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A method for screening and preserving bacterial genetic resources

A gene and bacterial technology, applied in the screening and preservation of bacterial genetic resources, can solve the problems of difficult direct application of haplotype bacterial analysis, high randomness, low accuracy, etc., to achieve stable methods, strong purpose, and low cost little effect

Active Publication Date: 2019-11-15
大襟岛海洋生物谷(深圳)有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Random and simple selection are mainly based on simple methods such as different colors of bacterial strains, plaque traits, and plaque sizes, which are more random and less accurate; the method of physiological and biochemical markers has the ability to distinguish the color, gas, and precipitation of physiological and biochemical reactions. Error, and the cycle is long, the accuracy is poor
[0004] The SRAP molecular marker method is mainly used in the construction of crop core germplasm, however, the plant is a diploid or polyploid species, and the type of markers selected in the analysis of the plant (phenotype, SSR molecular marker, single nucleotide polymorphism, etc.) are not suitable for analysis in bacteria, making it difficult to directly apply the method to bacterial analysis of haplotypes

Method used

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  • A method for screening and preserving bacterial genetic resources
  • A method for screening and preserving bacterial genetic resources
  • A method for screening and preserving bacterial genetic resources

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Isolation of strains

[0044] Take five 12-day cut-flower carnations in vases, cut off the stem ends of about 1cm, and use sterile scalpel tweezers to chop the stem ends and put them into test tubes, add sterilized deionized water until the stem ends are submerged , put the cork on the shaker and shake for 5 minutes. Dilute the bacterial solution by 10 -1 、10 -2、 10 -3 These three concentrations are labeled well. Dip the bacterial liquid with an inoculation needle, and do a streak separation on the LB medium, so as to achieve the purpose of isolating the bacterial species. Each sample has 10 replicates, which are marked and placed in an incubator for cultivation. After cultivating for 12 hours, observe the plate. If there is a single colony on the line marked by the inoculation needle, select the single colony, and then carry out streak plate culture to select a single strain to ensure the purity of the strain, and mark them in order, marked as S1, S2 , S3···S52, a...

Embodiment 2

[0047] Extract the total DNA of the bacterial classification obtained in Example 1, and the specific operations are as follows

[0048] (1) Pipette 2mL of bacterial liquid into a 2mL centrifuge tube, centrifuge at 10000rpm for 2min, and discard the supernatant.

[0049] (2) Add 0.5 mL of normal saline, resuspend the bacterial mass, centrifuge at 10,000 rpm for 2 min, and discard the supernatant.

[0050](3) Add 0.5mL extract, resuspend the bacterial mass, and bathe in water at 65°C for 30min,

[0051] (4) Take it out of the water bath and shake it well for 1 minute, then place it at -20°C for 10 minutes.

[0052] (5) Add 0.5mL chloroform / water-saturated phenol (volume ratio 1:1), shake well for 3 minutes and then centrifuge at 12000rpm for 10 minutes.

[0053] (6) Take about 0.4 mL of the supernatant, add an equal volume of isopropanol to gently mix dozens of times, and then stand at -20°C for more than 30 minutes.

[0054] (7) Discard the supernatant after centrifugation a...

Embodiment 3

[0057] SRAP PCR and establishment of matrix table: SRAPPCR was carried out using the strain DNA extracted in Example 2 as a template; the primer combinations used in PCR were: F1R1, F1R4, F2R1, F2R2, F3R1, F3R3;

[0058] Said:

[0059] F1 is TGAGTCCAAACCGGATA,

[0060] F2 is TGAGTCCAAACCGGAGC,

[0061] F3 is TGAGTCCAAACCGGAAG,

[0062] R1 is GACTGCGTACGAATTGAC,

[0063] R2 is GACTGCGTACGAATTTGA,

[0064] R3 is GACTGCGTACGAATTCAA,

[0065] R4 is GACTGCGTACGAATTAGC.

[0066] The amplification procedure of described PCR is:

[0067] (1) Preheat at 94°C for 5 minutes;

[0068] (2) Denaturation at 94°C for 45s, primer annealing at 35°C for 1min, extension at 72°C for 1min30s, 5 cycles;

[0069] (3) Denaturation at 94°C for 45s, primer annealing at 50°C for 1min, extension at 72°C for 1min45s, a total of 35 cycles;

[0070] (4) Extend at 72°C for 10 minutes and store at 4°C.

[0071] The PCR product was subjected to polyacrylamide electrophoresis, and the specific operatio...

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Abstract

The invention discloses a bacterial gene resource screening and storing method which includes the steps: (1) separating strains; (2) extracting DNA (deoxyribonucleic acid) of the strains; (3) building a matrix table; (4) acquiring a screening scheme; (5) storing representative bacterial strains. An SRAP (sequence-related amplified polymorphism) molecular marker method based on bacterial genome information adopts specific primers and is stable and high in purposefulness.

Description

technical field [0001] The invention relates to the field of microorganisms, in particular to a method for screening and preserving bacterial gene resources. Background technique [0002] Bacterial genetic resources are an important factor for maintaining bacterial diversity. Preserving a large number of bacterial genetic types and numbers requires a lot of manpower and material resources. Therefore, through the evaluation and analysis of bacterial genotypes, the repetitive types in the bacterial community can be removed with a minimum amount of The bacteria of maximally represent the total bacterial genetic resources. [0003] The traditional methods for screening different bacteria generally mainly adopt: 1 random selection, 2 simple selection, 3 methods of physiological and biochemical markers. Random and simple selection are mainly based on simple methods such as different colors of bacterial strains, plaque traits, and plaque sizes, which are more random and less accur...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6858C12Q1/04
CPCC12Q1/6858C12Q2537/165C12Q2565/125
Inventor 刘季平刘文周玲艳
Owner 大襟岛海洋生物谷(深圳)有限公司
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