Method for directed screening of soil target biological flora

A target microorganism and directional screening technology, which is applied in the field of directional screening of soil target microbial flora, can solve the problems of limited selection of selective medium, long cycle, and small number of target microbial flora, and achieve directional high-throughput rapid screening , simple operation, and shorten the strain identification period

Active Publication Date: 2018-10-02
HUATIAN NANJING ENG & TECH CORP MCC +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Due to the limited selection effect of selective medium, the number of isolated target microbial flora is small, especially for a large number of flora without suitable selective medium for use
Moreover, the traditional separation process is costly and takes a long time

Method used

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  • Method for directed screening of soil target biological flora
  • Method for directed screening of soil target biological flora
  • Method for directed screening of soil target biological flora

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] S1 Collect soil samples and prepare soil suspension: take 10g of fresh soil samples, put them in a sterile Erlenmeyer flask, add 90mL of sterile phosphate buffer (pH 6.5, containing 1g / L of potassium dihydrogen phosphate), and shake on a shaker 60min, 47kHz ultrasound once every 15min, 30s each time;

[0030] S2 Separation and cultivation: 10-fold gradient dilution of the soil suspension in step S1, take an appropriate amount of dilution (the dilution factor is 10, 10 2 、10 3 ) evenly spread on the culture medium plate containing fungal inhibitors (cycloheximide and thiabendazole, the concentrations are respectively 100 and 50 mg / L), cultivated in a 30°C incubator for 7 days, and the selected culture medium is low nutrient culture Base, in which, 1g / L potassium dihydrogen phosphate, 0.1g / L ammonium sulfate, 0.1g / L yeast extract, 20g / L agar, pH 6.5, use this medium to produce single colony; use a sterile toothpick to pick a single colony Streak and purify the colony on...

Embodiment 2

[0033] S1 Collect soil samples and prepare soil suspension: take 10g of fresh soil samples, put them in a sterile Erlenmeyer flask, add 90mL of sterile phosphate buffer (pH 6.5, containing 1g / L of potassium dihydrogen phosphate), and shake on a shaker 60min, 47kHz ultrasound once every 15min, 30s each time;

[0034] S2 Separation and cultivation: 10-fold gradient dilution of the soil suspension in step S1, take an appropriate amount of dilution (the dilution factor is 10, 10 2 、10 3 ) evenly spread on the culture medium plate containing fungal inhibitors (cycloheximide and thiabendazole, concentration respectively 100 and 50mg / L), cultivated in 30 ℃ incubator for 2 days, the selected culture medium is eutrophic culture Base, among them, tryptone 15.0g / L, soybean papain digest 5.0g / L, sodium chloride 5.0g / L, agar 15.0g / L, pH 7.1~pH 7.5; use a sterile toothpick to pick a single colony Streak and purify on the medium plate; use a sterile toothpick to pick the purified single co...

Embodiment 3

[0037] S1 Collect soil samples and prepare soil suspension: take 10g of fresh soil samples, put them in a sterile Erlenmeyer flask, add 90mL of sterile phosphate buffer (pH 6.5, containing 1g / L of potassium dihydrogen phosphate), and shake on a shaker 60min, 47kHz ultrasound once every 15min, 30s each time;

[0038] S2 Separation and cultivation: 10-fold gradient dilution of the soil suspension in step S1, take an appropriate amount of dilution (the dilution factor is 10, 10 2 、10 3 ) evenly spread on the culture medium plate containing fungal inhibitors (cycloheximide and thiabendazole, concentration respectively 100 and 50mg / L), cultivated in 30 ℃ incubator for 2 days, the selected culture medium is eutrophic culture Base, among them, tryptone 15.0g / L, soybean papain digest 5.0g / L, sodium chloride 5.0g / L, agar 15.0g / L, pH 7.1~pH 7.5; use a sterile toothpick to pick a single colony Streak and purify on the medium plate; use a sterile toothpick to pick the purified single co...

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Abstract

The invention discloses a method for directed screening of a soil target biological flora, wherein the method comprises the following steps: S1, sample collection; S2, isolation and culture; and S3, rapid screening by a polymerase chain reaction method with target microbial specific primers. A target flora in unknown bacterial floras is directly subjected to PCR rapid screening by the specific primers and a bacterial liquid. The whole screening process is simple to operate, saves time and labor, is fast, intuitive and efficient, and provides a fast and convenient method for high-throughput screening of the target flora in environmental samples.

Description

technical field [0001] The invention relates to the technical field of environmental microorganisms, in particular to a method for directional screening of soil target microbial flora. Background technique [0002] There are a large number of microorganisms in the soil, and there are various types. Microorganisms can degrade organic pollutants in the soil environment, and can also improve the efficiency of phytoremediation by promoting plant growth. Because the microorganisms added to the soil from exogenous sources need to compete with the indigenous microorganisms, the added microorganisms exist in the soil environment for a short time. Rapid screening of target microbial flora in soil samples can effectively improve the efficiency of phytoremediation and microbial remediation of contaminated soil. [0003] The traditional screening of target microbial flora mainly uses the selective medium method. Since the selection effect of the selective medium is often very limited...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/02C12N1/20C12Q1/686C12Q1/689C12Q1/04
CPCC12N1/02C12N1/20C12Q1/686C12Q1/689
Inventor 刘金光李明项萌冉景朱巧红高卫民夏溢焦朋朋程寒飞安忠义
Owner HUATIAN NANJING ENG & TECH CORP MCC
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