Fecal nucleic acid preserving fluid and preparation method and application thereof

A technology for preserving liquid and nucleic acid, applied in the field of microorganisms, can solve problems such as fecal sample collection and transportation constraints

Inactive Publication Date: 2020-10-27
SHANGHAI REALBIO TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] However, the collection and transportation of stool samples have been greatly constrained and needs to be improved

Method used

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  • Fecal nucleic acid preserving fluid and preparation method and application thereof
  • Fecal nucleic acid preserving fluid and preparation method and application thereof
  • Fecal nucleic acid preserving fluid and preparation method and application thereof

Examples

Experimental program
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Effect test

preparation example Construction

[0027] The present invention also provides a method for preparing a fecal nucleic acid preservation solution, comprising:

[0028] (1) Take the required Tris, adjust the pH to 5.0-10.0 with hydrochloric acid, and prepare the Tris-HCl buffer solution;

[0029] (2) Dissolve the required amount of EDTA in sterile deionized water, adjust the pH to 5.0-10.0 with hydrochloric acid, and prepare the EDTA solution;

[0030] (3) Dissolve the required amount of sodium chloride and sodium citrate in sterile deionized water, add Tris-HCl solution and EDTA solution to mix, adjust the pH value to 7.0-10.0 with NaOH, and autoclave at 121°C 30min;

[0031] (4) Add the required amount of absolute ethanol to the solution in step (3), and filter the microporous membrane to sterilize.

[0032] (5) Dispense the prepared preservation solution into sampling tubes, and store them under strict seal at room temperature.

Embodiment 1

[0035] In Example 1, the fresh stool samples of 5 volunteers were stored according to the following 5 conditions, and the influence of each storage condition on the nucleic acid of the stool samples was verified by nucleic acid extraction. The process is as follows:

[0036] First, fresh stool samples from 5 volunteers were collected and stored in accordance with the following 5 conditions:

[0037] Treatment group No. 1 (immediate extraction after sampling): about 0.2 g of fresh fecal samples were collected, and DNA extraction was performed immediately;

[0038] Treatment group No. 2 (direct cryopreservation for 7 days): about 0.2 g of fresh feces samples were collected, immediately frozen at -80°C, and stored for 7 days before DNA extraction;

[0039] Treatment group No. 3 (preservation solution at 40°C for 3 days and ultra-low temperature for 4 days): Collect about 0.2g of fresh feces sample, put it into an equal volume of preservation solution immediately, mix it upside d...

Embodiment 2

[0065] 1. Amplification and sequencing of ribosomal 16S rRNA gene V3-V4 region fragments

[0066] (1) Reagent: High-fidelity PCR polymerase system (Kapa Biosystems)

[0067] (2) Primer sequence:

[0068] Upstream primer (341F): 5'-ACTCCTACGGGAGGCAGCAG (SEQ ID NO: 1)

[0069] Downstream primer (806R): 5'-GGACTACHVGGGTWTCTAAT (SEQ ID NO:2), wherein V represents base A or base G or base C; W represents base A or T; H represents base A or base C or base T.

[0070] (3) Operation steps: Set the total PCR system to 30 μL, including 15 μL 2×KAPA HiFi HotStartReadyMix (Kapa Biosystems), 0.2 μM upstream and downstream primers, and 30-50 ng DNA template. Carry out the reaction according to the following PCR reaction conditions:

[0071] Table 1 PCR reaction conditions

[0072]

[0073] (4) The amplified fragments were purified by agarose gel electrophoresis, and sequenced in PE300 mode using the Illumina MiSeq sequencing platform.

[0074] 2. Analysis and comparison of sequenci...

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Abstract

The invention relates to a fecal nucleic acid preserving fluid as well as a preparation method and application thereof. The fecal nucleic acid preserving solution comprises a Tris-HCl buffer solutionwith the concentration being 0.01 to 1M, ethylenediaminetetraacetic acid (EDTA) with the concentration being 0.01 to 1M, sodium chloride with the concentration being 0.01 to 0.5 M, sodium citrate withthe concentration being 0.01 to 0.5 M and absolute ethyl alcohol with the volume concentration being 10 to 30 percent, wherein the pH value of the preservation solution is 7.0 to 10.0. The fecal nucleic acid preserving fluid provided by the invention can effectively maintain the stability of nucleic acid in feces, and is convenient for a user to sample at any time and transport at normal temperature. And downstream detection of intestinal flora is not influenced.

Description

technical field [0001] The invention relates to the technical field of microbes, in particular to a stool nucleic acid sample preservation solution and a preparation method and application thereof. Background technique [0002] The gut is one of the most important organs in the human body. According to reports, there are as many as 39 trillion bacterial cells in the human gut, which is 1.3 times the number of human cells, and the number of genes encoded by these bacteria is more than 100 times that of human genes. As a micro-ecological system, the human intestinal flora restricts and depends on each other to form an ecological balance in terms of quality and quantity. Intestinal flora with stable members and reasonable functions has an important positive impact on the normal physiological metabolism, nutrient absorption, immune maintenance, hormone balance, central nervous system maturity and even behavioral characteristics of the human body. The imbalance of intestinal fl...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/10
CPCC12N15/10
Inventor 王婷婷吴琼李佩璐秦楠
Owner SHANGHAI REALBIO TECH CO LTD
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