Intestinal flora testing experiments

Summary

From birth, a set of intestinal flora is produced and perfected through the process of food adaptation in the intestinal tract. The composition and proportion of each individual's intestinal flora are relatively unique, and the existence of normal intestinal flora plays a very important role in maintaining the normal structure and function of the host's intestinal tract, and has special immune protection and nutritional functions. Once the normal intestinal flora is damaged, the body's intestinal function will be greatly affected, and after rebuilding the intestinal flora again, although it may be different from the previous flora formula, a new homeostatic balance will be reached. The sequencing of 16s-rRNA, which is unique to the intestinal flora, can be used to analyze the specific composition and proportion of an individual's intestinal flora, and thus this test has become an important basic tool in the study of intestinal flora (Figure 8-3-12).

Principle

The basic principle of intestinal flora testing is that the sequencing of 16s-rRNA, which is unique to intestinal flora, can be used to analyze the specific composition and proportion of an individual's intestinal flora.

Operation method

Intestinal flora testing experiments

Principle

The basic principle of intestinal flora testing is that the sequencing of 16s-rRNA, which is unique to intestinal flora, can be used to analyze the specific composition and proportion of an individual's intestinal flora.

Materials and Instruments

Equipment:
Ion Torrent PGM, Ion sequencing kitv 2.0.
Reagents:
①Qiagen Stool Mini Kit ; ②Ion OneTouchTM 200 Template Kit.
Ion OneTouchTM 200 Template Kit; ①Qiagen Stool Mini Kit; ②Ion OneTouchTM 200 Template Kit.

Move

The basic process of a gut flora testing experiment can be divided into the following steps:

A Remove microorganisms from the specific gut of interest.

B Extract the genomic DNA of the gut microorganisms using the Qiagen Stool Mini Kit.

C Quantify the microbial genomic DNA.

D PCR amplification using the 16s-rRNA gene variable region.

E Gels were cut to recover the 16s-rRNA gene variable region PCR product.

F The 16s-rRNA gene variable region PCR product was library constructed using the Ion xpress plus gDNA and amplicon library preparation kit.

G Amplified libraries were quantified using Qubit 2.0 and Agilent Bioanalyzer 2100.

H Emulsion PCR and enrichment of libraries using the Ion OneTouchTM 200 Template Kit.

I Gut microbial 16s-rRNA libraries were sequenced using Ion Torrent PGM and Ion sequencing kitv 2.0.

J The sequencing results were compared with the RDP (Ribosomal Database Project) database to obtain the relative abundance and population distribution of the gut microflora. Examples of the results are shown in Figure 8-3-13.

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