (IBDV), duck virus 7 samples of enteritis virus (DEV) and duck viral hepatitis virus (DVH) cell culture samples were all pre...

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Abstract

The invention discloses a DTMUV non-diagnostic detection method based on metagenomics. The method comprises the following steps: (1) processing samples; (2) extracting DNA and RNA; (3) performing inverse transcription isothermal chain displacement amplification on the RNA samples; (4) preparing a nucleic acid library; (5) performing Ion Torrent sequencing; (6) analyzing bioinformatics; (7) and performing phylogenetic analysis and genetic distance calculation. The non-diagnostic detection method based on unknown pathogeny SPIA amplification combining with high-throughput sequencing can obtain nucleic acid information of a TMUV gene, and the method is an effective new method.

Description

technical field [0001] The invention relates to a non-diagnostic detection method for duck Tembusu virus based on metagenomics, belonging to the technical field of biological virus detection. Background technique [0002] Metagenomics was born in the 1990s. It refers to a new discipline that studies the sum of microbial genes by directly extracting total DNA and total RNA from the environment without going through the stage of microbial cultivation. The emergence of metagenomic technology has made The research on non-culturable microorganisms accounting for more than 99% of the total microorganisms has become a reality, and the detectable space of microbial genes has increased significantly. In general, the current application of metagenomic technology is mainly divided into two aspects: one is to screen functional genes , to develop proteins with the required functions; on the other hand, through the analysis of metagenomic libraries, to explore the interaction between micr...

Claims

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Application Information

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IPC IPC(8): G06F19/18
Inventor 孙涛邓明俊王超房保海徐彪
Owner INSPECTION & QUARANTINE TECH CENT SHANDONG ENTRY EXIT INSPECTION & QUARANTINE BUREAU
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