RNA (Ribonucleic Acid) virus metatranscriptome library building method based on nanopore sequencing platform and application

Active Publication Date: 2022-04-05
JIANGSU SIMCERE MEDICAL DEVICE CO LTD +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] Aiming at the difficult problem of RNA virus detection in clinical samples, the core problem or purpose to be solved by the present invention is to build a set of nanopore sequencing platform RNA virus macro transcription library The detection process is to improve the sensitivity of RNA virus detection on the nanopore sequencing platform, which meets the clinical needs for rapid detection of samples suspected of RNA virus infection, and is suitable for popularization and application

Method used

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  • RNA (Ribonucleic Acid) virus metatranscriptome library building method based on nanopore sequencing platform and application
  • RNA (Ribonucleic Acid) virus metatranscriptome library building method based on nanopore sequencing platform and application
  • RNA (Ribonucleic Acid) virus metatranscriptome library building method based on nanopore sequencing platform and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0070] Example 1 RNA virus removal process optimization

[0071] Considering that the samples contained a large amount of host DNA, RNA viruses were low in abundance, accounting for only a very small part. On the one hand, due to the interference of host DNA, only a very small part of the obtained sequencing data can be used for species identification; , which directly affects the detection sensitivity of the low-abundance RNA virus macrotranscriptional library, so consider incomplete removal of host DNA.

[0072] In this example, the incomplete removal of host DNA is performed on clinical influenza B virus samples (Influenza B virus for short, IVB) and rhinovirus samples (Rhinovirus for short, RhV), in order to screen out the incomplete removal of host DNA and Combinations with less viral impact.

[0073] The present invention sets two combinations respectively: combination ① saponin + DNase combination mode, the experimental purpose is to carry out host DNA dissociation an...

Embodiment 2

[0085] Example 2 The necessity of virus dehosting process for building a nanopore sequencing platform RNA virus macrotranscriptional library

[0086] In the process of sample extraction, in addition to a very small amount of RNA viruses, it also contains a large amount of human genome nucleic acid, other pathogenic DNA, and ribosomal RNA (rRNA). If these parts are not removed, it will occupy a large amount of sequencing space. It affects the detection of RNA; but at the same time, the de-hosting process will also remove some RNA viruses, which affects the load and detection of RNA viruses. Therefore, how to choose the de-host and the degree of removal in practice will affect the final result.

[0087] The purpose of this example is: according to the optimized viral host removal process reagent combination ② in Example 1, to test whether the host removal process is necessary for building the ONT platform RNA virus macrotranscriptional library for clinical samples.

[0088] Tabl...

Embodiment 3

[0095] Example 3 Screening of viral nucleic acid extraction reagents

[0096]The viral load of metagenomic clinical samples is low. In practice, the existing RNA viral nucleic acid extraction kits are not suitable for RNA viral nucleic acid extraction from metagenomic. In order to obtain high-quality sequencing results, high-quality RNA nucleic acid templates are required. The extraction effect and extraction time of RNA virus nucleic acid extraction kits from different manufacturers are quite different, which affects the effective detection of pathogens in the subsequent RNA virus sequencing stage. The effect of RNA virus nucleic acid extraction of pathogens varies from tens to thousands of times, which not only affects the sensitivity of the RNA virus metatranscriptomic process, but may even cause the missed detection of RNA pathogens to cause immeasurable and even life-threatening losses to the social economy and patients. Therefore, in this example, two RNA virus nucleic a...

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Abstract

The invention belongs to the field of gene sequencing, and particularly relates to an RNA (Ribonucleic Acid) virus metatranscriptome library building method of a nanopore sequencing platform. Aiming at the problems that a nanopore sequencing platform is low in flux, the host content of a clinical sample is high and a large amount of ribosome RNA is contained in total RNA, incomplete host cell DNA removal and ribosome RNA removal are carried out on the clinical sample through reverse enrichment and the like; aiming at the problems of low virus load of clinical samples and high diversity of RNA virus genomes, cDNA is actively and positively enriched by adopting single-primer isothermal amplification and the like. Through forward and reverse enrichment steps, a nanopore sequencing platform RNA virus macro transcriptome library building detection process is successfully built, the requirement for rapid identification of unknown RNA virus pathogens clinically is met, and the method is suitable for application and popularization.

Description

technical field [0001] The invention belongs to the field of gene sequencing, and in particular relates to a method and application of an RNA virus macro-transcription building library of a nanopore sequencing platform. Background technique [0002] Infectious diseases are an important cause of human disease and death, and have always received clinical attention. RNA viruses are the major pathogens in most severe cases of infection, such as the global pandemic of seasonal influenza and the worldwide pandemic of the novel coronavirus (SARS-CoV-2), which pose serious challenges to the world economy and human health Challenges, so that the accurate and rapid identification of pathogens has become a focus of attention. [0003] The main methods of virus identification in traditional clinical microbiology laboratories are virus isolation and culture, immunofluorescence detection and PCR molecular detection, but they have limitations. Virus isolation, culture and immunofluoresce...

Claims

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Application Information

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Patent Type & AuthorityApplications(China)
IPC IPC(8): C12Q1/6806C12Q1/6869C40B50/06
Inventor周水莲潘吾思戴岩李振童桢开李诗濛任用
OwnerJIANGSU SIMCERE MEDICAL DEVICE CO LTD