Novel method for detecting pathogenic microorganisms

Abstract

The invention discloses a novel method for detecting pathogenic microorganisms. The method comprises: a collection step of samples such as cerebrospinal fluid and alveolar lavage fluid; a collection steps of peripheral blood samples; a DNA extraction step; a library construction step; a sequencing step; and an analysis step. The DNA extraction step comprises: taking 400 [mu]l sample for detection,adding reaction reagents of buffer GB, Lysis Enzyme K, RNA, Carrier to the sample, conducting reaction at 56 DEG C for 10 minutes to obtain a reaction solution, purifying the reaction solution by silica gel membrane adsorption column to remove salt ions and organic reagents, and obtaining DNA with total amount and purity meeting the requirements. The library construction step comprises: conducting terminal repair, linker connection, library amplification and enrichment, purification and quality control of the DNA to complete the library construction. The sequencing step comprises: denaturingthe library into a single strand by NaOH, then binding the library to a sequencing chip, enriching each single strand DNA into a cluster by bridge amplification, and obtaining sequencing data by usingIllumina SBS sequencing method to read the sequence. The analysis step comprises: filtering the sequencing data, removing the human source and the linker sequence, and comparing the remaining data with a microbial reference database.

Description

technical field [0001] The invention relates to the fields of molecular biology and molecular genetics, in particular to a method for detecting novel pathogenic microorganisms based on a new generation sequencing technology. Background technique [0002] Traditional pathogenic microorganism identification technologies are mainly divided into two categories: methods based on cell culture such as morphological observation, cell physiological and biochemical characteristics, bacterial culture typing, gene chips, automated microbial analysis systems, etc., and methods based on specific primers / probes / antibodies Methods such as antigen-antibody reaction, PCR reaction detection, and various specific rapid detection systems for pathogenic microorganisms, etc. These technologies have played an important role in the daily identification of pathogenic microorganisms, but there are also certain shortcomings, such as the former needs to rely on cultivation, the cycle is long, and the id...

AI Technical Summary

Problems solved by technology

These technologies have played an important role in the daily identification of pathogenic microorganisms, but there are also certain shortcomings, such as the former needs to rely on cultivation, the cycle is long, and the identification accuracy is low; the latter requires certain prior knowledge of microbial sequences and cannot deal with unknown or mutated pathogens Wait

[0003] Therefore, genome sequencing of pathogenic microorganisms has gradually become an important tool and method in the field of clinical microbial detection. However, the current detection methods for pathogenic microorganisms are relatively scarce, and it is difficult to accurately detect the types of microorganisms in samples.

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