Model construction of human flora-associated piggy and molecular method for detecting flora in intestine tract of baby pig

A construction method and molecular detection technology, applied in the field of microorganisms, can solve problems such as laborious, time-consuming cultivation methods, and inability to reflect the diversity of microbial communities

Inactive Publication Date: 2006-10-04
SHANGHAI JIAO TONG UNIV
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  • Abstract
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  • Claims
  • Application Information

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Problems solved by technology

More and more studies have confirmed that the culture method is time-consuming, laborious and far from reflecting the diversity of the microbial community, so the establishment of a molecular biological detection method that does not rely on pure culture will b

Method used

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  • Model construction of human flora-associated piggy and molecular method for detecting flora in intestine tract of baby pig
  • Model construction of human flora-associated piggy and molecular method for detecting flora in intestine tract of baby pig
  • Model construction of human flora-associated piggy and molecular method for detecting flora in intestine tract of baby pig

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Experimental program
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Effect test

Embodiment 1

[0028] Example 1 Construction of human-sourced flora piglet model

[0029] (1) Preparation of healthy human feces bacteria suspension

[0030] Prepare PBS buffer solution (0.05M, pH7.4) containing 10% (vol / vol) glycerol, sterilize at 121° C. for 30 minutes, and place in an anaerobic tank for pre-reduction treatment for 24 hours. Collect freshly excreted healthy child or adult feces, suspend every 2 grams of feces in 40ml of the above-mentioned treated PBS buffer, let it stand for a while, draw the supernatant into a sterile centrifuge tube, this is the fecal bacteria suspension. The bacterial suspension can be directly inoculated into newborn piglets or quickly stored in a -70°C refrigerator, thawed before use, and repeated freezing and thawing should not be avoided.

[0031] (2) Caesarean section for pregnant sows to give birth

[0032] Caesarean section should be performed as close as possible to the time of natural delivery, preferably the day before the expected date of ...

Embodiment 2

[0039] Example 2 Molecular detection of the above-mentioned human flora (HFA) in the intestinal tract of piglets.

[0040] (1) Extraction of Fecal Microbial Genomic DNA

[0041] To detect the composition of intestinal flora by molecular microbiological methods, the total genomic DNA of high-quality intestinal microorganisms must be extracted first. HFA piglet feces DNA was extracted using the kit QIAamp DNA stool minikit (Qiagen, Hilden), and the method was according to the kit instructions. The obtained DNA was used with a DNA concentration analyzer DyNA Quant TM 200 fluorometer (Amersham Pharmacia Biotech, USA) was used to determine the concentration, and electrophoresed on 1% agarose gel to check its size and integrity.

[0042] (2) ERIC-PCR fingerprint analysis and community DNA hybridization

[0043] Continuous monitoring of the dynamic changes of gut microbiota composition in HFA piglets since birth by ERIC-PCR fingerprinting technique. ERIC-PCR system (25 μ l) incl...

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Abstract

Disclosed is a model human-source flora litter model construction and method for detecting flora in litter intestinal tract. The construction steps contain: (1) prepare healthy people fecal bacteria suspension; (2) paunch to farrowing sow to fetch litters; (3) breed the litters in 24-hour aero-ultra filter positive-pressure draft isolation room; (4) inoculate. The detecting steps contain: (1) extract the fecal microbe genome DNA; (2) analyze ERIC-PCR finger spectrum and community DNA crossing; (3) planting analyze bacillus in HFA litter intestinal tract flora; (4) planting analyze HFA bacteroide in HFA litter tract flora. This invention successfully plant human donator's bacillus and bacteroide in litter model intestinal tract. The finger spectrums comparing shows that the flora finger spectrum in litter model intestinal tract has homologous height with that of the human donator. This invention can be used to study the cross-correlation between intestinal tract flora and its host physiology and immune specificity, also it can be used to evaluate regulation and metabolism effect of functional foods or drugs to intestinal tract flora.

Description

technical field [0001] The invention relates to a method in the technical field of microorganisms, in particular to a construction of a piglet model of human-derived flora and a method for detecting flora molecules in the intestinal tract of piglets. Background technique [0002] The human gastrointestinal tract is populated by a very complex and active microbial community, including about 10 14 bacteria, equivalent to 10 times the total number of all tissue cells in the human body. A large number of studies have shown that this huge microbial flora forms a stable symbiotic system with humans, which is crucial to human health and disease conditions. Therefore, studying the structure and composition of the human gastrointestinal flora and the interaction between the flora and its host has always been a hot spot in the field of microbial ecology. In recent years, more and more commercially produced functional foods and drugs that regulate intestinal flora have emerged, and t...

Claims

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Application Information

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IPC IPC(8): A61K49/00C12Q1/68
Inventor 赵立平庞小燕华修国崔立
Owner SHANGHAI JIAO TONG UNIV
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