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Intestinal content sample preservation solution and preparation method

A technology for preserving solution and contents, which is applied in the field of intestinal contents sample preservation solution and preparation, and can solve the problems of influence of downstream test accuracy, complex components, sample loss, etc.

Active Publication Date: 2021-05-07
成都罗宁生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the composition of intestinal contents is complex and diverse, it contains a large number of nucleases and proteases, and also contains various inhibitors that inhibit nucleic acid extraction and PCR amplification, which has a great impact on the accuracy of subsequent downstream tests
[0003] Due to the complex composition of the intestinal content sample, coupled with the limitations of the sampling location and sample storage conditions, the nucleic acid cannot be purified immediately, and the microbial cells and intestinal exfoliation cells in the sample will be degraded and destroyed quickly, eventually leading to the accumulation of bacterial flora. The degree of change, resulting in the loss of samples
On the other hand, the guanidinium salt and dehydration fixative contained in the existing sample preservation solution will greatly damage the cell structure, resulting in a decrease in the availability of the sample, and the detergent and other components contained in it may also cause environmental damage. Pollution

Method used

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  • Intestinal content sample preservation solution and preparation method
  • Intestinal content sample preservation solution and preparation method
  • Intestinal content sample preservation solution and preparation method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 4

[0043] Contrast between embodiment four and the preservation solution of different brands

[0044] The preservation solution prepared in Example 1 is compared with existing preservation solutions of different brands, and the specific operations are as follows:

[0045] (1) Take 200 μl of different brands of preservation solutions, mix each preservation solution with 75 μl of ZymoBIOMICS Microbial Community Standard, and store in a dark place at 25°C for 15 days;

[0046] Preservation solution A: containing methanol, ethanol, sodium chloride, Tween and acetate;

[0047] Preservation solution B: containing ethanol, Triton X-100 and citrate;

[0048] Preservation solution C: containing guanidine salt (guanidine hydrochloride or guanidine isothiocyanate), citrate;

[0049] (2) Purify the total DNA using ZymoBIOMICS DNA Miniprep;

[0050] (3) Use ABM Bestaq DNA polymerase to amplify and detect the 16S gene (27F / 1492R primer) and ITS fragment (ITS1 / ITS4), and the detection result...

Embodiment 5

[0051] The influence of embodiment five different pH samples on total DNA extraction

[0052] Collect samples with different pH, use 3.5ml of the preservation solution prepared in Example 1 to fully mix with it, store at 25°C for 7 days, use Zymo Research ZymoBIOMICS DNA Miniprep Kit to purify the total DNA, the specific steps are as follows:

[0053] (1) Take an appropriate amount of sample on ZR BashingBead TM Add 750μl ZymoBIOMICS to Lysis Tubes TM Lysis Solution, tighten the tube cap;

[0054](2) Place the ZR BashingBead containing the sample TM Lysis Tubes were placed in a bead beater and vortexed for 5 minutes. If the bead beater is not used for vortex operation, the homogenization treatment time can be extended to 20 minutes;

[0055] (3) Take out ZR BashingBead after homogenization TM Lysis Tubes, centrifuged at 10000x g for 1min;

[0056] (4) Pipette 400μl supernatant to Zymo-Spin TM III-F Filter (filled in a 2ml collection tube), centrifuge at 8000xg for 1min,...

Embodiment 6

[0067] Example 6 Effect of Sample Storage Time on DNA Purification and Downstream Experiments

[0068] (1) Take 6 parts of 200 μl of the preservation solution prepared in Example 1, mix each part of the preservation solution with 75 μl of ZymoBIOMICS Microbial Community Standard, place it in a dark place, and store it at a constant temperature of 25°C for 0 days, 15 days, 30 days, 60 days, 180 days;

[0069] (2) Purify the total DNA using ZymoBIOMICS DNA Miniprep (the detailed steps are the same as in Example 5);

[0070] (3) Use ABM Bestaq DNA polymerase to amplify and detect the 16S gene (27F / 1492R primer) and ITS fragment (ITS1 / ITS4), and the detection results are as follows image 3 , Figure 4 shown. The amplified products in the linear phase were detected after separation on 1% agarose gel (5V / cm); the detection results were as follows: Figure 5 shown.

[0071] High-throughput sequencing was performed on the amplicon samples obtained from samples stored for 15 days...

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Abstract

The invention relates to the technical field of microbial detection of intestinal contents, in particular to a sample preservation solution of intestinal contents and a preparation method thereof, comprising the following raw materials: 2.6-4.5M ammonium sulfate, 0.15‰ methyl chloroisothiazolinone, 5-20mM lemon sodium phosphate, 1-10mM EDTA, 5mM sodium fluoride, 1‰Triton X-100, 2‰Tween-80, 0.5% bromophenol red, 0.02% xylene nitrile blue FF. The intestinal content sample preservation solution of the present invention, the preservation solution can ensure the integrity of the nucleic acid in the sample within a large temperature range, has good compatibility with all animal intestinal samples, and does not need to be frozen. The formula preservation solution is not limited by experimental conditions, and samples can be sampled and stored at any time and place, and can also be used for long-distance transportation of samples.

Description

technical field [0001] The invention relates to the technical field of microbial detection of intestinal contents, in particular to a preservation solution of intestinal contents samples and a preparation method thereof. Background technique [0002] The relationship between human microbiome and various non-chronic infectious diseases, such as metabolic diseases, is a hot topic in the field of biomedical research in recent years. A large number of microbial groups, through long-term co-evolution with the host, provide enzymes and biochemical metabolic pathways that the host does not have. Intestinal microbes can not only degrade indigestible nutrients in food, provide host vitamins and other nutrients, but also promote the differentiation and maturation of intestinal epithelial cells, activate the intestinal immune system to regulate host energy storage and metabolism. The human body and intestinal flora form a mutually beneficial symbiotic relationship. The physiological p...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/10
CPCC12N15/10
Inventor 孙梓健曾俊儒丁泽琴曾陈娟陈云慧曹丰弟
Owner 成都罗宁生物科技有限公司
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