Method for detecting enteritis pathogenic bacteria

Abstract

The invention relates to a method for detecting bacteria, in particular to a method for detecting enteritis pathogenic bacteria. The method comprises the following steps of: (1) extracting DNA (Deoxyribonucleic Acid) of one or more bacteria samples; (2) amplifying full length of 16S rDNA (ribosomal DNA) of a sample by using a primer of 16S rDNA of bacteria; (3) breaking an amplified PCR (Polymerase Chain Reaction) product; (4) repairing ends of the broken product from each bacteria sample and connecting deoxyadenosine at the end 3' of the product, and then connecting different PCR-free joints; (5) sequencing a connection product by using a second-generation sequencing technology to obtain a sequence of the connection product; and (6) comparing the sequence with 16S rDNA reference sequences of all existing bacteria to determine types of bacteria existing in each sample.

Description

technical field [0001] The invention belongs to the field of bacterial molecular biology, and in particular relates to a detection method for bacteria, especially enteritis pathogenic bacteria. Background technique [0002] In intestinal diseases, especially inflammatory bowel disease and enteritis, the etiology of many diseases is unknown. Studies have shown that most of these intestinal diseases of unknown etiology are related to the infection of pathogens, but so far no specific pathogens have been found to have a constant relationship with certain intestinal diseases. Some researchers believe that Mycobacterium paratuberculosis and measles virus are related to Crohn's disease in inflammatory bowel disease, but the evidence is not convincing. An alternative view of microbial pathogenicity has gained prominence in recent years, the idea that inflammatory bowel disease is caused by an abnormal immune response against the normal gut flora itself. In the animal model of inf...

AI Technical Summary

Problems solved by technology

[0007] Next-generation sequencing technology has been applied to the field of microbial species analysis, but it is generally based on one or several of the 10 variable regions (V6 region, V3 region, etc.) Primers for amplification of specific variable regions, which often result in the loss of certain species

Using full-length 16S rDNA combined with next-generation sequencing technology represented by IlluminaHiseq2000 to analyze bacterial species, the biggest obstacle lies in the short length measurement, and the Pair-End sequencing strategy can only achieve 200bp length measurement, because 16S rDNA is in different The similarity of the conserved regions between species is very large, which makes it still a challenge to assemble the 16S rDNA products of mixed amplification

At present, there is no report on the use of next-generation sequencing technology represented by Illumina Hiseq2000 to conduct 16S rDNA full-length sequencing and apply it to the research of clinical species classification

Images