Method for detecting target microbial genome RNA based on high-throughput sequencing technology

A high-throughput, microbial technology, applied in the field of high-throughput sequencing and microbial detection, can solve the problems of low RNA content in microbial genomes, RNA easily degradable background DNA content, and low detection sensitivity, so as to reduce RNA loss and improve microbial genomes. RNA ratio and the effect of improving detection sensitivity

Pending Publication Date: 2022-02-22
深圳华大因源医药科技有限公司 +2
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Problems solved by technology

[0005] The purpose of the present invention is to solve the technical problems of the existing microbial genome RNA high-throughput sequencing, the RNA is easy to degrade and the background DNA content is high, and the micr

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  • Method for detecting target microbial genome RNA based on high-throughput sequencing technology
  • Method for detecting target microbial genome RNA based on high-throughput sequencing technology
  • Method for detecting target microbial genome RNA based on high-throughput sequencing technology

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[0074] Example 1

[0075] The Karka virus is a bowel virus and a nucleic acid is a single strand RNA. This example is illustrated by detecting the Kosaki virus A16 type in the sample, and a method of detecting the microbial genomic RNA of the present invention based on a high-through-sequencing technique is illustrated. The Kessach virus A16 is derived from the clinical residual samples provided by the Applicant's Cooperative Hospital. The exemplary detection step of the method of the present invention will be described below.

[0076] 1. Collic virus A16 simulation sample formulation: Add HELA cells to physiological saline to form 10 5 Cell / ml of HeLa cell solution, divided into four aliquots, adding Carka viral A16 to viral loading, respectively, 1000 cp / ml, 500 cp / ml, 100 cp / ml, 50 cp / ml, respectively, to contain different Collic Virus A16 type simulation samples for viral loading.

[0077] 2. Nucleic acid extraction: use ViRal RNA Mini Kit extracts nucleic acid in a...

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Abstract

The invention belongs to the technical field of high-throughput sequencing and microbiological detection, and particularly relates to a method for detecting target microbiological genome RNA based on a high-throughput sequencing technology. The method comprises the following steps: carrying out nucleic acid extraction, DNA digestion, RNA purification, RNA fragmentation, inverse transcription synthesis of cDNA molecules, cDNA library construction, high-throughput sequencing and sequencing data analysis on a to-be-detected sample containing a target microorganism, and detecting the condition of the genome RNA of the target microorganism in the sample. In the DNA digestion step, digestion is carried out under mild conditions of incubation at 37 DEG C to 42 DEG C for 10 to 30 minutes using a DNA enzyme, and further, in the RNA fragmentation step, a thermal disruption means is used. According to the method, due to the adoption of a mild digestion means and a thermal interruption means, the microbial genome RNA can be efficiently and quickly enriched at low cost, the content of the microbial genome RNA is increased, and the success rate of constructing an RNA high-throughput sequencing library is increased, so that the detection sensitivity of the microbial genome RNA is improved.

Description

technical field [0001] The invention belongs to the technical field of high-throughput sequencing and microbial detection, and in particular relates to a method for detecting target microbial genome RNA based on high-throughput sequencing technology. Background technique [0002] The wide application of high-throughput sequencing technology in the field of microbial detection has made microbial detection more convenient and faster. This technology can accurately identify microorganisms (including newly discovered microbial species) that cannot be accurately identified by traditional detection methods, but there are still great difficulties in the detection of microbial genome RNA. On the one hand, there are a large number of highly active RNases in the environment, which can degrade RNA anytime and anywhere, and some microbial genome RNAs are single-stranded RNA, which are easy to degrade and have poor stability, resulting in generally low levels of microbial genome RNA. Fa...

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Application Information

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IPC IPC(8): C12Q1/6869C12Q1/6806
CPCC12Q1/6869C12Q1/6806C12Q2535/122C12Q2521/345C12Q2523/308C12Q2521/107
Inventor 马珍子林永权吴红龙
Owner 深圳华大因源医药科技有限公司
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