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Preparation method of mice intestinal flora alteration ERIC-PCR (Enterobacterial Repetitive Intergenic Consensus sequence-based Polymerase Chain Reaction) fingerprint spectrum

A technology for intestinal flora imbalance and fingerprinting, which is used in biochemical equipment and methods, microbial determination/inspection, resistance to vector-borne diseases, etc.

Inactive Publication Date: 2014-05-07
DALIAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

ERIC is a tandem repeat sequence that exists in a variety of bacterial genomes, mainly between intestinal bacterial genomes, and has not yet been found on bacterial phages and plasmids

Method used

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  • Preparation method of mice intestinal flora alteration ERIC-PCR (Enterobacterial Repetitive Intergenic Consensus sequence-based Polymerase Chain Reaction) fingerprint spectrum
  • Preparation method of mice intestinal flora alteration ERIC-PCR (Enterobacterial Repetitive Intergenic Consensus sequence-based Polymerase Chain Reaction) fingerprint spectrum

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Example 1 Modeling of intestinal flora imbalance in mice

[0028] After 7 days of adaptive feeding, 24 pure-line Balb / c mice were randomly divided into 2 groups, 12 in each group, all male, with a body weight of 20±2g, normal (normal saline, NS) group and model group . The mice in the normal group were gavaged with NS 0.35 mL / mouse / time, twice a day, and served as the control group. The dosage of each mouse in the model group was 28 mg of gentamicin sulfate and 70 mg of cephradine, respectively, and 0.35 mL / piece / time was administered after making a mixed antibiotic solution according to the dosage. Tract flora imbalance. The mixed antibiotic solution is prepared by mixing 40 bottles of gentamicin sulfate (80mg / 2mL / bottle) and 8 bottles of cephradine (1g / bottle). Both the normal group and the model group were treated continuously for 7 days.

Embodiment 2

[0029] Example 2 sample collection and processing

[0030] After the two groups of experimental mice were killed by cervical dislocation, they were soaked in 75% alcohol for a while, and the abdomen of the mice was dissected. A small amount of cecal contents were taken with a sterile spoon, and the weight of the cecal contents was kept at 0.15g, and stored at 4°C or It was directly used for the subsequent genome extraction operation, and a small amount of cecal content (0.01-0.10 g) was taken for routine flora analysis (as a reference for the ERIC-PCR identification method).

Embodiment 3

[0031] Example 3 routine flora analysis

[0032] The contents of the cecum were serially diluted 10 times with NS, that is, from 10 -1 to 10 -7, Bacteria were isolated and cultured by conventional methods. EMB and EC plates were cultured upright at 37°C for 18 hours, and LBS and BS plates were cultured upright at 37°C for 48 hours in anaerobic culture. The plates with a colony growth density of 10-100 were selected for colony counting, and each The colony counts of Enterobacter, Enterococcus, Lactobacillus and Bifidobacterium in grams of cecal contents (CFU / g), expressed as Lg10n / g, the data results of each group are expressed as mean±SE, and the statistical analysis adopts the mean of two samples t test. In the normal group, Enterobacter 3.68±0.28CFU / g, Enterococcus 3.65±0.34CFU / g, Lactobacillus 6.16±0.11CFU / g, Bifidobacterium 6.58±0.08CFU / g; in the model group, Enterobacter 5.29±0.21 CFU / g, Enterococcus 5.14±0.21CFU / g, Lactobacillus 4.83±0.21CFU / g, Bifidobacterium 4.89±0....

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Abstract

The invention provides a preparation method of a mice intestinal flora alteration ERIC-PCR (Enterobacterial Repetitive Intergenic Consensus sequence-based Polymerase Chain Reaction) fingerprint spectrum. The preparation method comprises the steps: moulding mice intestinal flora alteration caused by antibacterial-associated diarrhea, extracting a cecum content DNA, eliminating humic acid in the cecum content DNA, eliminating a PCR inhibitor in the cecum content DNA, performing ERIC-PCR amplification and native polyacrylamide gel electrophoresis (Native-PAGE), and comparing with a conventional flora analysis result so as to establish the ERIC-PCR fingerprint spectrum of the mice intestinal flora alteration. According to the preparation method, with a pure-series mice as a research object, the defect caused when a conventional bacterium culturing method is applied to the microflora analysis is solved. The preparation method can be used for rapid detection of the laboratory mice intestinal flora alteration, prevents the defects of complicated operation, long time consumption, large workload and the like of conventional bacterium separation and culturing, and is capable of accurately, rapidly and effectively detecting the mice intestinal flora distribution condition.

Description

technical field [0001] The invention belongs to the field of preparation of bacterial genome fingerprints, in particular to a method for preparing ERIC-PCR fingerprints of mouse intestinal flora imbalance. Background technique [0002] Dysbacteriosis is a problem often encountered in basic and clinical research. It is the initial factor of exogenous infection and endogenous infection, and it is difficult to control once it occurs. There are many factors that cause flora imbalance, including dietary factors, flora change factors, drug metabolism factors, age factors and gastrointestinal immune dysfunction factors. Therefore, research on the structural diversity and population dynamics of the intestinal flora is of vital significance for the treatment of dysbiosis. [0003] The most common and most familiar form of dysbiosis is the symptom of "diarrhea". Diarrhea is a manifestation of thin stools and increased frequency of defecation, and is a collective term for many simila...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/04
CPCC12Q1/04C12Q1/686C12Q2525/151C12Q2565/125Y02A50/30
Inventor 伦永志孙丽妲
Owner DALIAN UNIV
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