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Normal temperature preservation solution of intestinal microbial sample DNA

A technology for intestinal microbes, normal temperature preservation, applied in the field of biological sample preservation, can solve problems such as poor applicability and flexibility, inability to immediately cryopreservation, changes in the total amount, composition and relative abundance of microorganisms, and achieves the effect of simple operation.

Pending Publication Date: 2019-09-20
杭州高六博生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the biggest limitation of this method is that its applicability and flexibility are poor, and it also needs the support of large ultra-low temperature instruments
Subjects generally cannot store samples at low temperature immediately after collection, and the total amount, composition and relative abundance of microorganisms may change, which cannot accurately reflect the true status of the subject's intestinal flora

Method used

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  • Normal temperature preservation solution of intestinal microbial sample DNA
  • Normal temperature preservation solution of intestinal microbial sample DNA
  • Normal temperature preservation solution of intestinal microbial sample DNA

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0019] Embodiment 1: the source and processing mode of sample

[0020] The feces samples in this example were provided by 3 volunteers, and each volunteer provided 4 samples, which were stored in 4 different ways.

[0021] A. Room temperature for 3 hours: collect at least 0.2g of fresh fecal samples, place it in a sealed place at room temperature in the dark for 3 hours, then extract DNA, construct a library, and send it out for sequencing and other processing.

[0022] B. Freeze for 7 days: Collect at least 0.2g of fresh feces samples, seal and avoid light, and store at -80°C. Then perform DNA extraction, library construction, and outbound sequencing.

[0023] C. Invention formula for 7 days at room temperature: collect at least 0.2 g of fresh feces samples and completely immerse in the formula of the invention, seal and avoid light, and place at room temperature (25° C.) for 7 days. Then perform DNA extraction, library construction, and outbound sequencing.

Embodiment 2

[0024] Embodiment 2: experimental operation process

[0025] 1. Sample pretreatment

[0026] The sample of the invented formula at room temperature for 7 days needs to discard the preservation solution before DNA extraction. The specific operation is as follows: centrifuge at 10,000rpm for 5 minutes, discard the preservation solution, and retain the precipitate.

[0027] 2. DNA extraction

[0028] a. Add 1ml of InhibitEX Buffer and vortex for 1 minute or until the stool sample is completely mixed.

[0029] b. Heat incubate at 70°C for 5 minutes, vortex for 15 seconds.

[0030] c. Centrifuge at 20000×g for 1 minute.

[0031] d. Add 15 μl proteinase K to a new 2ml centrifuge tube.

[0032] e. Transfer 200 μl of the supernatant from step c to a 2 ml centrifuge tube containing proteinase K.

[0033] f. Add 200μl Buffer AL and vortex for 15 seconds.

[0034] g. Heat incubation at 70°C for 10 minutes and centrifuge briefly.

[0035] h. Add 200 μl alcohol (96–100%) to the lysa...

Embodiment 3

[0046] Embodiment 3: sample DNA extraction result

[0047] Determine the quality and integrity of sample DNA by agarose gel electrophoresis and NanoDrop, as shown in Table 1 and figure 1 As shown, the 260 / 280 ratio of the sample is relatively consistent, and the main band is clear. The results showed that high-quality DNA could be extracted under different treatment methods, and there was no significant difference.

[0048]

[0049] * Concentration unit: ng / μl

[0050] *Volume: 45μl

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Abstract

The invention belongs to the field of preservation of biological samples, and especially relates to a normal temperature preservation solution for intestinal microbial samples DNA. According to the invention, sterile water is used for dissolving 10-50 mM of disodium edetate (EDTA-2Na), 5-25 mM of a Tris-HCl buffer solution, 1-10 mM of tris(2- carboxyethyl)phosphine (TCEP), 140 mM of sodium chloride (NaCl), glycerol with a mass fraction of 10-20%, dimethyl sulfoxide (DMSO) with the mass fraction of 1-8% and sodium dodecyl sulfate (SDS) with the mass fraction of 0.1 %-0.5%, and a PH value is adjusted to 8.0. The intestinal microbial DNA preservation solution adopting the technical scheme can be stored and transported at room temperature for a long time, and is easy to operate; and the composition of the microorganisms can be stabilized by simply immersing a fresh stool sample in the preservation solution.

Description

technical field [0001] The invention belongs to the field of biological sample preservation, and in particular relates to a room temperature preservation solution for intestinal microbial sample DNA. Background technique [0002] Gut microbes, that is, a large number of microorganisms that rely on the human intestinal tract to survive. The number of microorganisms in the intestinal tract of an adult is about 1014, the number of genes is as high as 3.3 million, which is about 100 times the number of human genes, and the mass is about 1.2kg, which is very close to the mass of human liver. With the development of technologies such as metagenomics and metatranscriptomics, it has been confirmed that intestinal microbes are important participants in human metabolic reactions and can help the human body complete various physiological and biochemical reactions. For example: these microorganisms can provide the substrates, energy and enzymes required in the process of human metaboli...

Claims

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Application Information

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IPC IPC(8): C12N15/10
CPCC12N15/10
Inventor 李明定
Owner 杭州高六博生物科技有限公司