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

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...

AI Technical Summary

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 microbial genome RNA content is small, so the detection sensitivity is low, and a new microbial genome RNA is proposed. Enrichment method to increase the RNA content of microbial genomes, thereby improving detection sensitivity

Images