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
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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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