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Microorganism sample DNA storage solution

A technology of microorganisms and preservation solutions, applied in the field of biological sample preservation solutions, which can solve problems such as poor applicability, unsuitability for clinical testing, and unsuitability for DNA preservation

Inactive Publication Date: 2017-10-03
董克海
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] For the preservation of fecal microorganisms in the prior art, low-temperature freezing methods are often used. For example, the invention patent of Chinese Patent Publication No. CN102864138A discloses a method for extracting DNA from human feces. at -20°C to -80°C, however, the low-temperature freezing method requires the use of large-scale equipment, which has poor applicability and complicated operation, and is not suitable for extensive clinical testing; common DNA preservation solutions usually contain EDTA, although it can Used at room temperature, however, this DNA preservation solution is not suitable for DNA preservation of fecal microbial samples, and has limitations in use

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  • Microorganism sample DNA storage solution

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] Example 1 Sample source and group storage method

[0023] Due to the high biological preservation requirements of fecal microbial samples, a preferred embodiment of the present invention uses human fecal microbial samples as experimental samples to carry out the following experiments.

[0024] S1. Collect feces samples from 5 volunteers, collect 18 samples from each volunteer, and do 6 different treatments respectively, and repeat the test 3 times for each treatment;

[0025] S2, different processing methods

[0026] a. Freeze for one week (label 0): Collect at least 0.2g of feces samples, quick-freeze in liquid nitrogen, store in a sealed seal at -80°C, store for one week for DNA extraction, 16S rRNA gene V3 / V4 variable region amplification, library construction , sequencing, etc.;

[0027] b. Preservation solution for one week (label 1-3): Collect at least 0.2g of feces samples, use 3 different formulas of the present invention (the ingredients are shown in Table 1)...

Embodiment 2

[0034] Embodiment 2 Fecal sample processing method

[0035] The present embodiment provides a kind of processing method to above-mentioned stool sample:

[0036] S1. Pretreatment of samples in the control group:

[0037] Solid feces (corresponding to sample numbers 0 and 5):

[0038] Weigh 200mg of solid feces into a 2ml centrifuge tube, add 800ul stool DNA Buffer A, fully shake and mix for about 5min, and centrifuge at 1800g for 1min;

[0039] Take out 50ul of the resuspension in a clean 1.5ml centrifuge tube, add 800ul of Lysis-BindingBuffer, vortex and mix, and lyse at 70°C for 5min. After centrifugation for 5 minutes, transfer the supernatant to a clean 1.5ml centrifuge tube.

[0040] S2, experimental group sample pretreatment

[0041] Feces preservation solution (corresponding to sample number 1-4):

[0042] Take 200ul of semi-liquid feces and add no more than 10% (w / v) stool DNA Buffer A to dilute, fully shake for 5min, and take out 50ul of the resuspension into a c...

Embodiment 3

[0058] Example 3 Comparison of the diversity of the sample flora in different preservation methods

[0059] After the above-mentioned experimental operations, the sequence information of the intestinal flora of 5 volunteers under 6 treatments was obtained, and through bioinformatics analysis, the Shannon diversity index of the flora was calculated and compared. The Shannon index of each volunteer after testing is as follows: figure 1 As shown, the statistical results are shown in Table 3. The higher the value of the Shannon diversity index in the table, the higher the richness of the bacteria, and the p value is the difference between the sample numbered 0-4 and the sample numbered 5. The reliability analysis, the result shows, the sample of label 0-4 can basically satisfy the preservation of feces microbial sample DNA, yet compared with the preservation solution (label 4) of prior art, the treatment effect of microbial sample preservation solution of the present invention Th...

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Abstract

The present invention relates to a biological sample storage liquid, particularly to a microorganism sample DNA storage solution, wherein a 0.5-2 mM / L EDTA, saturated sodium chloride (NaCl), methanol with a mass fraction of 2-5%, glycerol with a mass fraction of 5-15%, fatty alcohol polyoxyethylene ether with a mass fraction of 5-10%, sodium dodecylsulfate (SDS) with a mass fraction of 1-5% and mixed mineral oil are dissolved in pure water, and the pH value of the obtained solution is adjusted to 6.0-9.0. According to the present invention, the microorganism sample DNA storage solution is suitable for the room temperature storage of microorganism samples, has characteristics of simple operation and good applicability, and can be used in large-scale sample detection.

Description

technical field [0001] The invention relates to a biological sample preservation solution, in particular to a microbial sample DNA preservation solution. Background technique [0002] Polymerase chain reaction (PCR) is an in vitro nucleic acid amplification technique widely used in molecular biology and medicine. The first step of PCR detection technology is the preparation of template DNA, that is, the extraction of DNA in the sample, which directly affects the result of PCR reaction. With the development of PCR technology, there have been many methods for extracting genomic DNA. However, it is necessary to quickly detect pathogenic microorganisms from on-site environmental samples such as soil, dust, and sewage, or human tissue samples such as feces. Due to the small amount of templates and the fact that the microorganisms are out of the original living environment, the original microorganisms often fail. Changes in abundance and DNA damage greatly reduce the sensitivity...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
CPCC12Q1/6806C12Q2527/125C12Q2523/113
Inventor 董克海
Owner 董克海
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